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  • 1. Aldick, Thomas
    et al.
    Bielaszewska, Martina
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Humpf, Hans-Ulrich
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Karch, Helge
    Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin2009Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 71, nr 6, s. 1496-508Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly), a putative EHEC virulence factor, belongs to the RTX (repeat-in-toxin) family whose members rapidly inactivate themselves by self-aggregation. By investigating the status of EHEC-Hly secreted extracellularly, we found the toxin both in a free, soluble form and associated, with high tendency and independently of its acylation status, to outer membrane vesicles (OMVs) extruded by EHEC. We compared the interaction of both toxin forms with erythrocytes using scanning electron microscopy and binding assays. The OMV-associated toxin was substantially (80 times) more stable under physiological conditions than the free EHEC-Hly as demonstrated by prolonged haemolytic activity (half-life time 20 h versus 15 min). The haemolysis was preceded by calcium-dependent binding of OMVs carrying EHEC-Hly to erythrocytes; this binding was mediated by EHEC-Hly. We demonstrate that EHEC-Hly is a biologically active cargo in OMVs with dual roles: a cell-binding protein and a haemolysin. These paired functions produce a biologically potent form of the OMV-associated RTX toxin and augment its potential towards target cells. Our findings provide a general concept for stabilization of RTX toxins and open new insights into the biology of these important virulence factors.

  • 2. Andersson, K
    et al.
    Carballeira Suarez, N
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Magnusson, K E
    Persson, C
    Stendahl, O
    Wolf-Watz, H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Fällman, M
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    YopH of Yersinia pseudotuberculosis interrupts early phosphotyrosine signalling associated with phagocytosis.1996Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 20, nr 5, s. 1057-69Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The PTPase YopH of Yersinia is essential to the ability of these bacteria to block phagocytosis. Wild-type Yersinia pseudotuberculosis, but not the yopH mutant strain, resisted phagocytosis by J774 cells. Ingestion of a yopH mutant was dependent on tyrosine kinase activity. Transcomplementation with wild-type yopH restored the anti-phagocytic effect, whereas introduction of the gene encoding the catalytically inactive yopHC403A was without effect. The PTPase inhibitor orthovanadate impaired the anti-phagocytic effect of the wild-type strain, further demonstrating the importance of bacteria-derived PTPase activity for this event. The ability to resist phagocytosis indicates that the effect of the bacterium is immediately exerted when it becomes associated with the phagocyte. Within 30 s after the onset of infection, wild-type Y. pseudotuberculosis caused a YopH-dependent dephosphorylation of phosphotyrosine proteins in J774 cells. Furthermore, interaction of the cells with phagocytosable strains led to a rapid and transient increase in tyrosine phosphorylation of paxillin and some other proteins, an event dependent on the presence of the bacterial surface-located protein invasin. Co-infection with the phagocytosable strain and the wild-type strain abolished the induction of tyrosine phosphorylation. Taken together, the present findings demonstrate an immediate YopH-mediated dephosphorylation of macrophage phosphotyrosine proteins, suggesting that this PTPase acts by preventing early phagocytosis-linked signalling in the phagocyte.

  • 3. Arnqvist, Anna
    et al.
    Olsén, A
    Normark, S
    Sigma S-dependent growth-phase induction of the csgBA promoter in Escherichia coli can be achieved in vivo by sigma 70 in the absence of the nucleoid-associated protein H-NS.1994Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 13, nr 6, s. 1021-32Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The stationary-phase-specific sigma factor sigma S (RpoS/KatF) is required for Escherichia coli to induce expression of fibronectin-binding curli organelles upon reaching stationary phase. We show that the csgA gene which encodes the curlin subunit protein belongs to a dicistronic operon, csgBA. The transcriptional start site of csgBA was determined and an AT-rich up-stream activating sequence (UAS) required for transcriptional activation was identified. The pcsgBA promoter is not specific for sigma S since the same promoter sequence can be used by E sigma 70 in vivo in a strain lacking nucleoid-associated protein H-NS and sigma S. Transcription remained growth-phase induced and dependent upon the UAS in such a double mutant. Furthermore, we demonstrate that an additional operon, hdeAB, which is also dependent upon sigma S for transcription, can be transcribed by E sigma 70 in vivo in the absence of H-NS by utilizing the phdeAB promoter. Two other genes known to be under the control of sigma S for expression, bolA and katE, remained transcriptionally silent in the absence of H-NS. It is suggested that a subset of E. coli promoters can be recognized by both E sigma S and E sigma 70 in vivo but H-NS interacting with these sequences prevents formation of successful transcription-initiation complexes with E sigma 70.

  • 4. Arnqvist, Anna
    et al.
    Olsén, A
    Pfeifer, J
    Russell, D G
    Normark, S
    The Crl protein activates cryptic genes for curli formation and fibronectin binding in Escherichia coli HB101.1992Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 6, nr 17, s. 2443-52Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Curli are thin, coiled, temperature-regulated fibres on fibronectin-binding Escherichia coli. The subunit protein of curli was highly homologous at its amino terminus to SEF-17, the subunit protein of thin, aggregative fimbriae of Salmonella enteritidis 27655 strain 3b, suggesting that these fibres form a novel class of surface organelles on enterobacteria. E. coli HB101 is non-curliated and unable to bind soluble, iodinated fibronectin. The phenotypically cryptic curlin subunit gene, csgA, in HB101 is transcriptionally activated by expressing the cytoplasmic Crl on a multicopy plasmid. Transcriptional activation of csgA by Crl was observed after growth at 26 degrees C but not at 37 degrees C, even though crl transcription was not thermoregulated. A deletion of the 39 carboxy-terminal residues abolished Crl activity, whereas a deletion of 10 residues at the C-terminus did not, implying that a region between residue 93 and 122 in the 132-amino-acid-residue large Crl protein is required for activating curli expression in E. coli HB101. crl is a normal housekeeping gene in E. coli and it is suggested that its gene product may either be a DNA-binding protein affecting chromatin structure as has been suggested for histone-like protein H1 or interact with specific regulatory protein(s) controlling transcription of genes required for curli formation and fibronectin binding.

  • 5.
    Balsalobre, Carlos
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Silván, José Manuel
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Berglund, Stina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Mizunoe, Yoshimitsu
    Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan.
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Nyunt Wai, Sun
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Release of the type I secreted α-haemolysin via outer membrane vesicles from Escherichia coli2006Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 59, nr 1, s. 99-112Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The α-haemolysin is an important virulence factor commonly expressed by extraintestinal pathogenic Escherichia coli. The secretion of the α-haemolysin is mediated by the type I secretion system and the toxin reaches the extracellular space without the formation of periplasmic intermediates presumably in a soluble form. Surprisingly, we found that a fraction of this type I secreted protein is located within outer membrane vesicles (OMVs) that are released by the bacteria. The α-haemolysin appeared very tightly associated with the OMVs as judged by dissociation assays and proteinase susceptibility tests. The α-haemolysin in OMVs was cytotoxically active and caused lysis of red blood cells. The OMVs containing the α-haemolysin were distinct from the OMVs not containing α-haemolysin, showing a lower density. Furthermore, they differed in protein composition and one component of the type I secretion system, the TolC protein, was found in the lower density vesicles. Studies of natural isolates of E. coli demonstrated that the localization of α-haemolysin in OMVs is a common feature among haemolytic strains. We propose an alternative pathway for the transport of the type I secreted α-haemolysin from the bacteria to the host cells during bacterial infections.

  • 6.
    Bernardo, Lisandro M D
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Johansson, Linda U M
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Solera, Dafne
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Skärfstad, Eleonore
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of σ54-dependent transcription2006Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 60, nr 3, s. 749-764Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The RNA polymerase-binding protein DksA is a cofactor required for guanosine tetraphosphate (ppGpp)-responsive control of transcription from sigma70 promoters. Here we present evidence: (i) that both DksA and ppGpp are required for in vivo sigma54 transcription even though they do not have any major direct effects on sigma54 transcription in reconstituted in vitro transcription and sigma-factor competition assays, (ii) that previously defined mutations rendering the housekeeping sigma70 less effective at competing with sigma54 for limiting amounts of core RNA polymerase similarly suppress the requirement for DksA and ppGpp in vivo and (iii) that the extent to which ppGpp and DksA affect transcription from sigma54 promoters in vivo reflects the innate affinity of the promoters for sigma54-RNA polymerase holoenzyme in vitro. Based on these findings, we propose a passive model for ppGpp/DksA regulation of sigma54-dependent transcription that depends on the potent negative effects of these regulatory molecules on transcription from powerful stringently regulated sigma70 promoters.

  • 7.
    Brady, L. Jeannine
    et al.
    Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA.
    Maddocks, Sarah E.
    School of Oral and Dental Sciences, University of Bristol, Bristol BS1 2LY, UK.
    Larson, Matthew R.
    Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
    Forsgren, Nina
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Kariologi.
    Persson, Karina
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Deivanayagam, Champion C.
    Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
    Jenkinson, Howard F.
    School of Oral and Dental Sciences, University of Bristol, Bristol BS1 2LY, UK.
    The changing faces of Streptococcus antigen I/II polypeptide family adhesins2010Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 77, nr 2, s. 276-286Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Streptococcus mutans antigen I/II (AgI/II) protein was one of the first cell wall-anchored adhesins identified in Gram-positive bacteria. It mediates attachment of S. mutans to tooth surfaces and has been a focus for immunization studies against dental caries. The AgI/II family polypeptides recognize salivary glycoproteins, and are also involved in biofilm formation, platelet aggregation, tissue invasion and immune modulation. The genes encoding AgI/II family polypeptides are found among Streptococcus species indigenous to the human mouth, as well as in Streptococcus pyogenes, S. agalactiae and S. suis. Evidence of functionalities for different regions of the AgI/II proteins has emerged. A sequence motif within the C-terminal portion of Streptococcus gordonii SspB (AgI/II) is bound by Porphyromonas gingivalis, thus promoting oral colonization by this anaerobic pathogen. The significance of other epitopes is now clearer following resolution of regional crystal structures. A new picture emerges of the central V (variable) region, predicted to contain a carbohydrate-binding trench, being projected from the cell surface by a stalk formed by an unusual association between an N-terminal α-helix and a C-terminal polyproline helix. This presentation mode might be important in determining functional conformations of other Gram-positive surface proteins that have adhesin domains flanked by α-helical and proline-rich regions.

    Ever since dental caries (tooth decay) was first shown to be caused by bacteria, there has been continued interest in developing vaccine or passive immunization protocols for its control or prevention (Lehner et al., 1980). Although dental caries is not fatal, and in developed countries caries is now considered to be largely avoidable through controlled diet and good oral hygiene, there remain significant problems with childhood disease, especially among indigent populations. Consequently, caries is one of the most common worldwide infectious diseases. Therefore, research continues towards employing vaccine formulations comprised of peptide components derived from surface proteins of Streptococcus mutans, a major agent associated with dental caries (Lehner et al., 1975). One of the most promising strategies seems to be delivery of peptides, derived from glucan-binding protein B (GbpB) and antigen I/II (AgI/II) protein, via a mucosal (nasal) route. The GbpB polypeptide binds extracellular glucans, thus promoting co-adhesion of S. mutans cells in the development of dental plaque (Taubman and Nash, 2006). The AgI/II protein (also named P1, SpaP, AgB or PAc) is a major surface protein that functions as an adhesin, attaching S. mutans to the saliva-coated tooth enamel surface (Koga et al., 1990; Kelly et al., 1995). Antibodies against SpaP and GbpB block adherence and co-adhesion, respectively, thus disrupting colonization of the oral cavity by S. mutans (Ma et al., 1990; 1998; Taubman and Nash, 2006).

    The terminology AgI/II derives from the identification of two major cell wall antigens I and II in S. mutans by Russell et al. (1980), and the subsequent recognition that AgII was a component of AgI. Following the discovery of AgI/II, it became apparent that genes encoding orthologous proteins were widely dispersed among the streptococci (Jenkinson and Demuth, 1997). The viridans Streptococcus AgI/II adhesins range in composition from 1310 to 1653 amino acid (aa) residues, while the Streptococcus agalactiae AgI/II proteins are smaller (826–932 aa residues) (Tettelin et al., 2005). The widespread distribution of these AgI/II protein genes across the streptococci is perhaps not surprising, given the complex streptococcal communities that exist on surfaces of the oro- and naso-pharynx and within the bacterial soup of saliva. It is interesting, though, that the AgI/II family polypeptide genes have not yet been discovered in Streptococcus pneumoniae, which might be by the fact that S. pneumoniae forms a distinct evolutionary cluster (Kilian et al., 2008).

  • 8. Bru, Samuel
    et al.
    Marc Martinez-Lainez, Joan
    Hernandez-Ortega, Sara
    Quandt, Eva
    Torres-Torronteras, Javier
    Marti, Ramon
    Canadell, David
    Arino, Joaquin
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Jimenez, Javier
    Clotet, Josep
    Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae2016Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 101, nr 3, s. 367-380Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4 Delta) or hydrolyse polyP (ppn1 Delta, ppx1 Delta) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.

  • 9. Bryan, Samantha J.
    et al.
    Burroughs, Nigel J.
    Shevela, Dmitriy
    Department of Mathematics and Natural Science, University of Stavanger, Stavanger, Norway.
    Yu, Jianfeng
    Rupprecht, Eva
    Liu, Lu-Ning
    Mastroianni, Giulia
    Xue, Quan
    Llorente-Garcia, Isabel
    Leake, Mark C.
    Eichacker, Lutz A.
    Schneider, Dirk
    Nixon, Peter J.
    Mullineaux, Conrad W.
    Localisation and interaction of the Vipp1 protein in cyanobacteria2014Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 94, s. 1179-1195Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Vipp1 protein is essential in cyanobacteria and chloroplasts for the maintenance of photosynthetic function and thylakoid membrane architecture. To investigate its mode of action we generated strains of the cyanobacteria Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942 in which Vipp1 was tagged with green fluorescent protein at the C-terminus and expressed from the native chromosomal locus. There was little perturbation of function. Live-cell fluorescence imaging shows dramatic relocalisation of Vipp1 under high light. Under low light, Vipp1 is predominantly dispersed in the cytoplasm with occasional concentrations at the outer periphery of the thylakoid membranes. High light induces Vipp1 coalescence into localised puncta within minutes, with net relocation of Vipp1 to the vicinity of the cytoplasmic membrane and the thylakoid membranes. Pull-downs and mass spectrometry identify an extensive collection of proteins that are directly or indirectly associated with Vipp1 only after high-light exposure. These include not only photosynthetic and stress-related proteins but also RNA-processing, translation and protein assembly factors. This suggests that the Vipp1 puncta could be involved in protein assembly. One possibility is that Vipp1 is involved in the formation of stress-induced localised protein assembly centres, enabling enhanced protein synthesis and delivery to membranes under stress conditions.

  • 10.
    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.

  • 11.
    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.

  • 12.
    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.

  • 13. Charpentier, E
    et al.
    Novak, R
    Tuomanen, E
    Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC.2000Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 37, nr 4, s. 717-26Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The ClpC ATPase is a subfamily of HSP100/Clp molecular chaperones-regulators of proteolysis. By screening a library of loss of function mutants for the ability to survive treatment with penicillin, we identified the gene clpC. The corresponding protein was identified as a ClpC ATPase, sharing strong peptide sequence identity with ClpC of Bacillus subtilis, Listeria monocytogenes and Lactococcus lactis. Northern blot experiments showed that expression of clpC was induced in response to high temperature (40-42 degrees C) versus 37 degrees C, suggesting that ClpC is a heat shock protein. Insertional duplication mutagenesis of clpC resulted in increased tolerance to high temperature; a result in contrast to other bacterial Clp proteases. The clpC-deficient mutant formed long chains and failed to undergo lysis after treatment with penicillin or vancomycin. The effect of the clpC mutation extended to deficiency of adherence to the human type II alveolar cells. Finally, the clpC disruption resulted in decreased genetic transformation. Western blot analysis demonstrated that the mutant failed to express pneumolysin and the choline-binding proteins LytA, CbpA, CbpE, CbpF, CbpJ. These results suggest that the heat shock protein ClpC plays an essential complex pleiotropic role in pneumococcal physiology, including cell growth under heat stress, cell division, autolysis, adherence and transformation.

  • 14.
    Cisneros, David A.
    et al.
    Molecular Genetics Unit, Department of Microbiology, Institut Pasteur, 75015, Paris, France; CNRS ERL3526, 75015 Paris, France..
    Pehau-Arnaudet, Gerard
    Francetic, Olivera
    Heterologous assembly of type IV pili by a type II secretion system reveals the role of minor pilins in assembly initiation2012Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 86, nr 4, s. 805-818Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In Gram-negative bacteria, type IV pilus assembly (T4PS) and type II secretion (T2SS) systems polymerize inner membrane proteins called major pilins or pseudopilins respectively, into thin filaments. Four minor pilins are required in both systems for efficient fibre assembly. Escherichia coli K-12 has a set of T4PS assembly genes that are silent under standard growth conditions. We studied the heterologous assembly of the E. coli type IV pilin PpdD by the Klebsiella oxytoca T2SS called the Pul system. PpdD pilus assembly in this context depended on the expression of the K. oxytoca minor pseudopilin genes pulHIJK or of the E. coli minor pilin genes ppdAB-ygdB-ppdC. The E. coli minor pilins restored assembly of the major pseudopilin PulG in a pulHIJK mutant, but not the secretion of the T2SS substrate pullulanase. Thus, minor pilins and minor pseudopilins are functionally interchangeable in initiating major pilin assembly, further extending the fundamental similarities between the two systems. The data suggest that, in both systems, minor pilins activate the assembly machinery through a common self-assembly mechanism. When produced together, PulG and PpdD assembled into distinct homopolymers, establishing major pilins as key determinants of pilus elongation and structure.

  • 15.
    Croxatto, Antony
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Pride, John
    Hardman, Andrea
    Williams, Paul
    Cámara, Miguel
    Milton, Debra L.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    A distinctive dual-channel quorum-sensing system operates in Vibrio anguillarum2004Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 52, nr 6, s. 1677-1689Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many bacterial cells communicate using diffusible signal molecules to monitor cell population density via a process termed quorum sensing. In marine Vibrio species, the Vibrio harveyi-type LuxR protein is a key player in a quorum-sensing phosphorelay cascade, which controls the expression of virulence, symbiotic and survival genes. Previously, we characterized Vibrio anguillarum homologues of LuxR (VanT) and LuxMN (VanMN) and, in this study, we have identified homologues of LuxPQ (VanPQ) and LuxOU (VanOU). In contrast to other Vibrio species, vanT was expressed at low cell density and showed no significant induction as the cell number increased. In addition, although the loss of VanO increased vanT expression, the loss of VanU, unexpectedly, decreased it. Both VanN and VanQ were required for repression of vanT even in a vanU mutant, suggesting an alternative route for VanNQ signal transduction other than via VanU. VanT negatively regulated its own expression by binding and repressing the vanT promoter and by binding and activating the vanOU promoter. The signal relay results in a cellular response as expression of the metalloprotease, empA, was altered similar to that of vanT in all the mutants. Consequently, the V. anguillarum quorum-sensing phosphorelay systems work differently from those of V. harveyi and may be used to limit rather than induce vanT expression.

  • 16.
    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.

  • 17.
    Dörr, Tobias
    et al.
    Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, USA ; Department of Microbiology and Immunobiology, Harvard Medical School and HHMI, Boston, MA, USA .
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). 3 Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain.
    Lam, Hubert
    Discovery Research, Sanofi Pasteur, Cambridge, MA, USA.
    Davis, Brigid M
    Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, USA ; Department of Microbiology and Immunobiology, Harvard Medical School and HHMI, Boston, MA, USA .
    Waldor, Matthew K
    Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, USA ; Department of Microbiology and Immunobiology, Harvard Medical School and HHMI, Boston, MA, USA .
    Substrate specificity of an elongation-specific peptidoglycan endopeptidase and its implications for cell wall architecture and growth of Vibrio cholerae2013Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 89, nr 5, s. 949-962Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial cell wall consists of peptidoglycan (PG), a sturdy mesh of glycan strands cross-linked by short peptides. This rigid structure constrains cell shape and size, yet is sufficiently dynamic to accommodate insertion of newly synthesized PG, which was long hypothesized, and recently demonstrated, to require cleavage of the covalent peptide cross-links that couple previously inserted material. Here, we identify several genes in Vibrio cholerae that collectively are required for growth - particularly elongation - of this pathogen. V. cholerae encodes three putative periplasmic proteins, here denoted ShyA, ShyB, and ShyC, that contain both PG binding and M23 family peptidase domains. While none is essential individually, the absence of both ShyA and ShyC results in synthetic lethality, while the absence of ShyA and ShyB causes a significant growth deficiency. ShyA is a D,d-endopeptidase able to cleave most peptide chain cross-links in V. cholerae's PG. PG from a ∆shyA mutant has decreased average chain length, suggesting that ShyA may promote removal of short PG strands. Unexpectedly, ShyA has little activity against muropeptides containing pentapeptides, which typically characterize newly synthesized material. ShyA's substrate-dependent activity may contribute to selection of cleavage sites in PG, whose implications for the process of side-wall growth are discussed.

  • 18.
    Edqvist, Petra J
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Bröms, Jeanette E
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Betts, Helen J
    Forsberg, Ake
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Pallen, Mark J
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinsk fakultet, Umeå Centre for Microbial Research (UCMR).
    Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.2006Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 59, nr 1, s. 31-44Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Non-flagellar type III secretion systems (T3SSs) transport proteins across the bacterial cell and into eukaryotic cells. Targeting of proteins into host cells requires a dedicated translocation apparatus. Efficient secretion of the translocator proteins that make up this apparatus depends on molecular chaperones. Chaperones of the translocators (also called class-II chaperones) are characterized by the possession of three tandem tetratricopeptide repeats (TPRs). We wished to dissect the relations between chaperone structure and function and to validate a structural model using site-directed mutagenesis. Drawing on a number of experimental approaches and focusing on LcrH, a class-II chaperone from the Yersinia Ysc-Yop T3SS, we examined the contributions of different residues, residue classes and regions of the protein to chaperone stability, chaperone-substrate binding, substrate stability and secretion and regulation of Yop protein synthesis. We confirmed the expected role of the conserved canonical residues from the TPRs to chaperone stability and function. Eleven mutations specifically abrogated YopB binding or secretion while three mutations led to a specific loss of YopD secretion. These are the first mutations described for any class-II chaperone that allow interactions with one translocator to be dissociated from interactions with the other. Strikingly, all mutations affecting the interaction with YopB mapped to residues with side chains projecting from the inner, concave surface of the modelled TPR structure, defining a YopB interaction site. Conversely, all mutations preventing YopD secretion affect residues that lie on the outer, convex surface of the triple-TPR cluster in our model, suggesting that this region of the molecule represents a distinct interaction site for YopD. Intriguingly, one of the LcrH double mutants, Y40A/F44A, was able to maintain stable substrates inside bacteria, but unable to secrete them, suggesting that these two residues might influence delivery of substrates to the secretion apparatus.

  • 19.
    Forslund, Anna-Lena
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Kuoppa, Kerstin
    Svensson, Kerstin
    Salomonsson, Emelie
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Johansson, Anders
    Byström, Mona
    Oyston, Petra C. F.
    Michell, Stephen L.
    Titball, Richard W.
    Noppa, Laila
    Frithz-Lindsten, Elisabet
    Forsman, Mats
    Forsberg, Åke
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Direct repeat-mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis2006Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 59, nr 6, s. 1818-1830Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis, the causative agent of tularaemia, is a highly infectious and virulent intracellular pathogen. There are two main human pathogenic subspecies, Francisella tularensis ssp. tularensis (type A), and Francisella tularensis ssp. holarctica (type B). So far, knowledge regarding key virulence determinants is limited but it is clear that intracellular survival and multiplication is one major virulence strategy of Francisella. In addition, genome sequencing has revealed the presence of genes encoding type IV pili (Tfp). One genomic region encoding three proteins with signatures typical for type IV pilins contained two 120 bp direct repeats. Here we establish that repeat-mediated loss of one of the putative pilin genes in a type B strain results in severe virulence attenuation in mice infected by subcutaneous route. Complementation of the mutant by introduction of the pilin gene in cis resulted in complete restoration of virulence. The level of attenuation was similar to that of the live vaccine strain and this strain was also found to lack the pilin gene as result of a similar deletion event mediated by the direct repeats. Presence of the pilin had no major effect on the ability to interact, survive and multiply inside macrophage-like cell lines. Importantly, the pilin-negative strain was impaired in its ability to spread from the initial site of infection to the spleen. Our findings indicate that this putative pilin is critical for Francisella infections that occur via peripheral routes.

  • 20.
    Francis, Matthew
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Aili, Margareta
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wiklund, Magda-Lena
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    A study of the YopD-LcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD2000Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 38, nr 1, s. 85-102Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersiniaouter protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic α-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic α-helix abolished the YopD–LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.

  • 21.
    Francis, Matthew S.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Aili, Margareta
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wiklund, Magda-Lena
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wolf-Watz, Hans
    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). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    A study of the YopD-LcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD2000Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 38, nr 1, s. 85-102Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersinia outer protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic alpha-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic alpha-helix abolished the YopD-LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.

  • 22.
    Francis, Matthew S
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lloyd, Scott A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The type III secretion chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis2001Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 42, nr 4, s. 1075-1093Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens secrete and subsequently translocate antihost effector proteins into target eukaryotic cells by a common type III secretion system (TTSS). In this process, YopD (Yersinia outer protein D) is essential to establish regulatory control of Yop synthesis and the ensuing translocation process. YopD function depends upon the non-secreted TTSS chaperone LcrH (low-calcium response H), which is required for presecretory stabilization of YopD. However, as a new role for TTSS chaperones in virulence gene regulation has been proposed recently, we undertook a detailed analysis of LcrH. A lcrH null mutant constitutively produced Yops, even when this strain was engineered to produce wild-type levels of YopD. Furthermore, the YopD-LcrH interaction was necessary to regain the negative regulation of virulence associated genes yops). This finding was used to investigate the biological significance of several LcrH mutants with varied YopD binding potential. Mutated LcrH alleles were introduced in trans into a lcrH null mutant to assess their impact on yop regulation and the subsequent translocation of YopE, a Rho-GTPase activating protein, across the plasma membrane of eukaryotic cells. Two mutants, LcrHK20E, E30G, I31V, M99V, D136G and LcrHE30G lost all regulatory control, even though YopD binding and secretion and the subsequent translocation of YopE was indistinguishable from wild type. Moreover, these regulatory deficient mutants showed a reduced ability to bind YscY in the two-hybrid assay. Collectively, these findings confirm that LcrH plays an active role in yop regulation that might be mediated via an interaction with the Ysc secretion apparatus. This chaperone-substrate interaction presents an innovative means to establish a regulatory hierarchy in Yersinia infections. It also raises the question as to whether or not LcrH is a true chaperone involved in stabilization and secretion of YopD or a regulatory protein responsible for co-ordinating synthesis of Yersinia virulence determinants. We suggest that LcrH can exhibit both of these activities.

  • 23.
    Francis, Matthew S
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    YopD of Yersinia pseudotuberculosis is translocated into the cytosol of HeLa epithelial cells: evidence of a structural domain necessary for translocation.1998Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 29, nr 3, s. 799-813Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Yersinia pseudotuberculosis YopB and YopD proteins are essential for translocation of Yop effector proteins into the target cell cytosol. YopB is suggested to mediate pore formation in the target cell plasma membrane, allowing translocation of Yop effector proteins, although the function of YopD is unclear. To investigate the role in translocation for YopD, a mutant strain in Y. pseudotuberculosis was constructed containing an in frame deletion of essentially the entire yopD gene. As shown recently for the Y. pestis YopD protein, we found that the in vitro low calcium response controlling virulence gene expression was negatively regulated by YopD. This yopD null mutant (YPIII/pIB621) was also non-cytotoxic towards HeLa cell monolayers, supporting the role for YopD in the translocation process. Although other constituents of the Yersinia translocase apparatus (YopB, YopK and YopN) are not translocated into the host cell cytosol, fractionation of infected HeLa cells allowed us to identify the cytosolic localization of YopD by the wild-type strain (YPIII/pIB102), but not by strains defective in either YopD or YopB. YopD was also identified by immunofluorescence in the cytoplasm of HeLa cell monolayers infected with a multiple yop mutant strain (YPIII/pIB29MEKA). These results demonstrate a dual function for YopD in negative regulation of Yop production and Yop effector translocation, including the YopD protein itself. To investigate whether an amphipathic domain near the C-terminus of YopD is involved in the translocation process, a mutant strain (YPIII/pIB155deltaD278-292) was constructed that is devoid of this region. Phenotypically, this small in frame deltayopD278-292 deletion mutant was indistinguishable from the yopD null mutant. The truncated YopD protein and Yop effectors were not translocated into the cytosol of HeLa cell monolayers infected with this mutant. The comparable regulatory and translocation phenotypes displayed by the small in frame deltayopD278-292 deletion and deltayopD null mutants suggest that regulation of Yop synthesis and Yop translocation are intimately coupled. We present an intriguing scenario to the Yersinia infection process that highlights the need for polarized translocation of YopD to specifically establish translocation of Yop effectors. These observations are contrary to previous suggestions that members of the translocase apparatus were not translocated into the host cell cytosol.

  • 24.
    Frithz-Lindsten, Elisabet
    et al.
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Holmström, Anna
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Jacobsson, Lars
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Soltani, Mehnam
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Olsson, Jan
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Rosqvist, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Functional conservation of the effector protein translocators PopB/YopB and PopD/YopD of Pseudomonas aeruginosa and Yersinia pseudotuberculosis.1998Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 29, nr 5, s. 1155-1165Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Virulent Yersinia species cause systemic infections in rodents, and Y. pestis is highly pathogenic for humans. Pseudomonas aeruginosa, on the other hand, is an opportunistic pathogen, which normally infects only compromised individuals. Surprisingly, these pathogens both encode highly related contact-dependent secretion systems for the targeting of toxins into eukaryotic cells. In Yersinia, YopB and YopD direct the translocation of the secreted Yop effectors across the target cell membrane. In this study, we have analysed the function of the YopB and YopD homologues, PopB and PopD, encoded by P. aeruginosa. Expression of the pcrGVHpopBD operon in defined translocation-deficient mutants (yopB/yopD) of Yersinia resulted in complete complementation of the cell contact-dependent, YopE-induced cytotoxicity of Y. pseudotuberculosis on HeLa cells. We demonstrated that the complementation fully restored the ability of Y. pseudotuberculosis to translocate the effector molecules YopE and YopH into the HeLa cells. Similar to YopB, PopB induced a lytic effect on infected erythrocytes. The lytic activity induced by PopB could be prevented if the erythrocytes were infected in the presence of sugars larger than 3 nm in diameter, indicating that PopB induced a pore of similar size compared with that induced by YopB. Our findings show that the contact-dependent toxin-targeting mechanisms of Y. pseudotuberculosis and P. aeruginosa are conserved at the molecular level and that the translocator proteins are functionally interchangeable. Based on these similarities, we suggest that the translocation of toxins such as ExoS, ExoT and ExoU by P. aeruginosa across the eukaryotic cell membrane occurs via a pore induced by PopB.

  • 25. Hammar, M
    et al.
    Arnqvist, Anna
    Bian, Z
    Olsén, A
    Normark, S
    Expression of two csg operons is required for production of fibronectin- and congo red-binding curli polymers in Escherichia coli K-12.1995Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 18, nr 4, s. 661-70Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Two divergently transcribed operons in Escherichia coli required for the expression of fibronectin- and Congo red-binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein. The csgBA operon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non-polar csgB mutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers. A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre. The promoter for the oppositely transcribed csgDEFG operon was identified by primer extension and shown, like the csgBA promoter, to be dependent upon the alternate stationary phase-specific sigma factor sigma s in wild-type cells, but not in mutants lacking the nucleoid associated protein H-NS. Insertions in csgD abolish completely trancription from the csgBA promoter. Therefore, any regulatory effect on the csgBA promoter might be secondary to events controlling the csgDEFG promoter and/or activation of CsgD. Insertions in csgE, csgF and csgG abolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein. No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a novel pathway.

  • 26.
    Holmström, Anna
    et al.
    Swedish Defence Research Agency, Division of CBRN Defence and Security, SE-901 82 Umeå, Sweden.
    Olsson, Jan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Cherepanov, Peter
    Maier, Elke
    Nordfelth, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Pettersson, Jonas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Benz, Roland
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Forsberg, Åke
    LcrV is a channel size-determining component of the Yop effector translocon of Yersinia2001Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 39, nr 3, s. 620-632Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Delivery of Yop effector proteins by pathogenic Yersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact‐dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127–195 of LcrV and to amino acids 106–173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size‐determining structural component of the Yop translocon.

  • 27.
    Holmström, Anna
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Petterson, Jonas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Rosqvist, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Håkansson, Sebastian
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Tafazoli, Farideh
    Department of Medical Microbiology, Linköping University, S-581 85 Linköping, Sweden.
    Fällman, Maria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Magnusson, Karl-Eric
    Department of Medical Microbiology, Linköping University, S-581 85 Linköping, Sweden.
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Department of Microbiology, National Defence Research Establishment, S-901 82 Umeå, Sweden..
    YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane.1997Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 24, nr 1, s. 73-91Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Introduction of anti-host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram-negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia, the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK-mutant strain was found to induce a more rapid YopE-mediated cytotoxic response in HeLa cells as well as in MDCK-1 cells compared to the wild-type strain. We found that this was the result of a cell-contact-dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB-dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK-mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild-type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell-associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.

  • 28. Howell, Matthew
    et al.
    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).
    Sundararajan, Kousik
    Daniel, Jeremy J.
    Lariviere, Patrick J.
    Goley, Erin D.
    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).
    Brown, Pamela J. B.
    Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division2019Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 111, nr 4, s. 1074-1092Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re-direct peptidoglycan biosynthesis to mid-cell during cell division in polar-growing Alphaproteobacteria are largely unknown. Here, we explore the role of early division proteins of Agrobacterium tumefaciens including three FtsZ homologs, FtsA and FtsW in the transition from polar growth to mid-cell growth and ultimately cell division. Although two of the three FtsZ homologs localize to mid-cell, exhibit GTPase activity and form co-polymers, only one, FtsZ(AT), is required for cell division. We find that FtsZ(AT) is required not only for constriction and cell separation, but also for initiation of peptidoglycan synthesis at mid-cell and cessation of polar peptidoglycan biosynthesis. Depletion of FtsZ(AT) in A. tumefaciens causes a striking phenotype: cells are extensively branched and accumulate growth active poles through tip splitting events. When cell division is blocked at a later stage by depletion of FtsA or FtsW, polar growth is terminated and ectopic growth poles emerge from mid-cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions to the regulation of polar growth and cell division.

  • 29. Jakubovics, NS
    et al.
    Strömberg, Nicklas
    Umeå universitet, Medicinsk fakultet, Odontologi, Kariologi.
    van Dolleweerd, CJ
    Kelly, CG
    Jenkinson, HF
    Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands.2005Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 55, nr 5, s. 1591-1605Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The antigen I/II (AgI/II) family polypeptides, ranging from 1310 to 1653 amino acid (aa) residues, are cell wall anchored adhesins expressed by most indigenous species of oral streptococci. The polypeptides interact with a wide range of host molecules, in particular salivary agglutinin glycoprotein (SAG or gp340), and with ligands on other oral bacteria. To determine the receptor recognition properties of six different AgI/II family polypeptides from strains of Streptococcus gordonii, Streptococcus intermedius and Streptococcus mutans, the genes were cloned and expressed on the surface of the surrogate host Lactococcus lactis. The S. gordonii SspA and SspB polypeptides mediated higher binding levels of L. lactis cells to surface immobilized gp340 than did S. intermedius Pas protein, or S. mutans SpaP or PAc proteins. However, the AgI/II proteins were all similar in their abilities to mediate aggregation of lactococci by fluid phase gp340. The SpaP(I) polypeptide from S. mutans Ingbritt, which was C-terminally truncated by approximately 400 aa residues, did not bind gp340. Lactococci expressing AgI/II proteins, including SpaP(I), were aggregated by a synthetic 16 aa residue peptide SRCRP2 derived from the aa repeat block sequences within gp340. In coaggregation assays, SspB from S. gordonii was unique in mediating coaggregation with only group A and group E strains of Actinomyces naeslundii. All the other AgI/II polypeptides mediated coaggregation with group C and group D strains of A. naeslundii. Analysis of chimeric protein constructs revealed that coaggregation specificity was determined by sequences within the N-terminal half of AgI/II protein. A synthetic peptide (20 aa residues), which defines a putative adhesion epitope within the C-terminal region of polypeptide, inhibited AgI/II-mediated aggregation by gp340 but did not affect coaggregation with A. naeslundii. These results suggest that different mechanisms operate in interactions of AgI/II family polypeptides with native gp340, gp340 SRCR domain peptide, and A. naeslundii. Specificity of these interactions appears to be determined by discontinuous but interacting regions of the polypeptides, thus providing flexibility in receptor recognition for streptococcal colonization of the human host.

  • 30. KALLA, R
    et al.
    LIND, LK
    Gustafsson, Petter
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    GENETIC-ANALYSIS OF PHYCOBILISOME MUTANTS IN THE CYANOBACTERIUM SYNECHOCOCCUS SPECIES PCC-63011989Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 3, nr 3, s. 339-347Artikel i tidskrift (Refereegranskat)
  • 31. Koshla, Oksana
    et al.
    Yushchuk, Oleksandr
    Stash, Iryna
    Dacyuk, Yuriy
    Myronovskyi, Maksym
    Jäger, Gunilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Sussmuth, Roderich D.
    Luzhetskyy, Andriy
    Byström, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Kirsebom, Leif A.
    Ostash, Bohdan
    Gene miaA for post-transcriptional modification of tRNAXXA is important for morphological and metabolic differentiation in Streptomyces2019Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 112, nr 1, s. 249-265Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Members of actinobacterial genus Streptomyces possess a sophisticated life cycle and are the deepest source of bioactive secondary metabolites. Although morphogenesis and secondary metabolism are subject to transcriptional co-regulation, streptomycetes employ an additional mechanism to initiate the aforementioned processes. This mechanism is based on delayed translation of rare leucyl codon UUA by the only cognate tRNA(UAA)(Leu) (encoded by bldA). The bldA-based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post-transcriptional tRNA modifications play a role in tRNA-based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)-N6)-dimethylallyltransferase and tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms(2)i(6)A37 residue in most of the A36-A37-containing tRNAs. We show that miaB and especially miaA null mutant of S. albus possess altered morphogenesis and secondary metabolism. We provide genetic evidence that miaA deficiency impacts translational level of gene expression, most likely through impaired decoding of codons UXX and UUA in particular.

  • 32.
    Li, Yunlong
    et al.
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Li, Lamei
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Huang, Li
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Francis, Matthew
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Hu, Yangbo
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Chen, Shiyun
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Yersinia Ysc-Yop type III secretion feedback inhibition is relieved through YscV-dependent recognition and secretion of LcrQ2014Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 91, nr 3, s. 494-507Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Human pathogenic Yersinia species share a virulence plasmid encoding the Ysc-Yop type III secretion system (T3SS). A plasmid-encoded anti-activator, LcrQ, negatively regulates the expression of this secretion system. Under inducible conditions, LcrQ is secreted outside of bacterial cells and this activates the T3SS, but the mechanism of targeting LcrQ for type III secretion remains largely unknown. In this study, we characterized the regulatory role of the export apparatus component YscV. Depletion or overexpression of YscV compromised Yop synthesis and this primarily prevented secretion of LcrQ. It followed that a lcrQ deletion reversed the repressive effects of excessive YscV. Further characterization demonstrated that the YscV residues 493–511 located within the C-terminal soluble cytoplasmic domain directly bound with LcrQ. Critically, YscV-LcrQ complex formation was a requirement for LcrQ secretion, since YscVΔ493–511 failed to secrete LcrQ. This forced a cytoplasmic accumulation of LcrQ, which predictably caused the feedback inhibition of Yops synthesis. Based on these observations, we proposed a model for the YscV-dependent secretion of LcrQ and its role in regulating Yop synthesis in Yersinia.

  • 33. Lind, Peter A
    et al.
    Tobin, Christina
    Berg, Otto G
    Kurland, Charles G
    Andersson, Dan I
    Compensatory gene amplification restores fitness after inter-species gene replacements.2010Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 75, nr 5Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Genes introduced by gene replacements and other types of horizontal gene transfer (HGT) represent a significant presence in many archaeal and eubacterial genomes. Most alien genes are likely to be neutral or deleterious upon arrival and their long-term persistence may require a mechanism that improves their selective contribution. To examine the fate of inter-species gene replacements, we exchanged three native S. typhimurium genes encoding ribosomal proteins with orthologues from various other microbes. The results show that replacement of each of these three genes reduces fitness to such an extent that it would provide an effective barrier against inter-species gene replacements in eubacterial populations. However, these fitness defects could be partially ameliorated by gene amplification that augmented the dosage of the heterologous proteins. This suggests that suboptimal expression is a common fitness constraint for inter-species gene replacements, with fitness costs conferred by either a lower expression level of the alien protein compared with the native protein or a requirement for an increased amount of the alien protein to maintain proper function. Our findings can explain the observation that duplicated genes are over-represented among horizontally transferred genes, and suggest a potential coupling between compensatory gene amplification after HGT and the evolution of new genes.

  • 34. Maddocks, Sarah E.
    et al.
    Wright, Christopher J.
    Nobbs, Angela H.
    Brittan, Jane L.
    Franklin, Linda
    Strömberg, Nicklas
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Kariologi.
    Kadioglu, Aras
    Jepson, Mark A.
    Jenkinson, Howard F.
    Streptococcus pyogenes antigen I/II-family polypeptide AspA shows differential ligand-binding properties and mediates biofilm formation2011Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 81, nr 4, s. 1034-1049Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The streptococcal antigen I/II (AgI/II)-family polypeptides are cell wall-anchored adhesins expressed by most indigenous oral streptococci. Proteins sharing 30-40% overall amino acid sequence similarities with AgI/II-family proteins are also expressed by Streptococcus pyogenes. The S. pyogenes M28_Spy1325 polypeptide (designated AspA) displays an AgI/II primary structure, with alanine-rich (A) and prolinerich (P) repeats flanking a V region that is projected distal from the cell. In this study it is shown that AspA from serotype M28 S. pyogenes, when expressed on surrogate host Lactococcus lactis, confers binding to immobilized salivary agglutinin gp-340. This binding was blocked by antibodies to the AspA-VP region. In contrast, the N-terminal region of AspA was deficient in binding fluid-phase gp-340, and L. lactis cells expressing AspA were not agglutinated by gp-340. Deletion of the aspA gene from two different M28 strains of S. pyogenes abrogated their abilities to form biofilms on saliva-coated surfaces. In each mutant strain, biofilm formation was restored by trans complementation of the aspA deletion. In addition, expression of AspA protein on the surface of L. lactis conferred biofilm-forming ability. Taken collectively, the results provide evidence that AspA is a biofilm-associated adhesin that may function in host colonization by S. pyogenes.

  • 35. Mangold, Monika
    et al.
    Siller, Maria
    Roppenser, Bernhard
    Vlaminckx, Bart J M
    Penfound, Tom A
    Klein, Reinhard
    Novak, Rodger
    Novick, Richard P
    Charpentier, Emmanuelle
    Synthesis of group A streptococcal virulence factors is controlled by a regulatory RNA molecule.2004Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 53, nr 5, s. 1515-1527Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The capacity of pathogens to cause disease depends strictly on the regulated expression of their virulence factors. In this study, we demonstrate that the untranslated mRNA of the recently described streptococcal pleiotropic effect locus (pel), which incidentally contains sagA, the structural gene for streptolysin S, is an effector of virulence factor expression in group A beta-haemolytic streptococci (GAS). Our data suggest that the regulation by pel RNA occurs at both transcriptional (e.g. emm, sic, nga) and post-transcriptional (e.g. SpeB) levels. We could exclude the possibility that the pel phenotype was linked to a polar effect on downstream genes (sagB-I). Remarkably, the RNA effector is regulated in a growth phase-dependent fashion and we provide evidence that pel RNA expression is induced by conditioned media.

  • 36. Mehlgarten, Constance
    et al.
    Jablonowski, Daniel
    Wrackmeyer, Uta
    Tschitschmann, Susan
    Sondermann, David
    Jäger, Gunilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Gong, Zhizhong
    Byström, Anders S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Schaffrath, Raffael
    Breunig, Karin D
    Elongator function in tRNA wobble uridine modification is conserved between yeast and plants2010Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 76, nr 5, s. 1082-1094Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator-dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator’s tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast-plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life.

  • 37. Meibom, Karin L
    et al.
    Dubail, Iharilalao
    Dupuis, Marion
    Barel, Monique
    Lenco, Juraj
    Stulik, Jiri
    Golovliov, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Charbit, Alain
    The heat-shock protein ClpB of Francisella tularensis is involved in stress tolerance and is required for multiplication in target organs of infected mice2008Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 67, nr 6, s. 1384-1401Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Intracellular bacterial pathogens generally express chaperones such as Hsp100s during multiplication in host cells, allowing them to survive potentially hostile conditions. Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. The ability of F. tularensis to multiply and survive in macrophages is considered essential for its virulence. Although previous mutant screens in Francisella have identified the Hsp100 chaperone ClpB as important for intracellular survival, no detailed study has been performed. We demonstrate here that ClpB of F. tularensis live vaccine strain (LVS) is important for resistance to cellular stress. Promoter analysis shows that the transcriptional start is preceded by a sigma32-like promoter sequence and we demonstrate that expression of clpB is induced by heat shock. This indicates that expression of clpB is dependent on the heat-shock response mediated by sigma32, the only alternative sigma-factor present in Francisella. Our studies demonstrate that ClpB contributes to intracellular multiplication in vitro, but is not essential. However, ClpB is absolutely required for Francisella to replicate in target organs and induce disease in mice. Proteomic analysis of membrane-enriched fractions shows that five proteins are recovered at lower levels in the mutant strain. The crucial role of ClpB for in vivo persistence of Francisella may be linked to its assumed function in reactivation of aggregated proteins under in vivo stress conditions.

  • 38.
    Mortezaei, Narges
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Epler, Chelsea
    Shao, Paul
    Shirdel, Mariam
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Yrkes- och miljömedicin.
    Bhupender, Singh
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    McVeigh, Annette
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Savarino, Stephen
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Bullitt, Esther
    Boston University School of Medicine.
    Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways2015Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 95, nr 1, s. 116-126Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pathogenic enterotoxigenic Escherichia coli (ETEC) are the major bacterial cause of diarrhea in young children in developing countries and in travelers, causing significant mortality in children. Adhesive fimbriae are a prime virulence factor for ETEC, initiating colonization of the small intestinal epithelium. Similar to other Gram-negative bacteria, ETEC express one or more diverse fimbriae, some assembled by the chaperone-usher pathway and others by the alternate chaperone pathway. Here, we elucidate structural and biophysical aspects and adaptations of each fimbrial type to its respective host niche. CS20 fimbriae are compared with colonization factor antigen I (CFA/I) fimbriae, which are two ETEC fimbriae assembled via different pathways, and with P-fimbriae from uropathogenic E.coli. Many fimbriae unwind from their native helical filament to an extended linear conformation under force, thereby sustaining adhesion by reducing load at the point of contact between the bacterium and the target cell. CFA/I fimbriae require the least force to unwind, followed by CS20 fimbriae and then P-fimbriae, which require the highest unwinding force. We conclude from our electron microscopy reconstructions, modeling and force spectroscopy data that the target niche plays a central role in the biophysical properties of fimbriae that are critical for bacterial pathophysiology.

  • 39. Mountain, H A
    et al.
    Byström, Anders S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Korch, C
    The general amino acid control regulates MET4, which encodes a methionine-pathway-specific transcriptional activator of Saccharomyces cerevisiae1993Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 7, nr 2, s. 215-228Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A met4 mutant of Saccharomyces cerevisiae was unable to transcribe a number of genes encoding enzymes of the methionine biosynthetic pathway. The sequence of the cloned MET4 gene allowed the previously sequenced flanking LEU4 and POL1 genes to be linked to MET4 into a 10,327 bp contiguous region of chromosome XIV. From the sequence and mapping of the transcriptional start points, MET4 is predicted to encode a protein of 634 amino acids (as opposed to 666 amino acids published by others) with a leucine zipper domain at the C-terminus, preceded by both acidic and basic regions. Thus, MET4 belongs to the family of basic leucine zipper trans-activator proteins. Disruption of MET4 resulted in methionine auxotrophy with no other phenotype. Transcriptional studies showed that MET4 was regulated by the general amino acid control and hence by another bZIP protein encoded by GCN4. GCN4 binding sequences are present between the divergently transcribed MET4 and LEU4 genes. Over-expression of MET4 resulted in leaky expression from the otherwise tightly regulated MET3 promoter under its control. The presence of consensus sequences for other potential regulatory elements in the MET4 promoter suggests a complex regulation of this gene.

  • 40.
    Müller, Claudia M
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Schneider, György
    Dobrindt, Ulrich
    Emödy, Levente
    Hacker, Jörg
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Differential effects and interactions of endogenous and horizontally acquired H-NS-like proteins in pathogenic Escherichia coli.2010Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 75, nr 2, s. 280-293Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The nucleoid-associated protein H-NS is important for gene regulation in Escherichia coli. We have studied H-NS interaction with StpA and an uncharacterized H-NS-like protein, Hfp, in the uropathogenic E. coli isolate 536 that expresses all three nucleoid-associated proteins. We found distinct interactions of the three proteins at the protein level, resulting in the formation of heteromers, as well as differences in their gene expression at the transcriptional level. Mutants lacking either StpA or Hfp alone did not exhibit a phenotype at 37 degrees C, which is consistent with a low level of expression at that temperature. Expression of the hfp and stpA genes was found to be induced by apparently diametrical conditions, and StpA and Hfp levels could be correlated to modulatory effects on the expression of different H-NS targets, the bgl operon and operons for virulence factors such as fimbriae and capsular polysaccharide. The hns/hfp and hns/stpA double mutants displayed severe growth defects at low and high temperatures respectively. Our findings demonstrated different requirements for the alternative H-NS/Hfp/StpA combinations under these growth conditions. We propose that Hfp and StpA have distinct functions and roles in a dynamic pool of nucleoid-associated proteins that is adapting to requirements in a particular environment.

  • 41. Novak, R
    et al.
    Braun, J S
    Charpentier, E
    Tuomanen, E
    Penicillin tolerance genes of Streptococcus pneumoniae: the ABC-type manganese permease complex Psa.1998Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 29, nr 5, s. 1285-1296Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Downregulation of the major autolysin in Streptococcus pneumoniae leads to penicillin tolerance, a feature that is characterized by the ability to survive but not grow in the presence of antibiotic. Screening a library of mutants in pneumococcal surface proteins for the ability to survive 10x minimum inhibitory concentration (MIC) of penicillin revealed over 10 candidate tolerance genes. One such mutant contained an insertion in the known gene psaA, which is part of the psa locus. This locus encodes an ABC-type Mn permease complex. Sequence analysis of adjacent DNA extended the known genetic organization of the locus to include two new open reading frames (ORFs), psaB, which encodes an ATP-binding protein, and psaC, which encodes a hydrophobic transmembrane protein. Mutagenesis of psaB, psaC, psaA and downstream psaD resulted in penicillin tolerance. Defective adhesion and reduced transformation efficiency, as reported previously for a psaA- mutant, were phenotypes shared by psaB-, psaC- and psaD- knockout mutants. Western blot analysis demonstrated that the set of mutants expressed RecA, but none of them showed translation of the autolysin gene, which is located downstream of recA. The addition of manganese (Mn) failed to correct the abnormal physiology. These results suggest that this ABC-type Mn permease complex has a pleiotropic effect on pneumococcal physiology including adherence and autolysis. These are the first genes suggested as being involved in triggering autolysin. The results raise the possibility that loss of function of PsaA, by vaccine-induced antibody for instance, may promote penicillin tolerance.

  • 42. Novak, R
    et al.
    Charpentier, E
    Braun, J S
    Park, E
    Murti, S
    Tuomanen, E
    Masure, R
    Extracellular targeting of choline-binding proteins in Streptococcus pneumoniae by a zinc metalloprotease.2000Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 36, nr 2, s. 366-376Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A genetic-based search for surface proteins of Streptococcus pneumoniae involved in adhesion identified a putative zinc metalloprotease (ZmpB). ZmpB shared high amino acid sequence similarities with IgA1 proteases of Gram-positive bacteria, but ZmpB had neither IgA1 nor IgA2 protease activity. Analysis of a family of surface-expressed proteins, the choline-binding proteins (Cbp's), in a zmpB-deficient mutant demonstrated a global loss of surface expression of CbpA, CbpE, CbpF and CbpJ. CbpA was detected within the cytoplasm. The zmpB-deficient mutant also failed to lyse with penicillin, a sign of lack of function of the Cbp LytA. Immunodetection studies revealed that the autolysin (LytA), normally located on the cell wall, was trapped in the cytoplasm colocalized with DNA and the transformation protein CinA. Trafficking of CinA and RecA to the cell membrane during genetic competence was also not observed in the zmpB-deficient mutant. These results suggest a protease dependent regulatory mechanism governing the translocation of CinA and the Cbp's LytA and CbpA of S. pneumoniae.

  • 43. Novak, R
    et al.
    Tuomanen, E
    Charpentier, E
    The mystery of psaA and penicillin tolerance in Streptococcus pneumoniae.2000Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 36, nr 6, s. 1504-1505Artikel i tidskrift (Refereegranskat)
  • 44.
    Olofsson, Annelie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Vallström, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Petzold, Katja
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Tegtmeyer, Nicole
    Schleucher, Jürgen
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Carlsson, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Haas, Rainer
    Backert, Steffen
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Arnqvist, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Biochemical and functional characterization of Helicobacter pylori vesicles2010Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 77, nr 6, s. 1539-1555Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Helicobacter pylori can cause peptic ulcer disease and/or gastric cancer. Adhesion of bacteria to the stomach mucosa is an important contributor to the vigor of infection and resulting virulence. H. pylori adheres primarily via binding of BabA adhesins to ABO/Lewis b (Leb) blood group antigens and the binding of SabA adhesins to sialyl-Lewis x/a (sLex/a) antigens. Similar to most Gram-negative bacteria, H. pylori continuously buds off vesicles and vesicles derived from pathogenic bacteria often include virulence-associated factors. Here we biochemically characterized highly purified H. pylori vesicles. Major protein and phospholipid components associated with the vesicles were identified with mass spectroscopy and NMR. A subset of virulence factors present was confirmed by immunoblots. Additional functional and biochemical analysis focused on the vesicle BabA and SabA adhesins and their respective interactions to human gastric epithelium. Vesicles exhibit heterogeneity in their protein composition, which were specifically studied in respect to the BabA adhesin. We also demonstrate that the oncoprotein, CagA, is associated with the surface of H. pylori vesicles. Thus, we have explored mechanisms for intimate H. pylori vesicle-host interactions and found that the vesicles carry effector-promoting properties that are important to disease development.

  • 45. Olsén, A
    et al.
    Arnqvist, Anna
    Hammar, M
    Sukupolvi, S
    Normark, S
    The RpoS sigma factor relieves H-NS-mediated transcriptional repression of csgA, the subunit gene of fibronectin-binding curli in Escherichia coli.1993Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 7, nr 4, s. 523-36Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Curli encoded by the curlin subunit gene, csgA, are fibronectin- and laminin-binding fibres expressed by many natural Escherichia coli and E. coli K-12 strains in response to low temperature, low osmolarity and stationary-phase growth conditions. Curli expression is dependent on RpoS, a sigma factor that controls many stationary phase-inducible genes. Many commonly used K-12 strains carry an amber mutation in rpoS. Strains able to form curli carry an amber suppressor whereas curli-negative E. coli K-12 strains, in general, are sup0. Introduction of SupD, SupE, or supF suppressors into sup0 strains resulted in expression of temperature-regulated curli. In curli-deficient, RpoS- E. coli K-12 strains, csgA is transcriptionally activated by mutations in hns, which encodes the histone-like protein H-NS. Curli expression, fibronectin binding, and csgA transcription remain temperature- and osmoregulated in such double mutants. Our data suggest that RpoS+ strains, and hence curli-proficient strains of E. coli K-12, are relieved for the transcriptional repression mediated by the H-NS protein upon accumulating RpoS as cells reach stationary phase.

  • 46.
    O'Neill, E
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Ng, L C
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Sze, C C
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Shingler, V
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Aromatic ligand binding and intramolecular signalling of the phenol-responsive sigma54-dependent regulator DmpR.1998Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 28, nr 1Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Pseudomonas-derived sigma54-dependent regulator DmpR has an amino-terminal A-domain controlling the specificity of activation by aromatic effectors, a central C-domain mediating an ATPase activity essential for transcriptional activation and a carboxy-terminal D-domain involved in DNA binding. In the presence of aromatic effectors, the DmpR protein promotes transcription from the -24, -12 Po promoter controlling the expression of specialized (methyl)phenol catabolic enzymes. Previous analysis of DmpR has led to a model in which the A-domain acts as an interdomain repressor of DmpR's ATPase and transcriptional promoting property until specific aromatic effectors are bound. Here, the autonomous nature of the A-domain in exerting its biological functions has been dissected by expressing portions of DmpR as independent polypeptides. The A-domain of DmpR is shown to be both necessary and sufficient to bind phenol. Analysis of phenol binding suggests one binding site per monomer of DmpR, with a dissociation constant of 16 microM. The A-domain is also shown to have specific affinity for the C-domain and to repress the C-domain mediated ATPase activity in vitro autonomously. However, physical uncoupling of the A-domain from the remainder of the regulator results in a system that does not respond to aromatics by its normal derepression mechanism. The mechanistic implications of aromatic non-responsiveness of autonomously expressed A-domain, despite its demonstrated ability to bind phenol, are discussed.

  • 47. O'Toole, R
    et al.
    Milton, Debra L
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, H
    Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum.1996Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 19, nr 3, s. 625-637Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The role of the flagellum and motility in the virulence of the marine fish pathogen Vibrio anguillarum was examined. Non-motile mutants were generated by transposon mutagenesis. Infectivity studies revealed that disruption of the flagellum and subsequent loss of motility correlated with an approximate 500-fold decrease in virulence when fish were inoculated by immersion in bacteria-containing water. However, the flagellar filament and motility were not required for pathogenicity following intraperitoneal injection of fish. The transposon-insertion site for six mutants was determined by cloning and sequencing of the Vibrio DNA flanking the transposon. V. anguillarum genes whose products showed strong homology to proteins with an established role in flagellum biosynthesis were identified. One of the aflagellate mutants had a transposon insertion in the rpoN gene of V. anguillarum. This rpoN mutant failed to grow at low concentrations of available iron and was avirulent by both the immersion and intraperitoneal modes of inoculation. A chemotaxis gene, cheR, was located upstream of one transposon insertion and an in-frame deletion was constructed in the coding region of this gene. The resulting non-chemotactic mutant exhibited wild-type pathogenicity when injected intra-peritoneally into fish but showed a decrease in virulence similar to that seen for the non-motile aflagellate mutants following immersion infection. Hence, chemotactic motility is a required function of the flagellum for the virulence of V. anguillarum.

  • 48. Park, Y I
    et al.
    Sandstrom, S
    Gustafsson, Petter
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Oquist, Gunnar
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Expression of the isiA gene is essential for the survival of the cyanobacterium Synechococcus sp PCC7942 by protecting photosystem II from excess light under iron limitation1999Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 32, nr 1, s. 123-129Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Iron deficiency is known to suppress primary productivity in both marine and freshwater ecosystems. In response to iron deficiency, certain cyanobacteria induce a chlorophyll (Chl)-protein complex, CP43 ', which is encoded by the isiA gene. The deduced amino-acid sequence of CP43 ' predicts some structural similarity to the CP43 polypeptide of photosystem II, but the function of CP43 ' remains uncertain. In order to assess its physiological role, the isiA gene of a cyanobacterium, Synechococcus sp. PCC7942, was inactivated by insertion mutagenesis (giving isiA(-) cells). Compared with isiA- cells, under iron deprivation, wild-type cells showed both lower rates of photosystem Ii-mediated O-2 evolution at limiting light irradiances and decreased yields of room temperature Chi fluorescence at various irradiances. These observations strongly suggest that the decreased photosystem II activity in wild-type cells with CP43 ' is attributable to increased non-radiative dissipation of light energy. In agreement with this hypothesis, isiA- cells were more susceptible to photoinhibition of photosynthesis than wild-type cells, resulting in much slower growth rates under iron limitation. Based on these results, we suggest that CP43' functions as a non-radiative dissipator of light energy, thus protecting photosystem II from excessive excitation under iron-deficient conditions.

  • 49.
    Persson, Cathrine
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Nordfelth, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Andersson, Kerstin
    Forsberg, Åke
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Localization of the Yersinia PTPase to focal complexes is an important virulence mechanism1999Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 33, nr 4, s. 828-838Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The protein tyrosine phosphatase YopH, produced by the pathogen Yersinia pseudotuberculosis, is an essential virulence determinant involved in antiphagocytosis. Upon infection, YopH is translocated into the target cell, where it recognizes focal complexes. Genetic analysis revealed that YopH harbours a region that is responsible for specific localization of this PTPase to focal complexes in HeLa cells and professional phagocytes. This region is a prerequisite for blocking an immediate-early Yersinia-induced signal within target cells. The region is also essential for antiphagocytosis and virulence, illustrating the biological significance of localization of YopH to focal complexes during Yersinia infection. These results also indicate that focal complexes play a role in the general phagocytic process.

  • 50.
    Persson, Cathrine
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Nordfelth, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Holmström, Anna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Håkansson, Sebastian
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Rosqvist, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Cell-surface-bound Yersinia translocate the protein tyrosine phosphatase YopH by a polarized mechanism into the target cell1995Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 18, nr 1, s. 135-150Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    YopH is translocated by cell-surface-bound bacteria through the plasma membrane to the cytosol of the HeLa cell. The transfer mechanism is contact dependent and polarizes the translocation to only occur at the contact zone between the bacterium and the target cell. More than 99% of the PTPase activity is associated with the HeLa cells. In contrast to the wild-type strain, the yopBD mutant cannot deliver YopH to the cytosol. Instead YopH is deposited in localized areas in the proximity of cell-associated bacteria. A yopN mutant secretes 40% of the total amount of YopH to the culture medium, suggesting a critical role of YopN in regulation of the polarized translocation. Evidence for a region in YopH important for its translocation through the plasma membrane of the target cell but not for secretion from the pathogen is provided.

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