- Volume 147, Issue 11, 2001
Volume 147, Issue 11, 2001
- Microbiology Comment
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- Antigens And Immunity
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Role of the polymorphic region 1 of the Bordetella pertussis protein pertactin in immunity
In several countries pertussis is re-emerging, despite a high vaccination coverage. It is suggested that antigenic divergence between Bordetella pertussis vaccine strains and circulating strains, in particular with respect to pertactin, has contributed to pertussis re-emergence. Polymorphism in pertactin is essentially limited to region 1, which is composed of repeats and is located adjacent to an Arg-Gly-Asp motif implicated in adherence. Evidence is provided for the immunological relevance of polymorphism in region 1. Region 1 was found to contain a B-cell epitope recognized in both humans and mice. Furthermore, variation in region 1 affected antibody binding and, in a mouse respiratory infection model, the efficacy of a whole-cell vaccine. Moreover, passive and active immunization indicated that region 1 confers protective immunity. An mAb directed against a linear conserved epitope conferred cross-immunity against isolates with distinct pertactin variants. The results indicate an important role of region 1 of pertactin in immunity.
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Intestinal infection of BALB/c mice with Yersinia enterocolitica O9 causes major modifications in phenotype and functions of spleen cells
More LessYersinia enterocolitica serotype O9 may cause a persistent intestinal infection with few or no symptoms in humans and in BALB/c mice. The present study demonstrated profound alterations in the immune status of BALB/c mice infected with Y. enterocolitica O9. Infected mice developed splenomegaly and phenotypic analysis of spleen cells revealed increases in CD3+ total T cells, CD4+ helper T cells, CD8+ cytotoxic T cells and CD11b+ phagocytic cells. Spleen cells from infected mice exhibited impaired responses to mitogens and suppressed the proliferation of normal splenocytes in response to mitogens. Suppression of responses to concanavalin A and heat-killed yersiniae was associated with increased production of gamma interferon and reactive nitrogen intermediates. Y. enterocolitica-infected mice resisted challenge with a lethal dose of the intracellular pathogen Listeria monocytogenes. These findings suggest that infection of mice with Y. enterocolitica O9 induces gamma-interferon-secreting cells that promote macrophage activation, mediating resistance to infection with L. monocytogenes, and macrophage production of reactive nitrogen intermediates, which results in in vitro inhibition of lymphocyte response to mitogens.
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- Bioenergetics And Transport
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Identification of the ABC protein SapD as the subunit that confers ATP dependence to the K+-uptake systems TrkH and TrkG from Escherichia coli K-12
The activity of the two almost identical K+-uptake systems, TrkH and TrkG, from Escherichia coli K-12 depends completely and partially on the presence of the trkE gene, respectively. trkE maps inside the sapABCDF operon, which encodes an ATP-binding cassette (ABC) transporter of unknown function from the subgroup of peptide-uptake systems. This study was carried out to clarify the role of sapABCDF gene products in the ATP dependence of the E. coli Trk systems. For this purpose ΔsapABCDF ΔtrkG and ΔsapABCDF ΔtrkH strains of E. coli containing plasmids with sap genes from either E. coli or Vibrio alginolyticus were used. All five plasmid-encoded E. coli Sap proteins were made in E. coli mini-cells. The presence of the ATP-binding SapD protein from either E. coli or V. alginolyticus alone was sufficient for stimulating the K+ transport activity of the TrkH and TrkG systems. K+-uptake experiments with Escherichia coli cells containing SapD variants with changes in the Walker A box Lys-46 residue, the Walker B box Asp-183 residue and the signature motif residues Gly-162 or Gln-165 suggested that adenine nucleotide binding to SapD rather than ATP hydrolysis by this subunit is required for the activity of the E. coli TrkH system. K+ transport via two plasmid-encoded Trk systems in a ΔsapABCDF E. coli strain remained dependent on both a high membrane potential and a high cytoplasmic ATP concentration, indicating that in E. coli ATP dependence of Trk activity can be independent of Sap proteins. These data are interpreted to mean that Trk systems can interact with an ABC protein other than SapD.
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Deletion of one of two Escherichia coli genes encoding putative Na+/H+ exchangers (ycgO) perturbs cytoplasmic alkali cation balance at low osmolarity
More LessTwo genes in the Escherichia coli genome, b4065 (yjcE) and b1191 (ycgO), are similar to genes encoding eukaryotic Na+/H+ exchangers. Mutants were constructed in which yjcE (GRN11), ycgO (GRF55) or both (GRD22) were inactivated. There was no change in respiration-driven Na+ efflux in any of the mutants when grown in media containing 50–500 mM Na+. The only striking finding was that growth of GRF55 was impaired at low osmolarity. In complex low-salt medium, GRF55 grew at a wild-type rate for three to four generations but then stopped; the growth was partially recovered after a pause, the length of which was dependent on salt concentration. Measurement of cytoplasmic alkali cations showed that an abrupt loss of about one-half of the intracellular K+ preceded the pause. When grown in low-salt medium with only 20 mM added Na+, GRF55 also lost the ability to maintain a sodium concentration gradient. However, this phenomenon appears to be a secondary effect of the ycgO deletion. The double mutant GRD22 has the same properties as GRF55; no additional effect was found. The data indicate that neither ycgO nor yjeE participates in respiration-driven Na+ extrusion. Instead, ycgO is required for growth at low osmolarity. Hence it is concluded that ycgO participates in cell volume regulation, and accordingly it is suggested that ycgO be renamed cvrA.
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- Development And Structure
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In vivo roles of the germination-specific lytic enzymes of Bacillus subtilis 168
More LessGermination of endospores of Bacillus subtilis involves the activities of several germination-specific lytic enzymes, including glucosaminidase and lytic transglycosylase. Another non-hydrolytic activity, likely to be due to an epimerase, also occurs. The effect of pH on enzyme activities and the overall germination rate was measured. Optimal germination occurred between pH 7–9; however, optimum glucosaminidase and epimerase activities were noted at pH 5. Conversely, the lytic transglycosylase activity was greatest at pH 8. Treatment of spores (15 min) with heat (90 °C) or NaOH (0·25 M) led to impaired cortex hydrolysis/modification, but with <20% loss in viability. Analysis of muropeptides in the germination exudate revealed a reduction of >85% in glucosaminidase and epimerase products, when compared to untreated spores. Conversely, lytic transglycosylase activity was increased by alkali or heat treatment, which was possibly due to increased substrate availability. FB101 (sleB) spores, which lack lytic transglycosylase activity, showed 90-fold greater loss in viability than the wild-type after 1 h at 90 °C. Similarly, 97% of FB101 (sleB) spores were unable to form a colony on nutrient agar after 130 min exposure to 0·25 M NaOH at 4 °C, whereas the wild-type was unaffected. This demonstrates the crucial role of the lytic transglycosylase in cortex hydrolysis of damaged spores. The respective targets of heat and alkali in spores and their role during germination are discussed.
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- Environmental Microbiology
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Individual-based modelling of biofilms
More LessUnderstanding the emergence of the complex organization of biofilms from the interactions of its parts, individual cells and their environment, is the aim of the individual-based modelling (IbM) approach. This IbM is version 2 of BacSim, a model of Escherichia coli colony growth, which was developed into a two-dimensional multi-substrate, multi-species model of nitrifying biofilms. It was compared with the established biomass-based model (BbM) of Picioreanu and others. Both models assume that biofilm growth is due to the processes of diffusion, reaction and growth (including biomass growth, division and spreading). In the IbM, each bacterium was a spherical cell in continuous space and had variable growth parameters. Spreading of biomass occurred by shoving of cells to minimize overlap between cells. In the BbM, biomass was distributed in a discrete grid and each species had uniform growth parameters. Spreading of biomass occurred by cellular automata rules. In the IbM, the effect of random variation of growth parameters of individual bacteria was negligible in contrast to the E. coli colony model, because the heterogeneity of substrate concentrations in the biofilm was more important. The growth of a single cell into a clone, and therefore also the growth of the less abundant species, depended on the randomly chosen site of attachment, owing to the heterogeneity of substrate concentrations in the biofilm. The IbM agreed with the BbM regarding the overall growth of the biofilm, due to the same diffusion-reaction processes. However, the biofilm shape was different due to the different biomass spreading mechanisms. The IbM biofilm was more confluent and rounded due to the steady, deterministic and directionally unconstrained spreading of the bacteria. Since the biofilm shape is influenced by the spreading mechanism, it is partially independent of growth, which is driven by diffusion-reaction. Chance in initial attachment events modifies the biofilm shape and the growth of single cells because of the high heterogeneity of substrate concentrations in the biofilm, which again results from the interaction of diffusion-reaction with spreading. This stresses the primary importance of spreading and chance in addition to diffusion-reaction in the emergence of the complexity of the biofilm community.
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Altered expression of two light-dependent genes in a microcystin-lacking mutant of Microcystis aeruginosa PCC 7806
Microcystin is a potent inhibitor of eukaryotic protein phosphatases and has been implicated in causing hepatotoxicity to humans and animals worldwide. It is produced primarily by the bloom-forming cyanobacterium Microcystis aeruginosa, although the function of the peptide in this micro-organism is unknown. In this study, a microcystin-related protein, MrpA, was identified using a microcystin-lacking mutant of M. aeruginosa, PCC 7806. Comparative two-dimensional protein electrophoresis showed that MrpA was strongly expressed in wild-type PCC 7806, but was not detectable in the mcyB mutant. MrpA showed similarity to the RhiA protein from Rhizobium leguminosarum, which is encoded by the rhiABC operon and controlled by quorum-sensing mediators. Sequencing of mrpA flanking regions in M. aeruginosa PCC 7806 revealed the presence of a rhiB homologue, mrpB, directly downstream of mrpA. Northern blot analyses of mrpA expression in cells exposed to different light conditions revealed a rapid decline of transcription under high light conditions. Most striking was a strong increase in transcript levels from cultures irradiated with blue light. The mrpA transcription level was strongly reduced in two independent microcystin-lacking mutants under all light conditions investigated.
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Role of biofilms in the survival of Legionella pneumophila in a model potable-water system
Legionellae can infect and multiply intracellularly in both human phagocytic cells and protozoa. Growth of legionellae in the absence of protozoa has been documented only on complex laboratory media. The hypothesis upon which this study was based was that biofilm matrices, known to provide a habitat and a gradient of nutrients, might allow the survival and multiplication of legionellae outside a host cell. This study determined whether Legionella pneumophila can colonize and grow in biofilms with and without an association with Hartmannella vermiformis. The laboratory model used a rotating disc reactor at a retention time of 6·7 h to grow biofilms on stainless steel coupons. The biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae and a Flavobacterium sp. The levels of L. pneumophila cells present in the biofilm were monitored for 15 d, with and without the presence of H. vermiformis, and it was found that, although unable to replicate in the absence of H. vermiformis, L. pneumophila was able to persist.
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- Genetics And Molecular Biology
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Transcriptional responses during outgrowth of Bacillus subtilis endospores
More LessThe Bacillus subtilis 168 genome contains an array of alternative σ factors, many of which play important roles in reprogramming expression during stress and sporulation. The role of the different σ factors during outgrowth, when the germinated endospore is converted back to a vegetative cell, is less well characterized. The activity of the alternative σ factors σB, σD and σH during endospore outgrowth was analysed by Northern blotting and lacZ reporter assays. While σD and σH were transcriptionally active during outgrowth, σB-dependent transcription was not observed until after the first cell division, when growth slowed. Using an IPTG-controllable copy of sigA, an optimal level of expression was required to maintain growth rate at the end of outgrowth. The genes encoding the putative extracytoplasmic function (ECF) σ factors σI, σV, σW, σZ and YlaC were insertionally inactivated using pMUTIN4. These strains, together with sigM and sigX mutants, were tested to determine their role and measure their expression during endospore outgrowth. Transcripts or β-galactosidase activity were observed for each of the ECF σ factors early after germination. With the exception of MJH003 (sigM), which showed an exacerbated salt stress defect, inactivation of the ECF σ factor genes did not affect outgrowth in the conditions tested.
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Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine
More LessWecA, an integral membrane protein that belongs to a family of polyisoprenyl phosphate N-acetylhexosamine-1-phosphate transferases, is required for the biosynthesis of O-specific LPS and enterobacterial common antigen in Escherichia coli and other enteric bacteria. WecA functions as an UDP-N-acetylglucosamine (GlcNAc):undecaprenyl-phosphate GlcNAc-1-phosphate transferase. A conserved short sequence motif (His-Ile-His-His; HIHH) and a conserved arginine were identified in WecA at positions 279–282 and 265, respectively. This region is located within a predicted cytosolic segment common to all bacterial homologues of WecA. Both HIHH279–282 and the Arg265 are reminiscent of the HIGH motif (His-Ile-Gly-His) and a nearby upstream lysine, which contribute to the three-dimensional architecture of the nucleotide-binding site among various enzymes displaying nucleotidyltransferase activity. Thus, it was hypothesized that these residues may play a role in the interaction of WecA with UDP-GlcNAc. Replacement of the entire HIHH motif by site-directed mutagenesis produced a protein that, when expressed in the E. coli wecA mutant MV501, did not complement the synthesis of O7 LPS. Membrane extracts containing the mutated protein failed to transfer UDP-GlcNAc into a lipid-rich fraction and to bind the UDP-GlcNAc analogue tunicamycin. Similar results were obtained by individually replacing the first histidine (H279) of the HIHH motif as well as the Arg265 residue. The functional importance of these residues is underscored by the high level of conservation of H279 and Arg265 among bacterial WecA homologues that utilize several different UDP-N-acetylhexosamine substrates.
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Actinobacillus actinomycetemcomitans harbours type IV secretion system genes on a plasmid and in the chromosome
More LessNine contiguous genes encoding a potential type IV secretion system have been identified in the chromosome of Actinobacillus actinomycetemcomitans strain VT747 and on a plasmid (pVT745) in strain VT745. Seven of these genes encode predicted proteins that share significant homology with type IV secretion proteins in Bordetella pertussis (ptl operon), Brucella melitensis biovar suis and Agrobacterium tumefaciens (virB operons), where they are involved in protein secretion, pathogen intracellular survival and multiplication, and DNA transport, respectively. Results of previous studies have demonstrated that pVT745 is a conjugative plasmid and that a secondary plasmid, pMMB67, can be mobilized from strain VT745. Given these results, it was hypothesized that (1) the type IV secretion genes on pVT745 are responsible for these two functions and (2) the type IV VT747 chromosomal genes also play a role in the transport of DNA. Wild-type and mutant strains of VT745 were evaluated for their conjugative abilities. Wild-type mating efficiency was 10−6 transconjugants per donor, while the mutant strain yielded no transconjugants. Wild-type VT745 harbouring a co-resident plasmid, pMMB67, mobilized pMMB67 at a frequency of 10−6, while VT747 was unable to mobilize this plasmid. These results support the hypothesis that the plasmid-encoded type IV secretion system on pVT745 is involved in DNA transport. However, the chromosomally encoded secretion system may not play a role in DNA transport in strain VT747. While the precise function of these chromosomal genes in strain VT747 has not been determined, Northern blot analyses demonstrated that these genes are expressed in both Act. actinomycetemcomitans strains VT745 and VT747.
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Molecular characterization of a chromosomal locus in Staphylococcus aureus that contributes to oxidative defence and is highly induced by the cell-wall-active antibiotic oxacillin
More LessPrevious studies employing two-dimensional gel electrophoresis and N-terminal protein sequencing have shown elevated synthesis of the enzyme methionine sulfoxide reductase (MsrA) in Staphylococcus aureus in response to cell-wall-active antibiotics. In the present study, the S. aureus msrA gene was cloned, overexpressed, purified as His-tagged MsrA and shown to have methionine sulfoxide reductase activity. The transcription of msrA was studied by assaying β-galactosidase activity in an msrA promoter::lacZ fusion strain and by Northern blot analysis. Transcription of msrA was increased by oxacillin; but not by a variety of other stresses including H2O2. Northern blot analysis revealed that the size of the msrA transcript was 2·3 kb, considerably larger than the 531 nt msrA ORF. The msrA transcription start site was mapped 25 nt upstream of the msrA start codon. Computer analysis from database sequences indicated at least three additional ORFs downstream of msrA. The deduced amino acid sequences of two of these three ORFs showed significant sequence homologies to PilB, and enzyme IIA of the phosphotransferase system, respectively. The third ORF could not be identified by homology searches. Northern blot hybridization with probes specific to the msrA downstream region indicated that the S. aureus msrA was transcribed as part of a polycistronic message. Interestingly, purified S. aureus PilB was shown to possess ∼∼28-fold higher methionine sulfoxide reductase activity than the MsrA. An insertional knockout mutation in the first gene of this operon resulted in increased susceptibility of the mutant to H2O2 compared to the parent strain, but not to oxacillin.
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Multiple evidence for widespread and general occurrence of type-III PHA synthases in cyanobacteria and molecular characterization of the PHA synthases from two thermophilic cyanobacteria: Chlorogloeopsis fritschii PCC 6912 and Synechococcus sp. strain MA19
T. Hai, S. Hein and A. SteinbüchelEleven different cyanobacteria were investigated with respect to their capabilities to synthesize poly-3-hydroxybutyrate [poly(3HB)] and the type of poly-β-hydroxyalkanoic acid (PHA) synthase accounting for the synthesis of this polyester. Several methods, including (i) Southern blot analysis using a phaC-specific DNA probe, (ii) Western blot analysis using specific polyclonal anti-PhaE antibodies raised in this study against PhaE of Synechocystis sp. strain PCC 6803, (iii) generation and sequence analysis of PCR products using phaC-specific oligonucleotides as primers, and/or (iv) cloning and sequence analysis of PHA synthase structural genes, were used to provide evidence for the presence of a type-III PHA synthase in the following cyanobacteria: Synechococcus sp. strains MA19 and PCC 6715, Chlorogloeopsis fritschii PCC 6912, Anabaena cylindrica SAG 1403-2, Cyanothece sp. strains PCC 7424, PCC 8303 and PCC 8801, and Gloeocapsa sp. strain PCC 7428. The screening was compared with corresponding studies using crude protein extracts and genomic DNA of Synechocystis sp. strain PCC 6803, as a positive control, which is so far the only cyanobacterium for which molecular data of the PHA synthase genes are available. No evidence for the presence of a type-III PHA synthase could be obtained for only three of the eleven investigated cyanobacteria (Stanieria sp. strain PCC 7437, Cyanothece sp. strain PCC 8955 and Gloeothece sp. strain PCC 6501). The entire PHA synthase structural genes of the two thermophilic cyanobacteria Synechococcus sp. strain MA19 and Chlorogloeopsis fritschii PCC 6912, and in addition a central region of the phaC gene of Cyanothece sp. strain PCC 8303, were cloned, sequenced and also heterologously expressed in Escherichia coli.
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Characterization of the Streptococcus gordonii chromosomal region immediately downstream of the glucosyltransferase gene
More LessThe Streptococcus gordonii glucosyltransferase gene, gtfG, is positively regulated by the upstream determinant rgg. In the present study, two ORFs, transcribed on the opposite DNA strand, were identified immediately downstream of gtfG. The first, designated dsg, shares a convergent putative transcriptional terminator with gtfG, and encodes a predicted 46 kDa transmembrane protein similar to the Yersinia enterocolitica TrsA involved in polysaccharide biosynthesis. Insertional inactivation of dsg resulted in only ∼∼60% of the parental level of glucosyltransferase activity. The 870 bp gene 5′ to dsg is similar to the gtfG regulatory determinant. Designated rggD, this rgg-like determinant downstream of gtfG encodes a putative 33·6 kDa cytoplasmic protein. Despite their sequence similarity, the functions of rgg and rggD appear specific. Strains in which rggD was insertionally inactivated and strains containing plasmid-borne rggD had parental levels of glucosyltransferase activity. Northern blot hybridization analyses showed ∼1·3 kb dsg-specific and ∼1·0 kb rggD-specific mRNA transcripts associated with this region; no polycistronic transcript was observed. Although rgg-like gene products have been demonstrated to function as positive transcriptional regulators of adjacent genes in several streptococcal species, Northern blot analysis suggested that rggD did not influence the transcription of dsg or the divergent downstream ylbN-like determinant under the conditions in the present study. Comparison of this S. gordonii chromosome region to other streptococcal genomes, which do not contain the rgg/rggD-flanked region involved in glucan synthesis, raised intriguing possibilities about the origins of this chromosomal region, and also suggested that rggD might regulate a distally located gene.
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Monomer–dimer control of the ColE1 P cer promoter
More LessXerCD-mediated recombination at cer converts multimers of plasmid ColE1 to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. In addition to XerCD, recombination requires the accessory factors ArgR and PepA. The promoter P cer , located centrally within cer, is also required for stable plasmid maintenance. P cer is active in plasmid multimers and directs transcription of a short RNA, Rcd, which appears to inhibit cell division. It has been proposed that Rcd is part of a checkpoint which ensures that multimer resolution is complete before the cell divides. This study has shown that ArgR does not act as a transcriptional repressor of P cer in plasmid monomers. P cer is unusual in that the −35 and −10 hexamers are separated by only 15 bp and this study has demonstrated that increasing this to a more conventional spacing results in elevated activity. An increase to 17 bp resulted in a 10- to 20-fold increase in activity, while smaller effects were seen when the spacer was increased to 16 bp or 18 bp. These observations are consistent with the hypothesis that P cer activation involves realignment of the −35 and −10 sequences within a recombinational synaptic complex. This predicts that a 17 bp spacer promoter derivative should be down-regulated by plasmid multimerization, and this is confirmed experimentally.
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A polyketide biosynthetic gene cluster from Streptomyces antibioticus includes a LysR-type transcriptional regulator
More LessIn the search for Type II polyketide synthases (PKSs) a DNA fragment was isolated from Streptomyces antibioticus ATCC 11891 (a producer of oleandomycin). DNA sequencing of the cloned fragment revealed six complete ORFs whose deduced products showed similarities to those of other genes known to be involved in polyketide biosynthesis. Several S. coelicolor strains mutated in different steps of actinorhodin biosynthesis (actI, actIII, actV A and actVII) were complemented by the cloned genes, suggesting that the isolated genes encode an aromatic polyketide of unknown structure and function. The cluster also contains a putative LysR-type transcriptional regulator (ORF0), which controls PKS gene expression in a heterologous host. DNA binding assays and transcriptional analysis suggest that the pathway-specific regulator for actinorhodin biosynthesis (actII-ORF4) is also involved in the expression of the cloned PKS in the host strain.
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N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli
More LessThe formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon. Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7–37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhlA9-2–DNA–formate complex was at least three times higher than that of the native protein–DNA–formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA− phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc–lac expression. The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for hyc promoter DNA in vitro. Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.
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Occurrence of two superoxide dismutases in Aeromonas hydrophila: molecular cloning and differential expression of the sodA and sodB genes
More LessAeromonas spp., considered as emerging opportunistic pathogens, belong to the family Vibrionaceae. Among the criteria currently used for their classification is the presence of a single FeSOD (iron-containing superoxide dismutase), which distinguishes them from Enterobacteriaceae. In this paper the cloning of the sodA and sodB genes encoding two different SODs in Aeromonas hydrophila ATCC 7966 is reported. The sodB gene encoded an FeSOD (196 amino acids, 21·5 kDa), was constitutively expressed and showed 75% homology with the E. coli FeSOD. The sodA gene encoded a protein of 206 amino acids (22·5 kDa) with MnSOD (manganese-containing SOD) activity and showed 55% homology with the Escherichia coli MnSOD. The MnSOD of A. hydrophila was detected only during the stationary phase of growth under high aeration or when induced by lack of iron. Nevertheless, paraquat had no detectable effect on its production. The amino-terminal part of the Mn-containing protein contained a putative signal sequence which could permit a periplasmic localization.
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- Pathogenicity And Medical Microbiology
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SvpA, a novel surface virulence-associated protein required for intracellular survival of Listeria monocytogenes
More LessA previously unknown protein, designated SvpA (surface virulence-associated protein) and implicated in the virulence of the intracellular pathogen Listeria monocytogenes, was identified. This 64 kDa protein, encoded by svpA, is both secreted in culture supernatants and surface-exposed, as shown by immunogold labelling of whole bacteria with an anti-SvpA antibody. Analysis of the peptide sequence revealed that SvpA contains a leader peptide, a predicted C-terminal transmembrane region and a positively charged tail resembling that of the surface protein ActA, suggesting that SvpA might partially reassociate with the bacterial surface by its C-terminal membrane anchor. An allelic mutant was constructed by disrupting svpA in the wild-type strain LO28. The virulence of this mutant was strongly attenuated in the mouse, with a 2 log decrease in the LD50 and restricted bacterial growth in organs as compared to the wild-type strain. This reduced virulence was not related either to a loss of adherence or to a lower expression of known virulence factors, which remained unaffected in the svpA mutant. It was caused by a restriction of intracellular growth of mutant bacteria. By following the intracellular behaviour of bacteria within bone-marrow-derived macrophages by confocal and electron microscopy studies, it was found that most svpA mutant bacteria remained confined within phagosomes, in contrast to wild-type bacteria which rapidly escaped to the cytoplasm. The regulation of svpA was independent of PrfA, the transcriptional activator of virulence genes in L. monocytogenes. In fact, SvpA was down-regulated by MecA, ClpC and ClpP, which are highly homologous to proteins of Bacillus subtilis forming a regulatory complex controlling the competence state of this saprophyte. The results indicate that: (i) SvpA is a novel factor involved in the virulence of L. monocytogenes, promoting bacterial escape from phagosomes of macrophages; (ii) SvpA is, at least partially, associated with the surface of bacteria; and (iii) SvpA is PrfA-independent and controlled by a MecA-dependent regulatory network.
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In vitro secretion kinetics of proteins from Legionella pneumophila in comparison to proteins from non-pneumophila species
More LessIt has been shown that the loss of PilD, a prepilin peptidase necessary for type IV pilus biogenesis and establishment of the type II secretion apparatus is associated with loss of virulence in Legionella pneumophila. L. pneumophila is the species most frequently associated with Legionnaires’ disease, but virulence factors unique to this species are not known, so the secretion kinetics of several pilD-dependent enzyme activities, including protease, acid phosphatase, phospholipase A (PLA) and lysophospholipase A (LPLA), of L. pneumophila and non-pneumophila species were compared during growth in BYE broth. Enzyme activity appeared during mid-exponential growth phase and reached maximal levels on entry into stationary growth phase. None of the enzyme activities were unique to L. pneumophila and it did not exclusively secrete the highest amounts of the hydrolytic proteins. However, the timing of PLA and LPLA secretion in L. pneumophila differed compared to other species. PLA activity was secreted prior to LPLA activity in L. pneumophila, which may lead to an accumulation of the cytotoxic agent lysophosphatidylcholine (LPC). In addition to L. pneumophila, several other Legionella species, including Legionella steigerwaltii and Legionella gormanii, were able to enrich for LPC due to a very potent PLA activity accompanied by only moderate LPLA activity. These species, in contrast to L. pneumophila, have not been shown to multiply within monocytic host cells. Thus none of the secreted enzymic activities investigated were unique to L. pneumophila, nor were they secreted at high concentrations. However, the timing of PLA and LPLA secretion may contribute to pathogenicity.
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Substrate-specific diffusion of select dicarboxylates through Chlamydia trachomatis PorB
More LessChlamydiae contain two porins, MOMP and PorB, that facilitate diffusion of solutes through the outer membrane. MOMP is a general porin that permits the diffusion of a wide variety of compounds including carbohydrates and amino acids. The relative inefficiency of PorB as a general porin and its low abundance in the outer membrane suggest that it may function as a substrate-specific porin. The tricarboxylic acid (TCA) cycle of chlamydiae is incomplete and to function would require the exogenous acquisition of 2-oxoglutarate or glutamate. A liposome-swelling assay for anions as well as an enzyme-linked liposome assay were used to demonstrate the efficient diffusion of dicarboxylates such as 2-oxoglutarate through PorB. These data demonstrate that PorB is a dicarboxylate-specific porin that may feed the chlamydial TCA cycle and provide chlamydiae with carbon and energy production intermediates.
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Binding to sulfatide and enterotoxicity of various Escherichia coli STb mutants
More LessBinding of the 48 amino acid polypeptide of the mature heat-stable Escherichia coli enterotoxin b (STb) to the functional receptor sulfatide (SFT) constitutes the first step in inducing secretory diarrhoea in the intestinal lumen of animals. The NMR structure of this toxin dictated the choice of amino acids for site-directed mutagenesis to delineate the binding site of STb to SFT. Amino acids facing the solvent either in the loop or the hydrophobic α-helix were selected. Seventeen site-specific mutants of STb toxin were produced and purified by high-pressure liquid chromatography. Enterotoxicity of the 17 mutants was determined using a rat loop assay and binding was evaluated using a microtitre plate binding assay. Both hydrophobic and electrostatic interactions are important for STb attachment. When mutations (F37K, I41S and M42S) were introduced into the hydrophobic α-helix to lessen hydrophobicity, binding activity and enterotoxicity decreased by more than sixfold. The loop defined by C21 and C36 also made specific contributions. Mutants generated at basic residues (K22, K23 and R29) within this region exhibited both reduced binding activities and reduced toxic activities. For all STb mutants constructed and analysed, when binding to SFT was reduced, a reduction in toxicity equivalent or greater was noted, indicating that binding to SFT is a step that precedes the toxic effect observed for STb toxin. Significantly, when the negatively charged D30 was substituted for either alanine or valine, the binding to SFT was about twice that of native STb, whereas the enterotoxicity was reduced by half.
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The elements of the locus of enterocyte effacement in human and wild mammal isolates of Escherichia coli: evolution by assemblage or disruption?
More LessEscherichia coli is an excellent model for studying the evolution of pathogenicity since within one species various genes can be found in pathogenic islands and plasmids causing a wide spectrum of virulence. A collection of 122 strains from different human and wild mammal hosts were analysed by PCR and Southern hybridization for the presence of a subset of the genes included in the LEE (locus of enterocyte effacement). In the PCR analysis, two markers (cesT/eae and espB genes) were found together in more strains (25·4%) than either were found alone. The cesT/eae gene was less frequently found alone (8·2%) than was the espB gene (15·6%). Four regions of the LEE were analysed in a subsample of 25 strains using Southern hybridization. The four regions were all present (44%), all absent (12%) or present in different combinations (44%) in a given strain. The flanking regions of the LEE showed the highest rate of hybridization (in 72% of the strains). The results indicate that the LEE is a dynamic genetic entity, both the complete gene cluster and the individual genes. The genes that comprise this locus seem to be horizontally acquired (or lost) in an independent way and may control other functions in non-pathogenic E. coli lineages. In this way, horizontal transfer may allow the gradual stepwise construction of gene cassettes facilitating coordinate regulation and expression of novel functions.
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Antibodies to α5β1 and αvβ3 integrins react with Candida albicans alcohol dehydrogenase
More LessIt has been hypothesized that Candida albicans possesses integrin-like receptors on its cell surface. This is because C. albicans binds numerous fluid-phase extracellular matrix (ECM) proteins on its cell surface and adheres to the same ECM proteins when immobilized. In addition, numerous antibodies to human integrins (receptors for ECM proteins) bind to the fungal cell surface and in so doing inhibit the binding of the respective proteins. To demonstrate the presence of such a cell surface integrin, a cDNA library of C. albicans yeast cells was screened with polyclonal antiserum to the human fibronectin receptor (α5β1 integrin). Clones isolated by this screening technique also reacted specifically to antiserum against the human vitronectin receptor (αvβ3 integrin). DNA sequence analysis of the cloned insert predicted a 350 aa protein (37 kDa). This predicted protein showed 75% homology at the nucleotide sequence level to alcohol dehydrogenase (ADH) of Saccharomyces cerevisiae. In vitro transcription/translation of the cloned inserts yielded a 37 kDa protein that was immunoprecipated with antibodies to the α5β1 and αvβ3 integrins and an antibody to a C. albicans fibronectin receptor. These antibodies and an mAb to the human vitronectin receptor demonstrated an antigen of −37 kDa present in the cell-wall preparations of C. albicans and in spent growth medium. All four antibodies reacted with authentic ADH. The possible significance of these results in relation to C. albicans adherence is discussed.
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- Physiology And Growth
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Glutamate synthase of Corynebacterium glutamicum is not essential for glutamate synthesis and is regulated by the nitrogen status
More LessThe Corynebacterium glutamicum gltB and gltD genes, encoding the large (α) and small (β) subunit of glutamate synthase (GOGAT), were investigated in this study. Using RT-PCR, a common transcript of gltB and gltD was shown. Reporter gene assays and Northern hybridization experiments revealed that transcription of this operon depends on nitrogen starvation. The expression of gltBD is under control of the global repressor protein AmtR as demonstrated by gel shift experiments and analysis of gltB transcription in an amtR deletion strain. In contrast to other bacteria, in C. glutamicum GOGAT plays no pivotal role; e.g. gltB and gltD inactivation did not result in growth defects when cells were grown in standard minimal medium and only a slight increase in the doubling time of the corresponding mutant strains was observed in the presence of limiting amounts of ammonia or urea. Additionally, mutant analyses revealed that GOGAT has no essential function in glutamate production by C. glutamicum.
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Effect of carbon starvation and proteolytic activity on stationary-phase acid tolerance of Streptococcus mutans
More LessPrevious research with Streptococcus mutans and other oral streptococci has demonstrated that the acid shock of exponential-phase cells (pH 7·5 to 5·5) resulted in the induction of an acid tolerance response (ATR) increasing survival at low pH (3·5–3·0). The current study was designed to determine whether two fresh isolates, H7 and BM71, and two laboratory strains, Ingbritt and LT11, were capable of a stationary-phase ATR as estimated by a survival test at pH 3·5 for 3 h. All four strains were unable to generate a stationary-phase ATR under control conditions at pH 7·5, with the exception of a burst of survivors in the transition between the exponential and stationary phases when the carbon source (glucose) was depleted. Adaptation at pH 5·5 resulted in the expected pH-dependent exponential-phase ATR, but only the fresh isolates exhibited a stationary-phase ATR at this pH. Glucose starvation of cells in complex medium was shown to enhance acid tolerance for the fresh isolates, but not the laboratory strains. This tolerance was, however, greatly diminished for all strains in a defined medium with a low concentration of amino acids. Growth of strain H7 in complex medium resulted in the formation of at least 56 extracellular proteins, nine of which were degraded in the early stationary phase following the induction of proteolytic activity during the transition period. No proteolytic activity was observed with strain LT11 and only 19 extracellular proteins/peptides were apparent in the medium with only one being degraded in the early stationary phase. Strain H7 was also shown to have two- to fourfold higher levels of intracellular glycogen in the stationary phase than strain LT11. These results suggest that S. mutans H7 possessed the required endogenous metabolism to support amino acid/peptide uptake in the early-stationary phase, which resulted in the formation of basic end products that, in turn, contributed to enhanced intracellular pH homeostasis.
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An analysis of multifactorial influences on the transcriptional control of ompF and ompC porin expression under nutrient limitation
More LessExpression of the major outer-membrane porins in Escherichia coli is transcriptionally controlled during nutrient limitation. Expression of ompF was more than 40-fold higher under glucose limitation than under nitrogen (ammonia) limitation in chemostat cultures at the same growth rate. In contrast, ompC expression was higher under N limitation. The basis of regulation by nutrient limitation was investigated using mutations affecting expression of porin genes. The influence of cyaA, rpoS, ackA and pta, as well as the two-component envZ-ompR system, was studied under glucose and N limitation in chemostat cultures. A major contributor to low ompF expression under N limitation was negative control by the RpoS sigma factor. RpoS levels were high under N limitation and loss of RpoS resulted in a 19-fold increase in ompF transcription, but little change was observed with ompC. Lack of RpoS under glucose limitation had a lesser stimulatory effect on ompF expression. Porin production was minimally dependent on EnvZ under N limitation due to OmpR phosphorylation by acetyl phosphate. Evidence obtained with pta and ackA mutants suggested that the acetyl phosphate level also regulates porins independently and indirectly via RpoS and other pathways. pta-envZ double mutants had a residual level of porin transcription, implicating alternative means of OmpR phosphorylation under nutrient limitation. Another critical factor in regulation was the level of cAMP, as a cyaA mutant hardly expressed ompF under glucose limitation but boosted ompC. In addition, the role of DNA-binding proteins encoded by hns and himA was tested under glucose limitation: the hns mutation reduced the glucose-limitation peak, but the himA mutation suppressed the hns effect, suggesting a complex web of interrelationships between the DNA-binding proteins. Indeed, multiple inputs and no single regulator were responsible for the high peak of ompF expression under glucose limitation.
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- Plant-Microbe Interactions
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Volatile antimicrobials from Muscodor albus, a novel endophytic fungus
More LessMuscodor albus is a recently described endophytic fungus obtained from small limbs of Cinnamomum zeylanicum (cinnamon tree). This xylariaceaous fungus effectively inhibits and kills certain other fungi, and bacteria, by virtue of a mixture of volatile compounds that it produces. The majority of these compounds were identified by gas chromatography/mass spectrometry and then made into an artificial mixture that mimicked the antibiotic effects of the mixture of volatile compounds given off by the fungus. Each of the five classes of volatile compounds produced by the fungus (alcohols, esters, ketones, acids and lipids) had some inhibitory effect against the test fungi and bacteria, but none was lethal. However, collectively they acted synergistically to kill a broad range of plant- and human-pathogenic fungi and bacteria. The most effective class of inhibitory compounds was the esters, of which 1-butanol, 3-methyl-, acetate was the most active biologically. This report describes the ecological implications and potential practical benefits of the ‘mycofumigation’ effects of M. albus.
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Molecular comparison of pathogenic bacteria from pear trees in Japan and the fire blight pathogen Erwinia amylovora
More LessSeveral strains of the genus Erwinia, which were isolated in Japan from pear trees with necrotic symptoms that resembled fire blight, and tentatively identified as Erwinia amylovora, were reinvestigated for their relationship to the fire blight pathogen. These isolates produced ooze on slices of immature pears and were mucoid on MM2Cu agar plates, but did not synthesize levan and did not give the expected PCR signals with several primer pairs specific for Erwinia amylovora. The isolates tested positive with PCR primers designed to detect the novel pear pathogen Erwinia pyrifoliae, which was isolated from Nashi pear trees in South Korea. The nucleotide sequence analysis of a DNA fragment preceding the gene cluster for exopolysaccharide synthesis revealed a closer relationship to Erwinia pyrifoliae than to Erwinia amylovora. Plasmid profiles, protein patterns and genomic DNA analysed by PFGE after XbaI and SpeI digestion were different than Erwinia amylovora. Experiments with strains of Erwinia amylovora isolated from raspberry (Rubus sp.), Erwinia mallotivora and Enterobacter pyrinus also did not reveal a relationship between these bacteria and the Japanese Erwinia strains. The latter are not identical to Erwinia pyrifoliae, but possess many similar features to this pathogen that causes Asian pear blight. It is concluded that pathogenic bacteria isolated in Japan from pear trees with symptoms resembling fire blight are possibly different from Erwinia amylovora.
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- Systematics And Evolution
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Phycobiliprotein genes of the marine photosynthetic prokaryote Prochlorococcus: evidence for rapid evolution of genetic heterogeneity
More LessProchlorococcus is a major photosynthetic prokaryote in nutrient-limited, open ocean environments and an important participant in the global carbon cycle. This phototroph is distinct from other members of the cyanobacterial lineage to which it belongs because it utilizes a chlorophyll a 2/b (2) light-harvesting complex as its major antenna, instead of phycobilisomes. Recently, genes encoding the phycobiliprotein phycoerythrin were identified in several Prochlorococcus isolates, thus making it the only extant photosynthetic prokaryote to possess a chlorophyll a/b antenna as well as phycobiliprotein genes. In order to understand the evolution of phycobiliproteins in this genus, the authors have sequenced the phycoerythrin genes of two isolates that are the most deeply branching in the Prochlorococcus lineage and share the highest degree of 16S rDNA sequence similarity to phycobilisome-containing marine Synechococcus. Sequence analyses suggest that within the Prochlorococcus lineage, the selective forces shaping the evolution of the phycoerythrin gene set have not been uniform. Although strains that are most closely related to marine Synechococcus possess genes (cpeB, cpeA) encoding both subunits of phycoerythrin, a more recently evolved strain is shown to lack cpeA and to possess a degenerate form of cpeB. Differences in phycoerythrin gene sequences between Prochlorococcus and Synechococcus appear to be consistent with a model of elevated mutation rates rather than relaxed selection. This suggests that although phycoerythrin is not a major constituent of the light-harvesting apparatus in Prochlorococcus, as it is in Synechococcus, the cpeB and cpeA genes are still under selection, albeit a different type of selection than in Synechococcus. The evolution of the Prochlorococcus light-harvesting antenna complex provides an important system for understanding the origins and scope of phylogenetic diversity in ocean ecosystems.
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Genetic polymorphism and taxonomic infrastructure of the Pleurotus eryngii species-complex as determined by RAPD analysis, isozyme profiles and ecomorphological characters
More LessThe Pleurotus eryngii species-complex includes populations of choice edible mushrooms, growing in the greater Mediterranean area in close association with different genera of plants of the family Apiaceae. Their distinct host-specialization served as the principal criterion for the discrimination of several taxa; however, the genetic relationships among the various P. eryngii ecotypes remain ambiguous. In the present study, 46 Pleurotus strains with a wide range of geographical origins were isolated from Eryngium spp., Ferula communis, Cachrys ferulacea, Thapsia garganica and Elaeoselinum asclepium subsp. asclepium, and were subjected to isozyme and random amplified polymorphic DNA-PCR (RAPD) analysis. The 16 enzyme activities tested were controlled by 28 loci, 11 of which were monomorphic. Host-exclusive zymograms for the Aph (acid phosphatase) and Phe-1 (dopa-phenoloxidase) loci were obtained from Pleurotus strains associated with C. ferulacea. Allele frequencies, genetic diversity and mean diversity were high for isolates from Eryngium spp. and Ferula communis. In RAPD analysis, the use of five primers allowed the production of 45 (out of 48) polymorphic bands, while four molecular markers specific for the identification of Pleurotus strains growing on E. asclepium subsp. asclepium and C. ferulacea were obtained. The Pleurotus strains produced 35 distinct electrophoretic types and 42 RAPD patterns, which independently permitted the separation of the fungal populations into five clusters in accordance with their host-specificity. In addition, the evaluation of the principal ecological and morphological characters provided further evidence for discriminating between P. nebrodensis growing on C. ferulacea and the rest of the host-associated populations. The latter represent taxa at the varietal level: P. eryngii var. eryngii, P. eryngii var. ferulae and P. eryngii var. elaeoselini. The position of taxa of dubious validity, such as P. hadamardii and P. fossulatus, is discussed in relation to the new findings. All Mediterranean Pleurotus populations growing on umbellifers seem to have recently diverged through a sympatric speciation process, that is based on both intrinsic reproductive barriers and extrinsic ecogeographical factors.
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