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Alvarez, Laura
Publications (10 of 16) Show all publications
Alvarez, L., Sanchez-Hevia, D., Sanchez, M. & Berenguer, J. (2019). A new family of nitrate/nitrite transporters involved in denitrification. International Microbiology, 22(1), 19-28
Open this publication in new window or tab >>A new family of nitrate/nitrite transporters involved in denitrification
2019 (English)In: International Microbiology, ISSN 1139-6709, E-ISSN 1618-1905, Vol. 22, no 1, p. 19-28Article in journal (Refereed) Published
Abstract [en]

Denitrifying bacteria carry out nitrate and nitrite respiration inside and outside the cell, respectively. In Thermus thermophilus, nitrate and nitrite transport processes are carried out by major facilitator superfamily (MFS) transporters. The sequence of the nar operon of nitrate-only respiring strains of T. thermophilus includes two tandemly organized MFS transporter genes (narK and narT) of the NarK1 and NarK2 families. Both can function as nitrate/nitrite antiporters, but NarK has been proposed as more specific for nitrate whereas NarT more specific for nitrite. In some nitrate- and nitrite-respiring strains of the same species, a single MFS transporter (NarO) belonging to a different MFS subfamily appears. To analyze the role of this single MFS in the same genetic context, we transferred the two types of nar operon to the aerobic strain HB27, and further included in both of them the ability to respire nitrite. The new denitrifying strains HB27dn, with two MFS, and HB27dp, with a single one, were used to isolate mutants devoid of transporters. Through in trans complementation experiments, we demonstrate that the NarO single MFS works efficiently in the transport of both nitrate and nitrite.

Place, publisher, year, edition, pages
Springer-Verlag New York, 2019
Keywords
Thermus, Denitrification, Nitrate, Nitrite, Transport, MFS
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-162498 (URN)10.1007/s10123-018-0023-0 (DOI)000464866200001 ()30810929 (PubMedID)
Available from: 2019-08-22 Created: 2019-08-22 Last updated: 2019-08-22Bibliographically approved
Murphy, S. G., Alvarez, L., Adams, M. C., Liu, S., Chappie, J. S., Cava, F. & Dorr, T. (2019). Endopeptidase Regulation as a Novel Function of the Zur-Dependent Zinc Starvation Response. mBio, 10(1), Article ID e02620-18.
Open this publication in new window or tab >>Endopeptidase Regulation as a Novel Function of the Zur-Dependent Zinc Starvation Response
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2019 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 1, article id e02620-18Article in journal (Refereed) Published
Abstract [en]

The cell wall is a strong, yet flexible, meshwork of peptidoglycan (PG) that gives a bacterium structural integrity. To accommodate a growing cell, the wall is remodeled by both PG synthesis and degradation. Vibrio cholerae encodes a group of three nearly identical zinc-dependent endopeptidases (EPs) that are predicted to hydrolyze PG to facilitate cell growth. Two of these (ShyA and ShyC) are conditionally essential housekeeping EPs, while the third (ShyB) is not expressed under standard laboratory conditions. To investigate the role of ShyB, we conducted a transposon screen to identify mutations that activate shyB transcription. We found that shyB is induced as part of the Zur-mediated zinc starvation response, a mode of regulation not previously reported for cell wall lytic enzymes. In vivo, ShyB alone was sufficient to sustain cell growth in low-zinc environments. In vitro, ShyB retained its D, D-endopeptidase activity against purified sacculi in the presence of the metal chelator EDTA at concentrations that inhibit ShyA and ShyC. This insensitivity to metal chelation is likely what enables ShyB to substitute for other EPs during zinc starvation. Our survey of transcriptomic data from diverse bacteria identified other candidate Zur-regulated EPs, suggesting that this adaptation to zinc starvation is employed by other Gram-negative bacteria. IMPORTANCE Bacteria encode a variety of adaptations that enable them to survive during zinc starvation, a condition which is encountered both in natural environments and inside the human host. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, we have identified a novel member of this zinc starvation response, a cell wall hydrolase that retains function and is conditionally essential for cell growth in low-zinc environments. Other Gram-negative bacteria contain homologs that appear to be under similar regulatory control. These findings are significant because they represent, to our knowledge, the first evidence that zinc homeostasis influences cell wall turnover. Anti-infective therapies commonly target the bacterial cell wall; therefore, an improved understanding of how the cell wall adapts to host-induced zinc starvation could lead to new antibiotic development. Such therapeutic interventions are required to combat the rising threat of drug-resistant infections.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY, 2019
Keywords
Gram-negative, Vibrio cholerae, cell wall, hydrolase, metalloproteins, peptidoglycan, zinc starvation
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-157540 (URN)10.1128/mBio.02620-18 (DOI)000460314300056 ()30782657 (PubMedID)
Available from: 2019-04-01 Created: 2019-04-01 Last updated: 2019-04-01Bibliographically approved
Hsu, Y.-P., Hall, E., Booher, G., Murphy, B., Radkov, A. D., Yablonowski, J., . . . VanNieuwenhze, M. S. (2019). Fluorogenic D-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays. Nature Chemistry, 11(4), 335-341
Open this publication in new window or tab >>Fluorogenic D-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays
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2019 (English)In: Nature Chemistry, ISSN 1755-4330, E-ISSN 1755-4349, Vol. 11, no 4, p. 335-341Article in journal (Refereed) Published
Abstract [en]

Peptidoglycan is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions, which construct peptide crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis and application of rotor-fluorogenic D-amino acids (RfDAAs), enabling real-time, continuous tracking of transpeptidation reactions. These probes allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D- and L,D-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-157944 (URN)10.1038/s41557-019-0217-x (DOI)000462046600011 ()30804500 (PubMedID)
Available from: 2019-04-18 Created: 2019-04-18 Last updated: 2019-04-18Bibliographically approved
Alvarez, L. & Cava, F. (2018). Bacterial Competition Assay Based on Extracellular D-amino Acid Production. Bio-protocol, 8(7), Article ID e2787.
Open this publication in new window or tab >>Bacterial Competition Assay Based on Extracellular D-amino Acid Production
2018 (English)In: Bio-protocol, E-ISSN 2331-8325, Vol. 8, no 7, article id e2787Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
BIO-PROTOCOL, 2018
Keywords
D-amino acid, Competition, Co-cultivation, Viability, D-amino acid oxidase (DAAO) assay
Identifiers
urn:nbn:se:umu:diva-157351 (URN)10.21769/BioProtoc.2787 (DOI)000457929800007 ()
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilThe Kempe Foundations
Available from: 2019-03-15 Created: 2019-03-15 Last updated: 2019-03-15Bibliographically approved
Alvarez, L., Aliashkevich, A., de Pedro, M. A. & Cava, F. (2018). Bacterial secretion of D-arginine controls environmental microbial biodiversity. The ISME Journal, 12(2), 438-450
Open this publication in new window or tab >>Bacterial secretion of D-arginine controls environmental microbial biodiversity
2018 (English)In: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 12, no 2, p. 438-450Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:umu:diva-144335 (URN)10.1038/ismej.2017.176 (DOI)000422779100013 ()29028003 (PubMedID)
Available from: 2018-02-08 Created: 2018-02-08 Last updated: 2018-06-09Bibliographically approved
Aliashkevich, A., Alvarez, L. & Cava, F. (2018). New Insights Into the Mechanisms and Biological Roles of D-Amino Acids in Complex Eco-Systems. Frontiers in Microbiology, 9, Article ID 683.
Open this publication in new window or tab >>New Insights Into the Mechanisms and Biological Roles of D-Amino Acids in Complex Eco-Systems
2018 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 683Article, review/survey (Refereed) Published
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.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2018
Keywords
D-amino acids, D-methionine, D-arginine, bacteria, cell wall, Vibrio cholerae
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-147303 (URN)10.3389/fmicb.2018.00683 (DOI)000429347700001 ()29681896 (PubMedID)
Available from: 2018-05-28 Created: 2018-05-28 Last updated: 2018-06-09Bibliographically approved
Chahlafi, Z., Alvarez, L., Cava, F. & Berenguer, J. (2018). The role of conserved proteins DrpA and DrpB in nitrate respiration of Thermus thermophilus. Environmental Microbiology, 20(10), 3851-3861
Open this publication in new window or tab >>The role of conserved proteins DrpA and DrpB in nitrate respiration of Thermus thermophilus
2018 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 20, no 10, p. 3851-3861Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
John Wiley & Sons, 2018
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-153131 (URN)10.1111/1462-2920.14400 (DOI)000447549700029 ()30187633 (PubMedID)2-s2.0-85054313930 (Scopus ID)
Available from: 2018-11-09 Created: 2018-11-09 Last updated: 2018-11-09Bibliographically approved
Alvarez, L., Quintáns, N. G., Blesa, A., Baquedano, I., Mencía, M., Bricio, C. & Berenguer, J. (2017). Hierarchical control of nitrite respiration by transcription factors encoded within mobile gene clusters of Thermus thermophilus. Genes, 8(12), Article ID 361.
Open this publication in new window or tab >>Hierarchical control of nitrite respiration by transcription factors encoded within mobile gene clusters of Thermus thermophilus
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2017 (English)In: Genes, ISSN 2073-4425, E-ISSN 2073-4425, Vol. 8, no 12, article id 361Article in journal (Refereed) Published
Abstract [en]

Denitrification in Thermus thermophilus is encoded by the nitrate respiration conjugative element (NCE) and nitrite and nitric oxide respiration (nic) gene clusters. A tight coordination of each cluster's expression is required to maximize anaerobic growth, and to avoid toxicity by intermediates, especially nitric oxides (NO). Here, we study the control of the nitrite reductases (Nir) and NO reductases (Nor) upon horizontal acquisition of the NCE and nic clusters by a formerly aerobic host. Expression of the nic promoters PnirS, PnirJ, and PnorC, depends on the oxygen sensor DnrS and on the DnrT protein, both NCE-encoded. NsrR, a nic-encoded transcription factor with an iron-sulfur cluster, is also involved in Nir and Nor control. Deletion of nsrR decreased PnorC and PnirJ transcription, and activated PnirS under denitrification conditions, exhibiting a dual regulatory role never described before for members of the NsrR family. On the basis of these results, a regulatory hierarchy is proposed, in which under anoxia, there is a pre-activation of the nic promoters by DnrS and DnrT, and then NsrR leads to Nor induction and Nir repression, likely as a second stage of regulation that would require NO detection, thus avoiding accumulation of toxic levels of NO. The whole system appears to work in remarkable coordination to function only when the relevant nitrogen species are present inside the cell.

Place, publisher, year, edition, pages
MDPI, 2017
Keywords
denitrification, nitrite respiration, regulation, thermophiles, Thermus thermophilus
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-144423 (URN)10.3390/genes8120361 (DOI)000419212400022 ()
Available from: 2018-02-07 Created: 2018-02-07 Last updated: 2018-06-09Bibliographically approved
Terceti, M. S., Rivas, A. J., Alvarez, L., Noia, M., Cava, F. & Osorio, C. R. (2017). rstB Regulates Expression of the Photobacterium damselae subsp damselae Major Virulence Factors Damselysin, Phobalysin P and Phobalysin C. Frontiers in Microbiology, 8, Article ID 582.
Open this publication in new window or tab >>rstB Regulates Expression of the Photobacterium damselae subsp damselae Major Virulence Factors Damselysin, Phobalysin P and Phobalysin C
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2017 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 582Article in journal (Refereed) Published
Abstract [en]

The marine pathogenic bacterium Photobacterium damselae subsp. damselae causes septicemia in marine animals and in humans. The pPHDD1 plasmid-encoded hemolysins damselysin (Dly) and phobalysin P (PhlyP), and the chromosome-encoded hemolysin phobalysin C (PhlyC) constitute its main virulence factors. However, the mechanisms by which expression of these three hemolysins is regulated remain unknown. Here we report the isolation of a mini-Tn10 transposon mutant which showed a strong impairment in its hemolytic activity. The transposon disrupted a putative sensor histidine kinase gene vda_000600 (rstB), which together with vda_000601 (rstA) is predicted to encode a putative two-component regulatory system. This system showed to be homologous to the Vibrio cholerae CarSR/VprAB and Escherichia coli RstAB systems. Reconstruction of the mutant by allelic exchange of rstB showed equal impairment in hemolysis, and complementation with a plasmid expressing rstAB restored hemolysis to wild-type levels. Remarkably, we demonstrated by promoter expression analyses that the reduced hemolysis in the rstB mutant was accompanied by a strong decrease in transcription activities of the three hemolysin genes dly (damselysin), hlyA(pl) (phobalysin P) and hlyA(ch) (phobalysin C). Thus, RstB, encoded in the small chromosome, regulates plasmid and chromosomal virulence genes. We also found that reduced expression of the three virulence genes correlated with a strong decrease in virulence in a sea bass model, demonstrating that RstB constitutes a master regulator of the three P. damselae subsp. damselae hemolysins and plays critical roles in the pathogenicity of this bacterium. This study represents the first evidence of a direct role of a RstAB-like system in the regulation of bacterial toxins.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2017
Keywords
RstAB, hemolysin, Photobacterium damselae, damselysin, phobalysin, vibriosis, CarSR
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-134818 (URN)10.3389/fmicb.2017.00582 (DOI)000398656700001 ()28443076 (PubMedID)
Available from: 2017-05-29 Created: 2017-05-29 Last updated: 2018-06-09Bibliographically approved
Dörr, T., Alvarez, L., Delgado, F., Davis, B. M., Cava, F. & Waldor, M. K. (2016). A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance. Proceedings of the National Academy of Sciences of the United States of America, 113(2), 404-409
Open this publication in new window or tab >>A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance
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2016 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 2, p. 404-409Article in journal (Refereed) Published
Abstract [en]

The bacterial cell wall is critical for maintenance of cell shape and survival. Following exposure to antibiotics that target enzymes required for cell wall synthesis, bacteria typically lyse. Although several cell envelope stress response systems have been well described, there is little knowledge of systems that modulate cell wall synthesis in response to cell wall damage, particularly in Gram-negative bacteria. Here we describe WigK/WigR, a histidine kinase/response regulator pair that enables Vibrio cholerae, the cholera pathogen, to survive exposure to antibiotics targeting cell wall synthesis in vitro and during infection. Unlike wild-type V. cholerae, mutants lacking wigR fail to recover following exposure to cell-wall-acting antibiotics, and they exhibit a drastically increased cell diameter in the absence of such antibiotics. Conversely, overexpression of wigR leads to cell slimming. Overexpression of activated WigR also results in increased expression of the full set of cell wall synthesis genes and to elevated cell wall content. WigKR-dependent expression of cell wall synthesis genes is induced by various cell-wall-acting antibiotics as well as by overexpression of an endogenous cell wall hydrolase. Thus, WigKR appears to monitor cell wall integrity and to enhance the capacity for increased cell wall production in response to damage. Taken together, these findings implicate WigKR as a regulator of cell wall synthesis that controls cell wall homeostasis in response to antibiotics and likely during normal growth as well.

Keywords
peptidoglycan, stress response, antibiotic tolerance, two component system, cell envelope
National Category
Cell and Molecular Biology Microbiology
Identifiers
urn:nbn:se:umu:diva-114884 (URN)10.1073/pnas.1520333113 (DOI)000367881500053 ()26712007 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilThe Kempe Foundations
Available from: 2016-04-27 Created: 2016-01-29 Last updated: 2018-06-07Bibliographically approved
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