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  • 1. Abraham, Nabil M.
    et al.
    Liu, Lei
    Jutras, Brandon Lyon
    Yadav, Akhilesh K.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Narasimhan, Sukanya
    Gopalakrishnan, Vissagan
    Ansari, Juliana M.
    Jefferson, Kimberly K.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Jacobs-Wagner, Christine
    Fikrig, Erol
    Pathogen-mediated manipulation of arthropod microbiota to promote infection2017Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 5, s. E781-E790Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Arthropods transmit diverse infectious agents; however, the ways microbes influence their vector to enhance colonization are poorly understood. Ixodes scapularis ticks harbor numerous human pathogens, including Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We now demonstrate that A. phagocytophilum modifies the I. scapularis microbiota to more efficiently infect the tick. A. phagocytophilum induces ticks to express Ixodes scapularis antifreeze glycoprotein (iafgp), which encodes a protein with several properties, including the ability to alter bacterial biofilm formation. IAFGP thereby perturbs the tick gut microbiota, which influences the integrity of the peritrophic matrix and gut barrier-critical obstacles for Anaplasma colonization. Mechanistically, IAFGP binds the terminal D-alanine residue of the pentapeptide chain of bacterial peptidoglycan, resulting in altered permeability and the capacity of bacteria to form biofilms. These data elucidate the molecular mechanisms by which a human pathogen appropriates an arthropod antibacterial protein to alter the gut microbiota and more effectively colonize the vector.

  • 2.
    Aliashkevich, Alena
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Alvarez, Laura
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    New Insights Into the Mechanisms and Biological Roles of D-Amino Acids in Complex Eco-Systems2018Ingår i: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 9, artikel-id 683Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    In the environment bacteria share their habitat with a great diversity of organisms, from microbes to humans, animals and plants. In these complex communities, the production of extracellular effectors is a common strategy to control the biodiversity by interfering with the growth and/or viability of nearby microbes. One of such effectors relies on the production and release of extracellular D-amino acids which regulate diverse cellular processes such as cell wall biogenesis, biofilm integrity, and spore germination. Non-canonical D-amino acids are mainly produced by broad spectrum racemases (Bsr). Bsr's promiscuity allows it to generate high concentrations of D-amino acids in environments with variable compositions of L-amino acids. However, it was not clear until recent whether these molecules exhibit divergent functions. Here we review the distinctive biological roles of D-amino acids, their mechanisms of action and their modulatory properties of the biodiversity of complex eco-systems.

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  • 3.
    Aliashkevich, Alena
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    LD-transpeptidases: the great unknown among the peptidoglycan cross-linkers2022Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 289, nr 16, s. 4718-4730Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The peptidoglycan (PG) cell wall is an essential polymer for the shape and viability of bacteria. Its protective role is in great part provided by its mesh-like character. Therefore, PG-cross-linking enzymes like the penicillin-binding proteins (PBPs) are among the best targets for antibiotics. However, while PBPs have been in the spotlight for more than 50 years, another class of PG-cross-linking enzymes called LD-transpeptidases (LDTs) seemed to contribute less to PG synthesis and, thus, has kept an aura of mystery. In the last years, a number of studies have associated LDTs with cell wall adaptation to stress including β-lactam antibiotics, outer membrane stability, and toxin delivery, which has shed light onto the biological meaning of these proteins. Furthermore, as some species display a great abundance of LD-cross-links in their cell wall, it has been hypothesized that LDTs could also be the main synthetic PG-transpeptidases in some bacteria. In this review, we introduce these enzymes and their role in PG biosynthesis and we highlight the most recent advances in understanding their biological role in diverse species.

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  • 4.
    Aliashkevich, Alena
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Howell, Matthew
    Division of Biological Sciences, University of Missouri, Columbia, MO, USA; Department of Biology and Environmental Science, Westminster College, Fulton, MO, USA.
    Brown, Pamela J. B.
    Division of Biological Sciences, University of Missouri, Columbia, MO, USA.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    D-canavanine affects peptidoglycan structure, morphogenesis and fitness in Rhizobiales2021Ingår i: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 23, nr 10, s. 5823-5836Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. Many bacteria alter their PG chemistry as a strategy to adapt their cell wall to external challenges. Therefore, identifying these environmental cues is important to better understand the interplay between microbes and their habitat. Here we used the soil bacterium Pseudomonas putida to uncover cell wall modulators from plant extracts and found canavanine (CAN), a non-proteinogenic amino acid. We demonstrated that cell wall chemical editing by CAN is licensed by P. putida BSAR, a broad-spectrum racemase which catalyzes production of DL-CAN from L-CAN, which is produced by many legumes. Importantly, D-CAN diffuses to the extracellular milieu thereby having a potential impact on other organisms inhabiting the same niche. Our results show that D-CAN alters dramatically the PG structure of Rhizobiales (e.g. Agrobacterium tumefaciens, Sinorhizobium meliloti), impairing PG crosslinkage and cell division. Using A. tumefaciens we demonstrated that the detrimental effect of D-CAN is suppressed by a single amino acid substitution in the cell division PG transpeptidase penicillin binding protein 3a. Collectively, this work highlights the role of amino acid racemization in cell wall chemical editing and fitness.

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  • 5.
    Aliashkevich, Alena
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Schiffthaler, Bastian
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Genetic dissection of LD-transpeptidation in Agrobacterium tumefaciensManuskript (preprint) (Övrigt vetenskapligt)
  • 6.
    Alvarez, Laura
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Aliashkevich, Alena
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    de Pedro, Miguel A.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Bacterial secretion of D-arginine controls environmental microbial biodiversity2018Ingår i: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 12, nr 2, s. 438-450Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacteria face tough competition in polymicrobial communities. To persist in a specific niche, many species produce toxic extracellular effectors to interfere with the growth of nearby microbes. These effectors include the recently reported non-canonical D-amino acids (NCDAAs). In Vibrio cholerae, the causative agent of cholera, NCDAAs control cell wall integrity in stationary phase. Here, an analysis of the composition of the extracellular medium of V. cholerae revealed the unprecedented presence of D-Arg. Compared with other D-amino acids, D-Arg displayed higher potency and broader toxicity in terms of the number of bacterial species affected. Tolerance to D-Arg was associated with mutations in the phosphate transport and chaperone systems, whereas D-Met lethality was suppressed by mutations in cell wall determinants. These observations suggest that NCDAAs target different cellular processes. Finally, even though virtually all Vibrio species are tolerant to D-Arg, only a few can produce this D-amino acid. Indeed, we demonstrate that D-Arg may function as part of a cooperative strategy in vibrio communities to protect non-producing members from competing bacteria. Because NCDAA production is widespread in bacteria, we anticipate that D-Arg is a relevant modulator of microbial subpopulations in diverse ecosystems.

  • 7.
    Alvarez, Laura
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Bacterial Competition Assay Based on Extracellular D-amino Acid Production2018Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 8, nr 7, artikel-id e2787Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacteria live in polymicrobial communities under tough competition. To persist in a specific niche many species produce toxic extracellular effectors as a strategy to interfere with the growth of nearby microbes. One of such effectors are the non-canonical D-amino acids. Here we describe a method to test the effect of D-amino acid production in fitness/survival of bacterial subpopulations within a community. Co-cultivation methods usually involve the growth of the competing bacteria in the same container. Therefore, within such mixed cultures the effect on growth caused by extracellular metabolites cannot be distinguished from direct physical interactions between species (e.g., T6SS effectors). However, this problem can be easily solved by using a filtration unit that allows free diffusion of small metabolites, like L- and D-amino acids, while keeping the different subpopulations in independent compartments. With this method, we have demonstrated that D-arginine is a bactericide effector produced by Vibrio cholerae, which strongly influences survival of diverse microbial subpopulations. Moreover, D-arginine can be used as a cooperative instrument in mixed Vibrio communities to protect non-producing members from competing bacteria.

  • 8.
    Alvarez, Laura
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cordier, Baptiste
    van Teeffelen, Sven
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Analysis of Gram-negative Bacteria Peptidoglycan by Ultra-performance Liquid Chromatography2020Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 10, nr 19, artikel-id e3780Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacteria are surrounded by a protective peptidoglycan cell wall. Provided that this structure and the enzymes involved are the preferred target for our most successful antibiotics, determining its structural and chemical complexity is of the highest interest. Traditionally, high-performance liquid chromatography (HPLC) analyses have been performed, but these methods are very time consuming in terms of sample preparation and chromatographic separation. Here we describe an optimized method for preparation of Gram-negative bacteria peptidoglycan and its subsequent analysis by ultra-performance liquid chromatography (UPLC). The use of UPLC in peptidoglycan analyses provides a dramatic reduction of the sample volume and hands-on time required and, furthermore, permits in-line mass spectrometry (MS) of the UPLC resolved muropeptides, thus facilitating their identification. This method improves our capability to perform high throughput analysis to better understand the cell- wall biology.

  • 9.
    Alvarez, Laura
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Espaillat, Akbar
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hermoso, Juan A.
    de Pedro, Miguel A.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Peptidoglycan Remodeling by the Coordinated Action of Multispecific Enzymes2014Ingår i: Microbial Drug Resistance, ISSN 1076-6294, E-ISSN 1931-8448, Vol. 20, nr 3, s. 190-198Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The peptidoglycan (PG) cell wall constitutes the main defense barrier of bacteria against environmental insults and acts as communication interface. The biochemistry of this macromolecule has been well characterized throughout the years but recent discoveries have unveiled its chemical plasticity under environmental stresses. Non-canonical D-amino acids (NCDAA) are produced and released to the extracellular media by diverse bacteria. Such molecules govern cell wall adaptation to challenging environments through their incorporation into the polymer, a widespread capability among bacteria that reveals the inherent catalytic plasticity of the enzymes involved in the cell wall metabolism. Here, we analyze the recent structural and biochemical characterization of Bsr, a new family of broad spectrum racemases able to generate a wide range of NCDAA. We also discuss the necessity of a coordinated action of PG multispecific enzymes to generate adequate levels of modification in the murein sacculus. Finally, we also highlight how this catalytic plasticity of NCDAA-incorporating enzymes has allowed the development of new revolutionary methodologies for the study of PG modes of growth and in vivo dynamics.

  • 10.
    Alvarez, Laura
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hernandez, Sara B.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cell Wall Biology of Vibrio cholerae2021Ingår i: Annual Review of Microbiology, ISSN 0066-4227, E-ISSN 1545-3251, Vol. 75, s. 151-174Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Most bacteria are protected from environmental offenses by a cell wall consisting of strong yet elastic peptidoglycan. The cell wall is essential for preserving bacterial morphology and viability, and thus the enzymes involved in the production and turnover of peptidoglycan have become preferred targets for many of our most successful antibiotics. In the past decades, Vibrio cholerae, the gram-negative pathogen causing the diarrheal disease cholera, has become a major model for understanding cell wall genetics, biochemistry, and physiology. More than 100 articles have shed light on novel cell wall genetic determinants, regulatory links, and adaptive mechanisms. Here we provide the first comprehensive review of V. cholerae's cell wall biology and genetics. Special emphasis is placed on the similarities and differences with Escherichia coli, the paradigm for understanding cell wall metabolism and chemical structure in gram-negative bacteria.

  • 11.
    Alvarez, Laura
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Hernandez, Sara B
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    de Pedro, Miguel A
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ultra-sensitive, high-resolution liquid chromatography methods for the high-throughput quantitative analysis of bacterial cell wall chemistry and structure2016Ingår i: Bacterial cell wall homeostasis: methods and protocols /edited by Hee-Jeon Hong / [ed] Hee-Jeon Hong, New York: Humana Press, 2016, Vol. 1440, s. 11-27Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    High-performance liquid chromatography (HPLC) analysis has been critical for determining the structural and chemical complexity of the cell wall. However this method is very time consuming in terms of sample preparation and chromatographic separation. Here we describe (1) optimized methods for peptidoglycan isolation from both Gram-negative and Gram-positive bacteria that dramatically reduce the sample preparation time, and (2) the application of the fast and highly efficient ultra-performance liquid chromatography (UPLC) technology to muropeptide separation and quantification. The advances in both analytical instrumentation and stationary-phase chemistry have allowed for evolved protocols which cut run time from hours (2-3 h) to minutes (10-20 min), and sample demands by at least one order of magnitude. Furthermore, development of methods based on organic solvents permits in-line mass spectrometry (MS) of the UPLC-resolved muropeptides. Application of these technologies to high-throughput analysis will expedite the better understanding of the cell wall biology.

  • 12. Amon, Jeremy D.
    et al.
    Yadav, Akhilesh K.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Analytical Chemistry Division, CSIR-Indian Institute of Toxicology Research, Lucknow, India.
    Ramirez-Guadiana, Fernando H.
    Meeske, Alexander J.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Rudner, David Z.
    SwsB and SafA Are Required for CwlJ-Dependent Spore Germination in Bacillus subtilis2020Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 202, nr 6, artikel-id e00668-19Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    When Bacillus subtilis spores detect nutrients, they exit dormancy through the processes of germination and outgrowth. A key step in germination is the activation of two functionally redundant cell wall hydrolases (SleB and CwlJ) that degrade the specialized cortex peptidoglycan that surrounds the spore. How these enzymes are regulated remains poorly understood. To identify additional factors that affect their activity, we used transposon sequencing to screen for synthetic germination defects in spores lacking SleB or CwlJ. Other than the previously characterized protein YpeB, no additional factors were found to be specifically required for SleB activity. In contrast, our screen identified SafA and YlxY (renamed SwsB) in addition to the known factors GerQ and CotE as proteins required for CwlJ function. SafA is a member of the spore's proteinaceous coat and we show that, like GerQ and CotE, it is required for accumulation and retention of CwlJ in the dormant spore. SwsB is broadly conserved among spore formers, and we show that it is required for CwlJ to efficiently degrade the cortex during germination. Intriguingly, SwsB resembles polysaccharide deacetylases, and its putative catalytic residues are required for its role in germination. However, we find no chemical signature of its activity on the spore cortex or in vitro. While the precise, mechanistic role of SwsB remains unknown, we explore and discuss potential activities. IMPORTANCE Spore formation in Bacillus subtilis has been studied for over half a century, and virtually every step in this developmental process has been characterized in molecular detail. In contrast, how spores exit dormancy remains less well understood. A key step in germination is the degradation of the specialized cell wall surrounding the spore called the cortex. Two enzymes (SleB and CwlJ) specifically target this protective layer, but how they are regulated and whether additional factors promote their activity are unknown. Here, we identified the coat protein SafA and a conserved but uncharacterized protein YlxY as additional factors required for CwlJ-dependent degradation of the cortex. Our analysis provides a more complete picture of this essential step in the exit from dormancy.

  • 13.
    Aurass, Philipp
    et al.
    Department of Enteropathogenic Bacteria and Legionella, Robert Koch Institute, Wernigerode, Germany; Department of Molecular Biology and Microbiology, Tufts University School of Medicine, MA, Boston, United States.
    Kim, Seongok
    Department of Molecular Biology and Microbiology, Tufts University School of Medicine, MA, Boston, United States.
    Pinedo, Victor
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Isberg, Ralph R.
    Department of Molecular Biology and Microbiology, Tufts University School of Medicine, MA, Boston, United States.
    Identification of genes required for long-term survival of Legionella Pneumophila in water2023Ingår i: mSphere, E-ISSN 2379-5042, Vol. 8, nr 2, artikel-id e0045422Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Long-term survival of Legionella pneumophila in aquatic environments is thought to be important for facilitating epidemic outbreaks. Eliminating bacterial colonization in plumbing systems is the primary strategy that depletes this reservoir and prevents disease. To uncover L. pneumophila determinants facilitating survival in water, a Tn-seq strategy was used to identify survival-defective mutants during 50-day starvation in tap water at 42°C. The mutants with the most drastic survival defects carried insertions in electron transport chain genes, indicating that membrane energy charge and/or ATP synthesis requires the generation of a proton gradient by the respiratory chain to maintain survival in the presence of water stress. In addition, periplasmically localized proteins that are known (EnhC) or hypothesized (lpg1697) to stabilize the cell wall against turnover were essential for water survival. To test that the identified mutations disrupted water survival, candidate genes were knocked down by CRISPRi. The vast majority of knockdown strains with verified transcript depletion showed remarkably low viability after 50-day incubations. To demonstrate that maintenance of cell wall integrity was an important survival determinant, a deletion mutation in lpg1697, in a gene encoding a predicted l,d-transpeptidase domain, was analyzed. The loss of this gene resulted in increased osmolar sensitivity and carbenicillin hypersensitivity relative to the wild type, as predicted for loss of an l,d-transpeptidase. These results indicate that the L. pneumophila envelope has been evolutionarily selected to allow survival under conditions in which the bacteria are subjected to long-term exposure to starvation and low osmolar conditions. IMPORTANCE Water is the primary vector for transmission of L. pneumophila to humans, and the pathogen is adapted to persist in this environment for extended periods of time. Preventing survival of L. pneumophila in water is therefore critical for prevention of Legionnaires' disease. We analyzed dense transposon mutation pools for strains with severe survival defects during a 50-day water incubation at 42°C. By tracking the associated transposon insertion sites in the genome, we defined a distinct essential gene set for water survival and demonstrate that a predicted peptidoglycan cross-linking enzyme, lpg1697, and components of the electron transport chain are required to ensure survival of the pathogen. Our results indicate that select characteristics of the cell wall and components of the respiratory chain of L. pneumophila are primary evolutionary targets being shaped to promote its survival in water.

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  • 14.
    Bernardo-Garcia, Noelia
    et al.
    Department of Crystallography and Structural Biology, Instituto de Química Física "Rocasolano", CSIC, Madrid, Spain.
    Sanchez-Murcia, Pedro
    Univ Alcala De Henares, Area Farmacol, Dept Ciencias Biomed, Unidad Asociada I D I,CSIC, Madrid, Spain.
    Gago, Federico
    Univ Alcala De Henares, Area Farmacol, Dept Ciencias Biomed, Unidad Asociada I D I,CSIC, Madrid, Spain.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Hermoso, Juan A.
    CSIC, Inst Quim Fis Rocasolano, Dept Crystallog & Struct Chem, Madrid, Spain.
    Structural Bioinformatics in Broad-Spectrum Racemases: a new path in anti-microbial research2016Ingår i: Current organic chemistry, ISSN 1385-2728, E-ISSN 1875-5348, Vol. 20, nr 11, s. 1222-1231Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    D-amino acids are essential components of the bacterial cell wall and play notable roles in microbiology as regulators, for example in sporulation, biofilm formation or interspecies communication. Racemases are the specific enzymes catalyzing the interconversion of L-amino acids to D-amino acids. While most of racemases are mono-specific, a family of broad-spectrum racemases that can racemize ten of the 19 natural chiral amino acids has been recently reported. These enzymes can interconvert radically different residues such as aliphatic and positively charged residues producing non-canonical D-amino acids. Crystal structures together with bioinformatics allowed identification of the residues defining the molecular footprint in broad-spectrum racemases, the specific features of their active sites and the structural basis of their promiscuity. Here we review the recent knowledge on this family compared with the well established of alanine racemases. This structural information is a prerequisite for the development of novel drugs against the important human pathogens for which broad-spectrum racemases play a key role.

  • 15. Bernardo-Garcia, Noelia
    et al.
    Sánchez-Murcia, Pedro A.
    Espaillat, Akbar
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Martínez-Caballero, Siseth
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Hermoso, Juan A.
    Gago, Federico
    Cold-induced aldimine bond cleavage by Tris in Bacillus subtilis alanine racemase2019Ingår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 17, nr 17, s. 4350-4358Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pyridoxal 5'-phosphate (PLP) is a versatile cofactor involved in a large variety of enzymatic processes. Most of PLP-catalysed reactions, such as those of alanine racemases (AlaRs), present a common resting state in which the PLP is covalently bound to an active-site lysine to form an internal aldimine. The crystal structure of BsAlaR grown in the presence of Tris lacks this covalent linkage and the PLP cofactor appears deformylated. However, loss of activity in a Tris buffer only occurred after the solution was frozen prior to carrying out the enzymatic assay. This evidence strongly suggests that Tris can access the active site at subzero temperatures and behave as an alternate racemase substrate leading to mechanism-based enzyme inactivation, a hypothesis that is supported by additional X-ray structures and theoretical results from QM/ MM calculations. Taken together, our findings highlight a possibly underappreciated role for a common buffer component widely used in biochemical and biophysical experiments.

  • 16.
    Blas-Galindo, Emilio
    et al.
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Cava, Felipe
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    López-Viñas, Eduardo
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Mendieta, Jesús
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Berenguer, José
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus2007Ingår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 73, nr 16, s. 5138-5145Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.

  • 17.
    Boamah, David
    et al.
    Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States.
    Gilmore, Michael C.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Bourget, Sarah
    Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States.
    Ghosh, Anushka
    Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States.
    Hossain, Mohammad J.
    Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States.
    Vogel, Joseph P.
    Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, United States.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    O'Connor, Tamara J.
    Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States.
    Peptidoglycan deacetylation controls type IV secretion and the intracellular survival of the bacterial pathogen Legionella pneumophila2023Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 120, nr 23, artikel-id  e2119658120Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Peptidoglycan is a critical component of the bacteria cell envelope. Remodeling of the peptidoglycan is required for numerous essential cellular processes and has been linked to bacterial pathogenesis. Peptidoglycan deacetylases that remove the acetyl group of the N-acetylglucosamine (NAG) subunit protect bacterial pathogens from immune recognition and digestive enzymes secreted at the site of infection. However, the full extent of this modification on bacterial physiology and pathogenesis is not known. Here, we identify a polysaccharide deacetylase of the intracellular bacterial pathogen Legionella pneumophila and define a two-tiered role for this enzyme in Legionella pathogenesis. First, NAG deacetylation is important for the proper localization and function of the Type IVb secretion system, linking peptidoglycan editing to the modulation of host cellular processes through the action of secreted virulence factors. As a consequence, the Legionella vacuole mis-traffics along the endocytic pathway to the lysosome, preventing the formation of a replication permissive compartment. Second, within the lysosome, the inability to deacetylate the peptidoglycan renders the bacteria more sensitive to lysozyme-mediated degradation, resulting in increased bacterial death. Thus, the ability to deacetylate NAG is important for bacteria to persist within host cells and in turn, Legionella virulence. Collectively, these results expand the function of peptidoglycan deacetylases in bacteria, linking peptidoglycan editing, Type IV secretion, and the intracellular fate of a bacterial pathogen.

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  • 18.
    Bolivar, Juan M
    et al.
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain.
    Cava, Felipe
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Mateo, Cesar
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain.
    Rocha-Martín, Javier
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain.
    Guisán, Jose M
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain.
    Berenguer, José
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Fernandez-Lafuente, Roberto
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain.
    Immobilization-stabilization of a new recombinant glutamate dehydrogenase from Thermus thermophilus2008Ingår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 80, nr 1, s. 49-58Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.

  • 19.
    Bolivar, Juan M
    et al.
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Cantoblanco, Madrid, Spain.
    Rocha-Martin, Javier
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Cantoblanco, Madrid, Spain.
    Mateo, Cesar
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Cantoblanco, Madrid, Spain.
    Cava, Felipe
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Berenguer, Jose
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Fernandez-Lafuente, Roberto
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Cantoblanco, Madrid, Spain.
    Guisan, Jose M
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Cantoblanco, Madrid, Spain.
    Coating of soluble and immobilized enzymes with ionic polymers: full stabilization of the quaternary structure of multimeric enzymes2009Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 10, nr 4, s. 742-747Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This paper shows a simple and effective way to avoid the dissociation of multimeric enzymes by coating their surface with a large cationic polymer (e.g., polyethylenimine (PEI)) by ionic exchange. As model enzymes, glutamate dehydrogenase (GDH) from Thermus thermophilus and formate dehydrogenase (FDH) from Pseudomonas sp. were used. Both enzymes are very unstable at acidic pH values due to the rapid dissociation of their subunits (half-life of diluted preparations is few minutes at pH 4 and 25 degrees C). GDH and FDH were incubated in the presence of PEI yielding an enzyme-PEI composite with full activity. To stabilize the enzyme-polymer composite, a treatment with glutaraldehyde was required. These enzyme-PEI composites can be crosslinked with glutaraldehyde by immobilizing previously the composite onto a weak cationic exchanger. The soluble GDH-PEI composite was much more stable than unmodified GDH at pH 4 and 30 degrees C (retaining over 90% activity after 24 h incubation) with no effect of the GDH concentration in the inactivation course. The composite could be very strongly, but reversibly, adsorbed on cationic exchangers. Similarly, FDH could be treated with PEI and glutaraldehyde after adsorption on cationic exchangers, This permitted a stabilized FDH preparation. In this way, the coating of the enzymes surfaces with PEI is used as a simple and efficient strategy to prevent enzyme dissociation of multimeric enzymes. These composites can be used as a soluble catalyst or reversibly immobilized onto a cationic exchanger (e.g., CM-agarose).

  • 20.
    Bueno, Emilio
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Pinedo, Victor
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Adaptation of Vibrio cholerae to Hypoxic Environments2020Ingår i: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 11, artikel-id 739Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Bacteria can colonize virtually any environment on Earth due to their remarkable capacity to detect and respond quickly and adequately to environmental stressors. Vibrio cholerae is a cosmopolitan bacterium that inhabits a vast range of environments. The V. cholerae life cycle comprises diverse environmental and infective stages. The bacterium is found in aquatic ecosystems both under free-living conditions or associated with a wide range of aquatic organisms, and some strains are also capable of causing epidemics in humans. In order to adapt between environments, V. cholerae possesses a versatile metabolism characterized by the rapid cross-regulation of energy-producing pathways. Low oxygen concentration is a key environmental factor that governs V. cholerae physiology. This article reviews the metabolic plasticity that enables V. cholerae to thrive on low oxygen concentrations and its role in environmental and host adaptation.

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  • 21.
    Bueno, Emilio
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Pinedo, Victor
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Shinde, Dhananjay D.
    Center for Staphylococcal Research, Department of Pathology and Microbiology, University of Nebraska Medical Centergrid.266813.8, NE, Omaha, United States.
    Mateus, André
    Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
    Typas, Athanasios
    Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
    Savitski, Mikhail M.
    Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
    Thomas, Vinai C.
    Center for Staphylococcal Research, Department of Pathology and Microbiology, University of Nebraska Medical Centergrid.266813.8, NE, Omaha, United States.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Transient glycolytic complexation of arsenate enhances resistance in the enteropathogen Vibrio cholerae2022Ingår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 13, nr 5, artikel-id e0165422Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The ubiquitous presence of toxic arsenate (AsV) in the environment has raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress-related systems to counter reactive oxygen species (ROS) production caused by intracellular AsV. Characterization of the var mutants suggested that these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions. IMPORTANCE Even though resistance to arsenate has been extensively investigated in environmental bacteria, how enteric pathogens tolerate this toxic compound remains unknown. Here, we found that the cholera pathogen V. cholerae exhibits increased resistance to arsenate compared to closely related enteric pathogens. Such resistance is promoted not by ArsC-dependent reduction of arsenate to arsenite but by an operon encoding an arsenate transporter (ArsJ), an alternative glyceraldehyde 3-phosphate dehydrogenase (VarG), and a putative, uncharacterized phosphatase (VarH). Mechanistically, we demonstrate that V. cholerae detoxifies arsenate by complexing it with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). 1As3PG is not transported outside the cell; instead, it is subsequently dissociated by VarH to enable extrusion of free arsenate through ArsJ. Collectively, this study proposes a novel mechanism for arsenate detoxification, entirely independent of arsenate reduction and arsenite extrusion, that enhances V. cholerae resistance to this metalloid compared to other enteric pathogens.

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  • 22.
    Bueno, Emilio
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sit, Brandon
    Waldor, Matthew K.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Anaerobic nitrate reduction divergently governs population expansion of the enteropathogen Vibrio cholerae2018Ingår i: Nature Microbiology, E-ISSN 2058-5276, Vol. 3, nr 12, s. 1346-1353Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To survive and proliferate in the absence of oxygen, many enteric pathogens can undergo anaerobic respiration within the host by using nitrate (NO3-) as an electron acceptor(1,2). In these bacteria, NO3- is typically reduced by a nitrate reductase to nitrite (NO2-), a toxic intermediate that is further reduced by a nitrite reductase(3). However, Vibrio cholerae, the intestinal pathogen that causes cholera, lacks a nitrite reductase, leading to NO2- accumulation during nitrate reduction 4(.) Thus, V. cholerae is thought to be unable to undergo NO3-(-)dependent anaerobic respiration(4). Here, we show that during hypoxic growth, NO3- reduction in V. cholerae divergently affects bacterial fitness in a manner dependent on environmental pH. Remarkably, in alkaline conditions, V. cholerae can reduce NO3- to support population growth. Conversely, in acidic conditions, accumulation of NO2- from NO3- reduction simultaneously limits population expansion and preserves cell viability by lowering fermentative acid production. Interestingly, other bacterial species such as Salmonella typhimurium, enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium also reproduced this pH-dependent response, suggesting that this mechanism might be conserved within enteric pathogens. Our findings explain how a bacterial pathogen can use a single redox reaction to divergently regulate population expansion depending on the fluctuating environmental pH.

  • 23.
    Bueno, Emilio
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sit, Brandon
    Waldor, Matthew K.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Genetic Dissection of the Fermentative and Respiratory Contributions Supporting Vibrio cholerae Hypoxic Growth2020Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 202, nr 24, artikel-id e00243-20Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Both fermentative and respiratory processes contribute to bacterial metabolic adaptations to low oxygen tension (hypoxia). In the absence of O-2 as a respiratory electron sink, many bacteria utilize alternative electron acceptors, such as nitrate (NO3-). During canonical NO3- respiration, NO3- is reduced in a stepwise manner to N-2 by a dedicated set of reductases. Vibrio cholerae, the etiological agent of cholera, requires only a single periplasmic NO3- reductase (NapA) to undergo NO3- respiration, suggesting that the pathogen possesses a noncanonical NO3- respiratory chain. In this study, we used complementary transposon-based screens to identify genetic determinants of general hypoxic growth and NO3- respiration in V. cholerae. We found that while the V. cholerae NO3- respiratory chain is primarily composed of homologues of established NO3- respiratory genes, it also includes components previously unlinked to this process, such as the Na+-NADH dehydrogenase Nqr. The ethanol-generating enzyme AdhE was shown to be the principal fermentative branch required during hypoxic growth in V. cholerae. Relative to single adhE or napA mutant strains, a V. cholerae strain lacking both genes exhibited severely impaired hypoxic growth in vitro and in vivo. Our findings reveal the genetic basis of a specific interaction between disparate energy production pathways that supports pathogen fitness under shifting conditions. Such metabolic specializations in V. cholerae and other pathogens are potential targets for antimicrobial interventions.

    IMPORTANCE Bacteria reprogram their metabolism in environments with low oxygen levels (hypoxia). Typically, this occurs via regulation of two major, but largely independent, metabolic pathways: fermentation and respiration. In this study, we found that the diarrheal pathogen Vibrio cholerae has a respiratory chain for NO3- that consists largely of components found in other NO3- respiratory systems but also contains several proteins not previously linked to this process. Both AdhE-dependent fermentation and NO3- respiration were required for efficient pathogen growth under both laboratory conditions and in an animal infection model. These observations provide a specific example of fermentative respiratory interactions and identify metabolic vulnerabilities that may be targetable for new antimicrobial agents in V. cholerae and related pathogens.

  • 24.
    Campbell, Christopher
    et al.
    Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Fingleton, Claire
    Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Zeden, Merve S.
    Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Bueno, Emilio
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gallagher, Laura A.
    Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Shinde, Dhananjay
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Ahn, Jongsam
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Olson, Heather M.
    Biological Sciences Division, Pacific Northwest National Laboratory, WA, Richland, United States.
    Fillmore, Thomas L.
    Biological Sciences Division, Pacific Northwest National Laboratory, WA, Richland, United States.
    Adkins, Joshua N.
    Biological Sciences Division, Pacific Northwest National Laboratory, WA, Richland, United States.
    Razvi, Fareha
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Bayles, Kenneth W.
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Fey, Paul D.
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Thomas, Vinai C.
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Clair, Geremy C.
    Biological Sciences Division, Pacific Northwest National Laboratory, WA, Richland, United States.
    O’gara, James P.
    Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Accumulation of succinyl coenzyme a perturbs the methicillin-resistant staphylococcus aureus (Mrsa) succinylome and is associated with increased susceptibility to beta-lactam antibiotics2021Ingår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 12, nr 3, artikel-id e00530-21Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Penicillin binding protein 2a (PBP2a)-dependent resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact β-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to β-lactam antibiotics.

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  • 25. Castanheira, Sonia
    et al.
    Cestero, Juan J.
    Rico-Perez, Gadea
    Garcia, Pablo
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ayala, Juan A.
    Graciela Pucciarelli, M.
    Garcia-del Portillo, Francisco
    A Specialized Peptidoglycan Synthase Promotes Salmonella Cell Division inside Host Cells2017Ingår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, nr 6, artikel-id e01685-17Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacterial cell division has been studied extensively under laboratory conditions. Despite being a key event in the bacterial cell cycle, cell division has not been explored in vivo in bacterial pathogens interacting with their hosts. We discovered in Salmonella enterica serovar Typhimurium a gene absent in nonpathogenic bacteria and encoding a peptidoglycan synthase with 63% identity to penicillin-binding protein 3 (PBP3). PBP3 is an essential cell division-specific peptidoglycan synthase that builds the septum required to separate daughter cells. Since S. Typhimurium carries genes that encode a PBP3 paralog-which we named PBP3(SAL)-and PBP3, we hypothesized that there are different cell division events in host and nonhost environments. To test this, we generated S. Typhimurium isogenic mutants lacking PBP3(SAL) or the hitherto considered essential PBP3. While PBP3 alone promotes cell division under all conditions tested, the mutant producing only PBP3(SAL) proliferates under acidic conditions (pH <= 5.8) but does not divide at neutral pH. PBP3(SAL) production is tightly regulated with increased levels as bacteria grow in media acidified up to pH 4.0 and in intracellular bacteria infecting eukaryotic cells. PBP3(SAL) activity is also strictly dependent on acidic pH, as shown by beta-lactam antibiotic binding assays. Live-cell imaging microscopy revealed that PBP3(SAL) alone is sufficient for S. Typhimurium to divide within phagosomes of the eukaryotic cell. Additionally, we detected much larger amounts of PBP3(SAL) than those of PBP3 in vivo in bacteria colonizing mouse target organs. Therefore, PBP3(SAL) evolved in S. Typhimurium as a specialized peptidoglycan synthase promoting cell division in the acidic intraphagosomal environment. IMPORTANCE During bacterial cell division, daughter cells separate by a transversal structure known as the division septum. The septum is a continuum of the cell wall and therefore is composed of membrane(s) and a peptidoglycan layer. To date, actively growing bacteria were reported to have only a "cell division-specific" peptidoglycan synthase required for the last steps of septum formation and consequently, essential for bacterial life. Here, we discovered that Salmonella enterica has two peptidoglycan synthases capable of synthesizing the division septum. One of these enzymes, PBP3(SAL), is present only in bacterial pathogens and evolved in Salmonella to function exclusively in acidic environments. PBP3(SAL) is used preferentially by Salmonella to promote cell division in vivo in mouse target organs and inside acidified phagosomes. Our data challenge the concept of only one essential cell division-specific peptidoglycan synthase and demonstrate that pathogens can divide in defined host locations using alternative mechanisms.

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  • 26.
    Castán, Pablo
    et al.
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Zafra, Olga
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Moreno, Renata
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    de Pedro, Miguel A
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Vallés, Cristina
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Cava, Felipe
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Caro, Eddy
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Schwarz, Heinz
    Max Planck Institut für Entwicklungsbiologie, Tübingen, Germany.
    Berenguer, José
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    The periplasmic space in Thermus thermophilus: evidence from a regulation-defective S-layer mutant overexpressing an alkaline phosphatase2002Ingår i: Extremophiles, ISSN 1431-0651, E-ISSN 1433-4909, Vol. 6, nr 3, s. 225-232Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The presence of a periplasmic space within the cell envelope of Thermus thermophilus was analyzed in a mutant (HB8(Delta)UTR1) defective in the regulation of its S-layer (surface crystalline layer). This mutant forms round multicellular bodies (MBs) surrounded by a common envelope as the culture approaches the stationary phase. Confocal microscopy revealed that the origin of the MBs is the progressive detachment of the external layers coupled with the accumulation of NH(2)-containing material between the external envelopes and the peptidoglycan. A specific pattern of proteins was found as soluble components of the intercellular space of the MBs by a single fractionation procedure, suggesting that they are periplasmic-like components. To demonstrate this, we cloned a gene ( phoA) from T. thermophilus HB8 encoding a signal peptide-wearing alkaline phosphatase (AP), and engineered it to be overexpressed in the mutant from a shuttle vector. Most of the AP activity (>80%) was found as a soluble component of the MBs' intercellular fraction. All these data indicate that Thermus thermophilus actually has a periplasmic space which is functionally similar to that of Proteobacteria. The potential application of the HB8(Delta)UTR1 mutant for the overexpression of periplasmic thermophilic proteins is discussed.

  • 27.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Biology of Vibrio cholerae: editorial overview2017Ingår i: International Microbiology, ISSN 1139-6709, E-ISSN 1618-1905, Vol. 20, nr 3Artikel i tidskrift (Övrigt vetenskapligt)
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  • 28.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Divergent functional roles of D-amino acids secreted by Vibrio cholerae2017Ingår i: International Microbiology, ISSN 1139-6709, E-ISSN 1618-1905, Vol. 20, nr 3, s. 149-150Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The L-forms of amino acids are used in all kingdoms of life to synthesize proteins. However, the bacterium Vibrio cholerae, the causative agent of cholera, produces D-amino acids which are released to the environment at millimolar concentrations. We baptized these D-amino acids as non-canonical D-amino acids (NCDAAs) since they are different from those (i.e. D-alanine and D-glutamate) normally present in the bacterial cell wall. In V. cholerae, production of NCDAAs relies on the BsrV enzyme, a periplasmic broad spectrum racemase. BsrV multispecific activity, produces of a wide range of distinct D-amino acids. Using a combination of genetics and molecular physiology approaches we have demonstrated that NCDAAs target different cellular processes which may function as part of a cooperative strategy in vibrio communities to protect non-producing members from competing bacteria. Because NCDAA production is widespread in bacteria, we anticipate that NCDAAs are relevant modulators of microbial subpopulations in diverse ecosystems.

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  • 29.
    Cava, Felipe
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    de Pedro, Miguel A.
    Peptidoglycan plasticity in bacteria: emerging variability of the murein sacculus and their associated biological functions2014Ingår i: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 18, s. 46-53Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The peptidoglycan (PG) sacculus once thought to be just a reinforcing, static and uniform structure, is fast becoming recognized as a dynamic cell constituent involved in every aspect of bacterial physiology. Recent advances showed that in addition to 'classical' tasks - as an essential element to define bacterial shape, size, division and resistance to osmotic stress the sacculus plays very important roles in many other fields. The very few chemical and structural changes that were once considered as bizarre, or maybe exotic exceptions, are now universally accepted as fundamental pieces in bacterial cell wall adaptation to different kinds of environmental stresses; immune response; intra-specific and inter-specific signalling and antibiotics, just to mention a few. Most, if not all, of these implications are a consequence of the enormous adaptability of PG metabolism to cope with changing conditions, a characteristic for which the term plasticity is proposed. Here we overview and comment on a number of recent contributions on the cell wall adaptive responses to environmental challenges that has greatly impacted the already high complexity of the PG biology field. These new evidences have revived the interest in PG plasticity as an exciting and trendy topic in current microbiology which considers this variability as the trustworthy picture of bacterial PG in nature.

  • 30.
    Cava, Felipe
    et al.
    Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School and HHMI, Boston, MA, USA ; CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    de Pedro, Miguel A
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Lam, Hubert
    Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School and HHMI, Boston, MA, USA.
    Davis, Brigid M
    Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School and HHMI, Boston, MA, USA.
    Waldor, Matthew K
    Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School and HHMI, Boston, MA, USA.
    Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids2011Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 30, nr 16, s. 3442-3453Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Production of non-canonical D-amino acids (NCDAAs) in stationary phase promotes remodelling of peptidoglycan (PG), the polymer that comprises the bacterial cell wall. Impairment of NCDAAs production leads to excessive accumulation of PG and hypersensitivity to osmotic shock; however, the mechanistic bases for these phenotypes were not previously determined. Here, we show that incorporation of NCDAAs into PG is a critical means by which NCDAAs control PG abundance and strength. We identified and reconstituted in vitro two (of at least three) distinct processes that mediate NCDAA incorporation. Diverse bacterial phyla incorporate NCDAAs into their cell walls, either through periplasmic editing of the mature PG or via incorporation into PG precursor subunits in the cytosol. Production of NCDAAs in Vibrio cholerae requires the stress response sigma factor RpoS, suggesting that NCDAAs may aid bacteria in responding to varied environmental challenges. The widespread capacity of diverse bacteria, including non-producers, to incorporate NCDAAs suggests that these amino acids may serve as both autocrine- and paracrine-like regulators of chemical and physical properties of the cell wall in microbial communities.

  • 31.
    Cava, Felipe
    et al.
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    de Pedro, Miguel A
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Schwarz, Heinz
    Max Plank Institut für Entwicklungsbiologie, Tübingen, Germany.
    Henne, Anke
    Goettingen Genomics Laboratory, Institute for Microbiology and Genetics, Germany.
    Berenguer, José
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Binding to pyruvylated compounds as an ancestral mechanism to anchor the outer envelope in primitive bacteria2004Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 52, nr 3, s. 677-690Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Electron microscopy of isolated cell walls of the ancient bacterium Thermus thermophilus revealed that most of the peptidoglycan (PG) surface, apart from the septal region, was shielded against specific alphaPG antibodies. On the other hand, an antiserum raised against S-layer-attached cell wall fragments (alphaSAC) bound to most of the surface except for the septal regions. Treatments with alpha-amylase and pronase E made the entire cell wall surface uniformly accessible to alphaPG and severely decreased the binding of alphaSAC. We concluded that a layer of strongly bound secondary cell wall polymers (SCWPs) covers most of the cell wall surface in this ancient bacterium. A preliminary analysis revealed that such SCWPs constitute 14% of the cell wall and are essentially composed of sugars. Enzyme treatments of the cell walls revealed that SCWP was required in vitro for the binding of the S-layer protein through the S-layer homology (SLH) motif. The csaB gene was necessary for the attachment of the S-layer-outer membrane (OM) complex to the cell wall in growing cells of T. thermophilus. In vitro experiments confirmed that cell walls from a csaB mutant bound to the S-layer with a much lower affinity ( approximately 1/10) than that of the wild type. CsaB was found to be required for pyruvylation of components of the SCWP and for immunodetection with alpha-SAC antiserum. Therefore, the S-layer-OM complex of T. thermophilus binds to the cell wall through the SLH motif of the S-layer protein via a strong interaction with a highly immunogenic pyruvylated component of the SCWP. Immuno-cross-reactive compounds were detected with alphaSAC on cell walls of other Thermus spp. and in the phylogenetically related microorganism Deinococcus radiodurans. These results imply that the interaction between the SLH motif and pyruvylated components of the cell wall arose early during bacterial evolution as an ancestral mechanism for anchoring proteins and outer membranes to the cell walls of primitive bacteria.

  • 32.
    Cava, Felipe
    et al.
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    de Pedro, Miguel Angel
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Blas-Galindo, Emilio
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Waldo, Geoffrey S
    Bioscience Division, Los Alamos National Laboratory, USA.
    Westblade, Lars F
    The Rockefeller University, New York, USA.
    Berenguer, José
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Expression and use of superfolder green fluorescent protein at high temperatures in vivo: a tool to study extreme thermophile biology2008Ingår i: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 10, nr 3, s. 605-613Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Superfolder GFP (sGFP) is a variant of the Green Fluorescent Protein that folds efficiently when fused to poorly folded proteins. In this study, we show that sGFP, but not enhanced GFP, is functional in vivo at 70 degrees C in the extreme thermophile Thermus thermophilus (Tth); thus, permitting the use of sGFP as a localization tag in vivo. We created a suite of plasmids that allow the expression of carboxy-terminal sGFP fusion proteins in both Escherichia coli and Tth. In order to demonstrate the facility of sGFP as an in vivo localization tag in Tth, we tagged GroES (the small subunit of the bacterial GroES/GroEL chaperone), NarC (a membrane component of the nitrate respiration apparatus) and PhoA (a TAT-secreted periplasmic protein), and visualized the distribution of the sGFP fusion proteins using confocal microscopy. Fusions to NarC and PhoA produced enzymatically active proteins that complemented both the narC and the phoA strains respectively. Observation of the distribution of the GroES-sGFP protein by confocal microscopy revealed a homogeneous fluorescence in the cells, which is in full agreement with the cytoplasmic nature of GroES, whereas the NarC-sGFP protein was localized to the membrane. Finally, a combination of confocal microscopy and biochemistry revealed that PhoA is localized in the periplasm. We suggest that sGFP will be broadly applicable in characterizing various extreme thermophile systems.

  • 33.
    Cava, Felipe
    et al.
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Hidalgo, Aurelio
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Berenguer, José
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Thermus thermophilus as biological model2009Ingår i: Extremophiles, ISSN 1431-0651, E-ISSN 1433-4909, Vol. 13, nr 2, s. 213-31Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Thermus spp is one of the most wide spread genuses of thermophilic bacteria, with isolates found in natural as well as in man-made thermal environments. The high growth rates, cell yields of the cultures, and the constitutive expression of an impressively efficient natural competence apparatus, amongst other properties, make some strains of the genus excellent laboratory models to study the molecular basis of thermophilia. These properties, together with the fact that enzymes and protein complexes from extremophiles are easier to crystallize have led to the development of an ongoing structural biology program dedicated to T. thermophilus HB8, making this organism probably the best so far known from a protein structure point view. Furthermore, the availability of plasmids and up to four thermostable antibiotic selection markers allows its use in physiological studies as a model for ancient bacteria. Regarding biotechnological applications this genus continues to be a source of thermophilic enzymes of great biotechnological interest and, more recently, a tool for the over-expression of thermophilic enzymes or for the selection of thermostable mutants from mesophilic proteins by directed evolution. In this article, we review the properties of this organism as biological model and its biotechnological applications.

  • 34.
    Cava, Felipe
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kuru, Erkin
    Brun, Yves V.
    de Pedro, Miguel A.
    Modes of cell wall growth differentiation in rod-shaped bacteria2013Ingår i: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 16, nr 6, s. 731-737Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A bacterial cell takes on the challenge to preserve and reproduce its shape at every generation against a substantial internal pressure by surrounding itself with a mechanical support, a peptidoglycan cell wall. The enlargement of the cell wall via net incorporation of precursors into the pre-existing wall conditions bacterial growth and morphology. However, generation, reproduction and/or modification of a specific shape requires that the incorporation takes place at precise locations for a defined time period. Much has been learnt in the past few years about the biochemistry of the peptidoglycan synthesis process, but topological approaches to the understanding of shape generation have been hindered by a lack of appropriate techniques. Recent technological advances are paving the way for substantial progress in understanding the mechanisms of bacterial morphogenesis. Here we review the latest developments, focusing on the impact of new techniques on the precise mapping of cell wall growth sites.

  • 35.
    Cava, Felipe
    et al.
    Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, USA.
    Lam, Hubert
    Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, USA.
    de Pedro, Miguel A
    Centro de Biología Molecular "Severo Ochoa" Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Spain.
    Waldor, Matthew K
    Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, USA.
    Emerging knowledge of regulatory roles of D-amino acids in bacteria2011Ingår i: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 68, nr 5, s. 817-831Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The D-enantiomers of amino acids have been thought to have relatively minor functions in biological processes. While L-amino acids clearly predominate in nature, D-amino acids are sometimes found in proteins that are not synthesized by ribosomes, and D-Ala and D-Glu are routinely found in the peptidoglycan cell wall of bacteria. Here, we review recent findings showing that D-amino acids have previously unappreciated regulatory roles in the bacterial kingdom. Many diverse bacterial phyla synthesize and release D-amino acids, including D-Met and D-Leu, which were not previously known to be made. These noncanonical D-amino acids regulate cell wall remodeling in stationary phase and cause biofilm dispersal in aging bacterial communities. Elucidating the mechanisms by which D-amino acids govern cell wall remodeling and biofilm disassembly will undoubtedly reveal new paradigms for understanding how extracytoplasmic processes are regulated as well as lead to development of novel therapeutics.

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  • 36.
    Cava, Felipe
    et al.
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Laptenko, Oleg
    Department of Cell Biology, UMDNJ-SOM, Stratford, USA.
    Borukhov, Sergei
    Department of Cell Biology, UMDNJ-SOM, Stratford, USA.
    Chahlafi, Zahra
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Blas-Galindo, Emilio
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Gómez-Puertas, Paulino
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Berenguer, José
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Control of the respiratory metabolism of Thermus thermophilus by the nitrate respiration conjugative element NCE2007Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 64, nr 3, s. 630-646Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The strains of Thermus thermophilus that contain the nitrate respiration conjugative element (NCE) replace their aerobic respiratory chain by an anaerobic counterpart made of the Nrc-NADH dehydrogenase and the Nar-nitrate reductase in response to nitrate and oxygen depletion. This replacement depends on DnrS and DnrT, two homologues to sensory transcription factors encoded in a bicistronic operon by the NCE. DnrS is an oxygen-sensitive protein required in vivo to activate transcription on its own dnr promoter and on that of the nar operon, but not required for the expression of the nrc operon. In contrast, DnrT is required for the transcription of these three operons and also for the repression of nqo, the operon that encodes the major respiratory NADH dehydrogenase expressed during aerobic growth. Thermophilic in vitro assays revealed that low DnrT concentrations allows the recruitment of the T. thermophilus RNA polymerase sigma(A) holoenzyme to the nrc promoter and its transcription, whereas higher DnrT concentrations are required to repress transcription on the nqo promoter. In conclusion, our data show a complex autoinducible mechanism by which DnrT functions as the transcriptional switch that allows the NCE to take the control of the respiratory metabolism of its host during adaptation to anaerobic growth.

  • 37.
    Cava, Felipe
    et al.
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Zafra, Olga
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Berenguer, José
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    A cytochrome c containing nitrate reductase plays a role in electron transport for denitrification in Thermus thermophilus without involvement of the bc respiratory complex2008Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, nr 2, s. 507-518Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bc(1) respiratory complex III constitutes a key energy-conserving respiratory electron transporter between complex I (type I NADH dehydrogenase) and II (succinate dehydrogenase) and the final nitrogen oxide reductases (Nir, Nor and Nos) in most denitrifying bacteria. However, we show that the expression of complex III from Thermus thermophilus is repressed under denitrification, and that its role as electron transporter is replaced by an unusual nitrate reductase (Nar) that contains a periplasmic cytochrome c (NarC). Several lines of evidence support this conclusion: (i) nitrite and NO are as effective signals as nitrate for the induction of Nar; (ii) narC mutants are defective in anaerobic growth with nitrite, NO and N2O; (iii) such mutants present decreased NADH oxidation coupled to these electron acceptors; and (iv) complementation assays of the mutants reveal that the membrane-distal heme c of NarC was necessary for anaerobic growth with nitrite, whereas the membrane-proximal heme c was not. Finally, we show evidence to support that Nrc, the main NADH oxidative activity in denitrification, interacts with Nar through their respective membrane subunits. Thus, we propose the existence of a Nrc-Nar respiratory super-complex that is required for the development of the whole denitrification pathway in T. thermophilus.

  • 38.
    Cava, Felipe
    et al.
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Zafra, Olga
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    da Costa, Milton S
    Departamento de Bioquímica, Universidade de Coimbra, Portugal.
    Berenguer, José
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    The role of the nitrate respiration element of Thermus thermophilus in the control and activity of the denitrification apparatus2008Ingår i: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 10, nr 2, s. 522-533Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The nitrate conjugative element (NCE) encodes the ability to respire nitrate in the facultative Thermus thermophilus NAR1 strain. This process is carried out by two heterotetrameric enzymes that catalyse the oxidation of NADH (Nrc) and the reduction of nitrate (Nar), whose expression is activated by the NCE-encoded transcription factors DnrS and DnrT. We report the presence of NCE in other facultative strains of T. thermophilus and analyse its role in subsequent steps of the denitrification pathway. We encountered that nrc mutants of denitrifying strains show a decrease in anaerobic growth rates not only with nitrate, but also with nitrite, NO and N(2)O, which is concomitant to their lower NADH oxidation activities in vitro. We show that nitrate, nitrite and NO are activating signals for transcription of nrc in these strains. Finally, we demonstrate that DnrS and DnrT are required for anaerobic growth not only with nitrate, but also with nitrite, NO and N(2)O. These data allow us to conclude that: (i) Nrc constitutes the main electron donor for the four reductases of the denitrification pathway, and (ii) the NCE controls the expression of the whole denitrification pathway and the repression of the aerobic respiration through the transcription factors DnrS and DnrT.

  • 39.
    Cava, Felipe
    et al.
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Zafra, Olga
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Magalon, Axel
    Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France.
    Blasco, Francis
    Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France.
    Berenguer, José
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    A new type of NADH dehydrogenase specific for nitrate respiration in the extreme thermophile Thermus thermophilus2004Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 44, s. 45369-45378Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A four-gene operon (nrcDEFN) was identified within a conjugative element that allows Thermus thermophilus to use nitrate as an electron acceptor. Three of them encode homologues to components of bacterial respiratory chains: NrcD to ferredoxins; NrcF to iron-sulfur-containing subunits of succinate-quinone oxidoreductase (SQR); and NrcN to type-II NADH dehydrogenases (NDHs). The fourth gene, nrcE, encodes a membrane protein with no homologues in the protein data bank. Nitrate reduction with NADH was catalyzed by membrane fractions of the wild type strain, but was severely impaired in nrc::kat insertion mutants. A fusion to a thermophilic reporter gene was used for the first time in Thermus spp. to show that expression of nrc required the presence of nitrate and anoxic conditions. Therefore, a role for the nrc products as a new type of membrane NDH specific for nitrate respiration was deduced. Consistent with this, nrc::kat mutants grew more slowly than the wild type strain under anaerobic conditions, but not in the presence of oxygen. The oligomeric structure of this Nrc-NDH was deduced from the analysis of insertion mutants and a two-hybrid bacterial system. Attachment to the membrane of NrcD, NrcF, and NrcN was dependent on NrcE, whose cytoplasmic C terminus interacts with the three proteins. Interactions were also detected between NrcN and NrcF. Inactivation of nrcF produced solubilization of NrcN, but not of NrcD. These data lead us to conclude that the Nrc proteins form a distinct third type of bacterial respiratory NDH.

  • 40. Chahlafi, Zahra
    et al.
    Alvarez, Laura
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Berenguer, José
    The role of conserved proteins DrpA and DrpB in nitrate respiration of Thermus thermophilus2018Ingår i: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 20, nr 10, s. 3851-3861Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In many Thermus thermophilus strains, nitrate respiration is encoded in mobile genetic regions, along with regulatory circuits that modulate its expression based on anoxia and nitrate presence. The oxygen‐responsive system has been identified as the product of the dnrST (dnr) operon located immediately upstream of the nar operon (narCGHJIKT), which encodes the nitrate reductase (NR) and nitrate/nitrite transporters. In contrast, the nature of the nitrate sensory system is not known. Here, we analyse the putative nitrate‐sensing role of the bicistronic drp operon (drpAB) present downstream of the nar operon in most denitrifying Thermus spp. Expression of drp was found to depend on the master regulator DnrT, whereas the absence of DrpA or DrpB increased the expression of both DnrS and DnrT and, concomitantly, of the NR. Absence of both proteins made expression from the dnr and nar operons independent of nitrate. Polyclonal antisera allowed us to identify DrpA as a periplasmic protein and DrpB as a membrane protein, with capacity to bind to the cytoplasmic membrane. Here, we propose a role for DrpA/DrpB as nitrate sensors during denitrification.

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  • 41.
    Chan, Helena
    et al.
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Taib, Najwa
    Department of Microbiology, Unit Evolutionary Biology of the Microbial Cell, Paris, France; Hub Bioinformatics and Biostatistics, Department of Computational Biology, USR 3756 CNRS, Paris, France.
    Gilmore, Michael C.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Mohamed, Ahmed M T
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Hanna, Kieran
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Luhur, Johana
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Nguyen, Hieu
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Hafiz, Elham
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gribaldo, Simonetta
    Department of Microbiology, Unit Evolutionary Biology of the Microbial Cell, Paris, France; Hub Bioinformatics and Biostatistics, Department of Computational Biology, USR 3756 CNRS, Paris, France.
    Rudner, David
    Department of Microbiology, MA, Boston, United States.
    Rodrigues, Christopher D A
    School of Life Sciences, University of Warwickgrid.7372.1, Coventry, United Kingdom.
    Genetic screens identify additional genes implicated in envelope remodeling during the engulfment stage of bacillus subtilis sporulation2022Ingår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 13, nr 5, artikel-id e0173222Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    During bacterial endospore formation, the developing spore is internalized into the mother cell through a phagocytic-like process called engulfment, which involves synthesis and hydrolysis of peptidoglycan. Engulfment peptidoglycan hydrolysis requires the widely conserved and well-characterized DMP complex, composed of SpoIID, SpoIIM, and SpoIIP. In contrast, although peptidoglycan synthesis has been implicated in engulfment, the protein players involved are less well defined. The widely conserved SpoIIIAH-SpoIIQ interaction is also required for engulfment efficiency, functioning like a ratchet to promote membrane migration around the forespore. Here, we screened for additional factors required for engulfment using transposon sequencing in Bacillus subtilis mutants with mild engulfment defects. We discovered that YrvJ, a peptidoglycan hydrolase, and the MurA paralog MurAB, involved in peptidoglycan precursor synthesis, are required for efficient engulfment. Cytological analyses suggest that both factors are important for engulfment when the DMP complex is compromised and that MurAB is additionally required when the SpoIIIAH-SpoIIQ ratchet is abolished. Interestingly, despite the importance of MurAB for sporulation in B. subtilis, phylogenetic analyses of MurA paralogs indicate that there is no correlation between sporulation and the number of MurA paralogs and further reveal the existence of a third MurA paralog, MurAC, within the Firmicutes. Collectively, our studies identify two new factors that are required for efficient envelop remodeling during sporulation and highlight the importance of peptidoglycan precursor synthesis for efficient engulfment in B. subtilis and likely other endospore-forming bacteria. IMPORTANCE In bacteria, cell envelope remodeling is critical for cell growth and division. This is also the case during the development of bacteria into highly resistant endospores (spores), known as sporulation. During sporulation, the developing spore becomes internalized inside the mother cell through a phagocytic-like process called engulfment, which is essential to form the cell envelope of the spore. Engulfment involves both the synthesis and hydrolysis of peptidoglycan and the stabilization of migrating membranes around the developing spore. Importantly, although peptidoglycan synthesis has been implicated during engulfment, the specific genes that contribute to this molecular element of engulfment have remained unclear. Our study identifies two new factors that are required for efficient envelope remodeling during engulfment and emphasizes the importance of peptidoglycan precursor synthesis for efficient engulfment in the model organism Bacillus subtilis and likely other endospore-forming bacteria. Finally, our work highlights the power of synthetic screens to reveal additional genes that contribute to essential processes during sporulation.

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  • 42. Chauhan, Deepika
    et al.
    Srivastava, Pulkit Anupam
    Ritzl, Barbara
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Yennamalli, Ragothaman M.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Priyadarshini, Richa
    Amino Acid-Dependent Alterations in Cell Wall and Cell Morphology of Deinococcus indicus DR12019Ingår i: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 10, artikel-id 1449Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Deinococcus radiodurans exhibits growth medium-dependent morphological variation in cell shape, but there is no evidence whether this phenomenon is observed in other members of the Deinococcaceae family. In this study, we isolated a red-pigmented, aerobic, Deinococcus indicus strain DR1 from Dadri wetland, India. This D. indicus strain exhibited cell-morphology transition from rod-shaped cells to multi-cell chains in a growth-medium-dependent fashion. In response to addition of 1% casamino acids in the minimal growth medium, rod-shaped cells formed multi-cell chains. Addition of all 20 amino acids to the minimal medium was able to recapitulate the phenotype. Specifically, a combination of L-methionine, L-lysine, L-aspartate, and L-threonine caused morphological alterations. The transition from rod shape to multi-cell chains is due to delay in daughter cell separation after cell division. Minimal medium supplemented with L-ornithine alone was able to cause cell morphology changes. Furthermore, a comparative UPLC analysis of PG fragments isolated from D. indicus cells propagated in different growth media revealed alterations in the PG composition. An increase in the overall cross-linkage of PG was observed in muropeptides from nutrient-rich TSB and NB media versus PYE medium. Overall our study highlights that environmental conditions influence PG composition and cell morphology in D. indicus.

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  • 43.
    Chautard, Hélène
    et al.
    Biométhodes SA, Evry, France.
    Blas-Galindo, Emilio
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Menguy, Thierry
    Biométhodes SA, Evry, France.
    Grand'Moursel, Laure
    Biométhodes SA, Evry, France.
    Cava, Felipe
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Berenguer, José
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Delcourt, Marc
    Biométhodes SA, Evry, France.
    An activity-independent selection system of thermostable protein variants2007Ingår i: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 4, nr 11, s. 919-921Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We describe an activity-independent method for the selection of thermostable mutants of any protein. It is based on a fusion construct comprising the protein of interest and a thermostable antibiotic resistance reporter, in such a way that thermostable mutants provide increased resistance in a thermophile. We isolated thermostable mutants of three human interferons and of two enzymes to demonstrate the applicability of the system.

  • 44. Cuenca, Miguelangel
    et al.
    Pfister, Simona P.
    Buschor, Stefanie
    Bayramova, Firuza
    Hernandez, Sara B.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kuru, Erkin
    Van Nieuwenhze, Michael S.
    Brun, Yves V.
    Coelho, Fernanda M.
    Hapfelmeier, Siegfried
    D-Alanine-Controlled Transient Intestinal Mono-Colonization with Non-Laboratory-Adapted Commensal E. coli Strain HS2016Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 11, nr 3, artikel-id e0151872Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Soon after birth the mammalian gut microbiota forms a permanent and collectively highly resilient consortium. There is currently no robust method for re-deriving an already microbially colonized individual again-germ-free. We previously developed the in vivo growth-incompetent E. coli K-12 strain HA107 that is auxotrophic for the peptidoglycan components D-alanine (D-Ala) and meso-diaminopimelic acid (Dap) and can be used to transiently associate germ-free animals with live bacteria, without permanent loss of germ-free status. Here we describe the translation of this experimental model from the laboratory-adapted E. coli K-12 prototype to the better gut-adapted commensal strain E. coli HS. In this genetic background it was necessary to complete the D-Ala auxotrophy phenotype by additional knockout of the hypothetical third alanine racemase metC. Cells of the resulting fully auxotrophic strain assembled a peptidoglycan cell wall of normal composition, as long as provided with D-Ala and Dap in the medium, but could not proliferate a single time after D-Ala/Dap removal. Yet, unsupplemented bacteria remained active and were able to complete their cell cycle with fully sustained motility until immediately before autolytic death. Also in vivo, the transiently colonizing bacteria retained their ability to stimulate a live-bacteria-specific intestinal Immunoglobulin (Ig) A response. Full D-Ala auxotrophy enabled rapid recovery to again-germ-free status. E. coli HS has emerged from human studies and genomic analyses as a paradigm of benign intestinal commensal E. coli strains. Its reversibly colonizing derivative may provide a versatile research tool for mucosal bacterial conditioning or compound delivery without permanent colonization.

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  • 45.
    Dai, Yunfei
    et al.
    Department of Biology, Northeastern University, MA, Boston, United States.
    Pinedo, Victor
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Tang, Amy Y.
    Department of Biology, Northeastern University, MA, Boston, United States.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Geisinger, Edward
    Department of Biology, Northeastern University, MA, Boston, United States.
    A New Class of Cell Wall-Recycling L,D-Carboxypeptidase Determines β-Lactam Susceptibility and Morphogenesis in Acinetobacter baumannii2021Ingår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 12, nr 6, artikel-id e0278621Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The hospital-acquired pathogen Acinetobacter baumannii possesses a complex cell envelope that is key to its multidrug resistance and virulence. The bacterium, however, lacks many canonical enzymes that build the envelope in model organisms. Instead, A. baumannii contains a number of poorly annotated proteins that may allow alternative mechanisms of envelope biogenesis. We demonstrated previously that one of these unusual proteins, ElsL, is required for maintaining a characteristic short rod shape and for withstanding antibiotics that attack the septal cell wall. Curiously, ElsL is composed of a leaderless YkuD-family domain usually found in secreted, cell wall-modifying L,D-transpeptidases (LDTs). Here, we show that, rather than being an LDT, ElsL is actually a new class of cytoplasmic L,D-carboxypeptidase (LDC) that provides a critical step in cell wall recycling previously thought to be missing from A. baumannii. Absence of ElsL impairs cell wall integrity, morphology, and intrinsic resistance due to buildup of murein tetrapeptide precursors, toxicity of which is bypassed by preventing muropeptide recycling. Multiple pathways in the cell become sites of vulnerability when ElsL is inactivated, including L,D-cross-link formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on envelope physiology. We thus reveal a novel class of cell wall-recycling LDC critical to growth and homeostasis of A. baumannii and likely many other bacteria.

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  • 46.
    de Pedro, Miguel A.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Structural constraints and dynamics of bacterial cell wall architecture2015Ingår i: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 6, artikel-id 449Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The peptidoglycan wall (PG) is a unique structure which confers physical strength and defined shape to bacteria. It consists of a net-like macromolecule of peptide interlinked glycan chains overlying the cell membrane. The structure and layout of the PG dictates that the wall has to be continuously modified as bacteria go through division, morphological differentiation, and adaptive responses. The PG is poorly known in structural terms. However, to understand morphogenesis a precise knowledge of glycan strand arrangement and of local effects of the different kinds of subunits is essential. The scarcity of data led to a conception of the PG as a regular, highly ordered structure which strongly influenced growth models. Here, we review the structure of the PG to define a more realistic conceptual framework. We discuss the consequences of the plasticity of murein architecture in morphogenesis and try to define a set of minimal structural constraints that must be fulfilled by any model to be compatible with present day information.

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  • 47.
    del Peso Santos, Teresa
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Alvarez, Laura
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sit, Brandon
    Howard Hughes Medical Institute, Brigham and Women’s Hospital Division of Infectious Diseases and Harvard Medical School Department of Microbiology and Immunobiology, MA, Boston, United States.
    Irazoki, Oihane
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Blake, Jonathon
    Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
    Warner, Benjamin R.
    Department of Microbiology, The Ohio State University, OH, Columbus, United States; Center for RNA Biology, The Ohio State University, OH, Columbus, United States.
    Warr, Alyson R.
    Howard Hughes Medical Institute, Brigham and Women’s Hospital Division of Infectious Diseases and Harvard Medical School Department of Microbiology and Immunobiology, MA, Boston, United States.
    Bala, Anju
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Benes, Vladimir
    Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
    Waldor, Matthew K.
    Howard Hughes Medical Institute, Brigham and Women’s Hospital Division of Infectious Diseases and Harvard Medical School Department of Microbiology and Immunobiology, MA, Boston, United States.
    Fredrick, Kurt
    Department of Microbiology, The Ohio State University, OH, Columbus, United States; Center for RNA Biology, The Ohio State University, OH, Columbus, United States.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    BipA exerts temperature-dependent translational control of biofilm-associated colony morphology in Vibrio cholerae2021Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 10, artikel-id e60607Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Adaptation to shifting temperatures is crucial for the survival of the bacterial pathogen Vibrio cholerae. Here, we show that colony rugosity, a biofilm-associated phenotype, is regulated by temperature in V. cholerae strains that naturally lack the master biofilm transcriptional regulator HapR. Using transposon-insertion mutagenesis, we found the V. cholerae ortholog of BipA, a conserved ribosome-associated GTPase, is critical for this temperature-dependent phenomenon. Proteomic analyses revealed that loss of BipA alters the synthesis of >300 proteins in V. cholerae at 22˚C, increasing the production of biofilm-related proteins including the key transcriptional activators VpsR and VpsT, as well as proteins important for diverse cellular processes. At low temperatures, BipA protein levels increase and are required for optimal ribosome assembly in V. cholerae, suggesting that control of BipA abundance is a mechanism by which bacteria can remodel their proteomes. Our study reveals a remarkable new facet of V. cholerae’s complex biofilm regulatory network.

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  • 48.
    Delerue, Thomas
    et al.
    Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, MD, Bethesda, United States.
    Anantharaman, Vivek
    National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, MD, Bethesda, United States.
    Gilmore, Michael C.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Popham, David L.
    Department of Biological Sciences, Virginia Tech, VA, Blacksburg, United States.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Aravind, L.
    National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, MD, Bethesda, United States.
    Ramamurthi, Kumaran S.
    Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, MD, Bethesda, United States.
    Bacterial developmental checkpoint that directly monitors cell surface morphogenesis2022Ingår i: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 57, nr 3, s. 344-360Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacillus subtilis spores are encased in two concentric shells: an outer proteinaceous “coat” and an inner peptidoglycan “cortex,” separated by a membrane. Cortex assembly depends on coat assembly initiation, but how cells achieve this coordination across the membrane is unclear. Here, we report that the protein SpoVID monitors the polymerization state of the coat basement layer via an extension to a functional intracellular LysM domain that arrests sporulation when coat assembly is initiated improperly. Whereas extracellular LysM domains bind mature peptidoglycan, SpoVID LysM binds to the membrane-bound lipid II peptidoglycan precursor. We propose that improper coat assembly exposes the SpoVID LysM domain, which then sequesters lipid II and prevents cortex assembly. SpoVID defines a widespread group of firmicute proteins with a characteristic N-terminal domain and C-terminal peptidoglycan-binding domains that might combine coat and cortex assembly roles to mediate a developmental checkpoint linking the morphogenesis of two spatially separated supramolecular structures.

  • 49. Desmarais, Samantha M.
    et al.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    de Pedro, Miguel A.
    Huang, Kerwyn Casey
    Isolation and preparation of bacterial cell walls for compositional analysis by Ultra Performance Liquid Chromatography2014Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 83, s. UNSP e51183-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan's central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

  • 50.
    Desmarais, Samantha M
    et al.
    Department of Bioengineering, Stanford University, Stanford, CA, USA.
    De Pedro, Miguel A
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Huang, Kerwyn Casey
    Department of Bioengineering, Stanford University, Stanford, CA, USA ; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
    Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly2013Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 89, nr 1, s. 1-13Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The peptidoglycan (PG) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. A quantitative understanding of the relationships between PG architecture, morphogenesis, immune system activation and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell wall synthesis and cell growth. High-performance liquid chromatography (HPLC) has played an important role in our understanding of the structural and chemical complexity of the cell wall by providing an analytical method to quantify differences in chemical composition. Here, we present a primer on the basic chemical features of wall structure that can be revealed through HPLC, along with a description of the applications of HPLC PG analyses for interpreting the effects of genetic and chemical perturbations to a variety of bacterial species in different environments. We describe the physical consequences of different PG compositions on cell shape, and review complementary experimental and computational methodologies for PG analysis. Finally, we present a partial list of future targets of development for HPLC and related techniques.

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