- Volume 150, Issue 2, 2004
Volume 150, Issue 2, 2004
- Microbiology Comment
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- Cell And Developmental Biology
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Differential surface localization and temperature-dependent expression of the Candida albicans CSH1 protein
More LessCell-surface hydrophobicity (CSH) in Candida albicans contributes to virulence and can be conveniently regulated in planktonic cultures by altering growth temperature. The CSH1 gene is the first candidate gene that has been demonstrated to play a role in affecting the CSH phenotype. However, the primary amino acid sequence of the CSH1 gene product suggests that the protein should be restricted to the cytoplasm. A majority of the protein appears to demonstrate that localization. Cell-surface biotinylation and limited glucanase digestion were used to determine and estimate the relative amount of Csh1p in the extracellular compartment in comparison to the cytoplasmic pool. Additionally, Western and Northern blotting were used to assess expression of the CSH1 gene under different growth conditions. Compared with cells grown at 23 °C, the total cellular levels of Csh1p are significantly greater at elevated growth temperatures. Detection of Csh1p on the cell surface correlates with the level of overall protein expression. The temperature-dependent regulation and surface presentation of Csh1p suggests a mechanism for regulating the CSH phenotype.
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Light-regulated asexual reproduction in Paecilomyces fumosoroseus
The entomopathogenic fungus Paecilomyces fumosoroseus has been successfully used in the control of several insect pests. Asexually produced spores (conidia) are the means for dispersal and transmission of the entomopathogen; upon contact with the insect cuticle they germinate and penetrate the host. In model fungal systems it has been found that phototropism, resetting of the circadian rhythm, the induction of carotenogenesis and the development of reproductive structures are controlled by blue light. The effect of light quality on conidial yield of P. fumosoroseus was investigated. Incubation in total darkness resulted in continued vegetative growth and lack of reproductive structures. In contrast, growth of the fungus in continuous illumination or under a night–day regime resulted in prolific formation of conidiophores bearing abundant mature conidia. Conidiation was photoinduced in competent mycelia by a single pulse of blue light and colonies were competent only after they had grown at least 72 h under total darkness. The fluence–response curves generated with blue light indicated that the minimal fluence required for the photomorphogenetic response was 180 μmol m−2 and the half-maximal response was at 400 μmol m−2. A fluence of 540 μmol m−2 was enough to saturate the system, inducing the maximum production of 2·12×108 conidia per colony. Higher light intensities markedly decreased conidiation, suggesting the occurrence of a process of adaptation. The authors propose the existence of a dual light-perception system with at least two photoreceptors in P. fumosoroseus, one promoting and one inhibiting conidiation.
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alr0117, a two-component histidine kinase gene, is involved in heterocyst development in Anabaena sp. PCC 7120
Degang Ning and Xudong XuAnabaena sp. PCC 7120 was mutagenized by transposon Tn5-1087b, generating a mutant whose heterocysts lack the envelope polysaccharide layer. The transposon was located between nucleotides 342 and 343 of alr0117, a 918 bp gene encoding a histidine kinase for a two-component regulatory system. Complementation of the mutant with a DNA fragment containing alr0117 and targeted inactivation of the gene confirmed that alr0117 is involved in heterocyst development. RT-PCR showed that alr0117 was constitutively expressed in the presence or absence of a combined-nitrogen source. hepA and patB, the two genes turned on during wild-type heterocyst development, were no longer activated in an alr0117-null mutant. The two-component signal transduction system involving alr0117 may control the formation of the envelope polysaccharide layer and certain late events essential to the function of heterocysts.
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- Biochemistry And Molecular Biology
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Expression and complexity of the PRT1 multigene family of Pneumocystis carinii
Pneumocystis carinii has a multigene family, PRT1, that encodes proteins with homology to KEX2-like proteases. PRT1 genes cluster with MSG genes near the telomeres and, like MSG, PRT1 proteins seem to be surface-expressed. The clustering of PRT1 and MSG genes suggested that expression of the two multigene families might be coordinated. Studying gene expression in P. carinii has been hampered by the lack of a culture system, and by lack of clonality in P. carinii populations in naturally infected rats, the host of this fungus. Heterogeneity can be reduced, however, by low-dose intratracheal inoculation, which can produce P. carinii populations dominated by organisms derived from a single progenitor. To study PRT1 expression, nude rats were inoculated with approximately 10 P. carinii each. The clonality of the P. carinii populations from inoculated rats was assessed by analysis of the UCS locus, a site in the genome that is known to be very heterogeneous in naturally infected rats, but nearly homogeneous in rats infected by low-dose intratracheal inoculation. Each of the populations had the same MSG gene at the UCS locus in at least 80 % of the organisms. To investigate PRT1 gene expression, RNA was amplified using primers that amplify numerous PRT1 genes. Seventy-four cloned cDNAs were sequenced, including at least 12 clones from each population of P. carinii. Many differently expressed PRT1 sequences were identified in each population, and a total of 45 different sequences were detected. However, the same PRT1 sequence was present in 15 of 74 plasmids and was found in 3 of the 5 P. carinii populations, suggesting that some PRT1 genes may be either more commonly expressed or expressed at a higher level. These data show that many members of the PRT1 gene family can be expressed in populations of P. carinii derived from few progenitors and suggest that the regulation of this family is different from that governing expression of the MSG gene family.
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Phospholipid synthesis in Borrelia burgdorferi: BB0249 and BB0721 encode functional phosphatidylcholine synthase and phosphatidylglycerolphosphate synthase proteins
More LessPhospholipids are an important component of bacterial membranes. Borrelia burgdorferi differs from many other bacteria in that it contains only two major membrane phospholipids: phosphatidylglycerol (PG) and phosphatidylcholine (PC). B. burgdorferi appears to lack enzymes required for synthesis of PC through the well-described methylation pathway. However, B. burgdorferi does contain a gene (BB0249) with significant identity to a recently described phosphatidylcholine synthase gene (pcs) of Sinorhizobium meliloti. B. burgdorferi also contains a gene (BB0721) with significant identity to the gene (pgs) encoding phosphatidylglycerolphosphate synthase, an enzyme in the synthetic pathway of PG. Activity of BB0249 was confirmed by cloning the gene into Escherichia coli, which does not produce PC. Transformation with a plasmid carrying BB0249 resulted in production of PC by E. coli, but only in the presence of exogenously supplied choline, as would be predicted for a Pcs. Because loss of Pgs activity is lethal to E. coli, activity of BB0721 was confirmed by the ability of BB0721 to complement an E. coli Pgs− mutant. A plasmid containing BB0721 was transformed into a Pgs− mutant of E. coli containing a copy of the native gene on a temperature-regulated plasmid. The temperature-regulated plasmid was exchanged for a plasmid containing BB0721 and it was shown that BB0721 was able to replace the lost Pgs function and restore bacterial growth. This study has established the existence and function of two critical enzymes in the synthesis of PC and PG in B. burgdorferi. Understanding of the biosynthetic pathways of PC and PG in B. burgdorferi is the first step in delineating the role of these phospholipids in the pathogenesis of Lyme disease.
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Biochemical characterization of a Rhizobium etli monovalent cation-stimulated acyl-coenzyme A carboxylase with a high substrate specificity constant for propionyl-coenzyme A
More LessBiotin has a profound effect on the metabolism of rhizobia. It is reported here that the activities of the biotin-dependent enzymes acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) and propionyl-coenzyme A carboxylase (PCC; EC 6.4.1.3) are present in all species of the five genera comprising the Rhizobiaceae which were examined. Evidence is presented that the ACC and PCC activities detectable in Rhizobium etli extracts are catalysed by a single acyl-coenzyme A carboxylase. The enzyme from R. etli strain 12-53 was purified 478-fold and displayed its highest activity with propionyl-CoA as substrate, with apparent K m and V max values of 0·064 mM and 2885 nmol min−1 (mg protein)−1, respectively. The enzyme carboxylated acetyl-CoA and butyryl-CoA with apparent K m values of 0·392 and 0·144 mM, respectively, and V max values of 423 and 268 nmol min−1 (mg protein)−1, respectively. K+, or Cs+ markedly activated the enzyme, which was essentially inactive in their absence. Electrophoretic analysis indicated that the acyl-CoA carboxylase was composed of a 74 kDa biotin-containing α subunit and a 45 kDa biotin-free β subunit, and gel chromatography indicated a total molecular mass of 620 000 Da. The strong kinetic preference of the enzyme for propionyl-CoA is consistent with its participation in an anaplerotic pathway utilizing this substrate.
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CtaG is required for formation of active cytochrome c oxidase in Bacillus subtilis
More LessThe Gram-positive bacterium Bacillus subtilis contains two respiratory oxidases of the haem-copper superfamily: cytochrome aa 3, which is a quinol oxidase, and cytochrome caa 3, which is a cytochrome c oxidase. Cytochrome c oxidase uniquely contains a di-copper centre, CuA. B. subtilis CtaG is a membrane protein encoded by the same gene cluster as that which encodes the subunits of cytochrome c oxidase. The role of B. subtilis CtaG and orthologous proteins present in many other Gram-positive bacteria has remained unexplored. The sequence of CtaG is unrelated to that of CtaG/Cox11p of proteobacteria and eukaryotic cells. This study shows that B. subtilis CtaG is essential for the formation of active cytochrome caa 3 but is not required for assembly of the core subunits I and II with haem in the membrane and it has no role in the synthesis of active cytochrome aa 3. B. subtilis YpmQ, a homologue to Sco1p of eukaryotic cells, is also a membrane-bound cytochrome c oxidase-specific assembly factor. Properties of CtaG- and YpmQ-deficient mutants were compared. Cells lacking YpmQ showed a low cytochrome c oxidase activity and this defect was suppressed by the supplementation of the growth medium with copper ions. It has previously been proposed that YpmQ/Sco1p is involved in synthesis of the CuA centre. The results of this study are consistent with this proposal but the exact role of YpmQ in assembly of cytochrome c oxidase remains to be elucidated.
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The fate of extracellular proteins tagged by the SsrA system of Bacillus subtilis
More LessIn bacteria, SsrA, a highly conserved RNA molecule, functions in a mechanism meant to rescue stalled ribosomes. In this process, a peptide tag encoded by SsrA is cotranslationally added to truncated polypeptides, thereby targeting these molecules for proteolytic degradation, at least when they stay inside the cell. This study examined the fate of two extracellular proteins that were tagged by the SsrA system of Bacillus subtilis. Gene constructs encoding human interleukin-3 (hIL-3) fused to a signal peptide and B. subtilis α-amylase, both lacking an in-frame stop codon, were used as models to achieve ribosome stalling and activation of the SsrA system. Introduction of these gene constructs into B. subtilis led to tagging of the gene products by SsrA RNA. The tagged protein products bound to antibodies that were raised against the proteolysis tag encoded by B. subtilis SsrA [(A)GKTNSFNQNVALAA]. The apolar C-terminal SsrA-tag does not function as a specific signal for proteolytic degradation of SsrA-tagged amylase directly after trans-translation or during the secretion process. Also, SsrA-tagged amylase appeared to be very stable once outside the cell. In contrast, hIL-3 molecules tagged with the native, apolar SsrA-tag were considerably less stable than hIL-3 molecules that received a negatively charged control tag. Not one specific protease, but several non-specific proteases seem to play a role in the rapid degradation of SsrA-tagged hIL-3. The polarity of the C-terminus of heterologous hIL-3 protein proved to be an important determinant for protein stability when produced by B. subtilis. As observed previously in Escherichia coli and B. subtilis, SsrA tagging also occurs frequently in normally growing Gram-positive bacilli and it appears that intracellular proteins are the predominant natural substrates of SsrA.
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- Environmental Microbiology
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Interactions between intracellular Na+ levels and saxitoxin production in Cylindrospermopsis raciborskii T3
More LessSaxitoxin (STX) is the most potent representative among the paralytic shellfish poisoning (PSP) toxins, which are highly selective Na+ channel-blocking alkaloids. This study investigated, in cultures of the cyanobacterium Cylindrospermopsis raciborskii T3, the effects of pH, salt, amiloride and lidocaine hydrochloride on total cellular levels of Na+ and K+ ions and STX accumulation. Both Na+ levels and intracellular STX concentrations increased exponentially in response to rising alkalinity. NaCl inhibited cyanobacterial growth at a concentration of 10 mM. In comparison with osmotically stressed controls, however, NaCl promoted STX accumulation in a dose-dependent manner. A correlation was seen in the time-course of both total cellular Na+ levels and intracellular STX for NaCl, amiloride and lidocaine exposure. The increase in cellular Na+ induced by NaCl at 10 mM was coupled with a proportional accumulation of STX. The two Na+ channel-blocking agents amiloride and lidocaine had opposing effects on both cellular Na+ levels and STX accumulation. Amiloride at 1 mM reduced ion and toxin concentrations, while lidocaine at 1 μM increased the total cellular Na+ and STX levels. The effects of the channel-blockers were antagonistic and dependent on an alkaline pH. The results presented suggest that, in C. raciborskii T3, STX is responsive to cellular Na+ levels. This may indicate that either STX metabolism or the toxin itself could be linked to the maintenance of cyanobacterial homeostasis. The results also enhance the understanding of STX production and the ecology of PSP toxin-producing cyanobacteria.
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Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate
More LessA gene cluster containing a gene for maleylacetate reductase (EC 1.3.1.32) was cloned from Ralstonia eutropha 335T (DSM 531T), which is able to utilize 4-fluorobenzoate as sole carbon source. Sequencing of this gene cluster showed that the R. eutropha 335T maleylacetate reductase gene, macA, is part of a novel gene cluster, which is not related to the known maleylacetate-reductase-encoding gene clusters. It otherwise comprises a gene for a hypothetical membrane transport protein, macB, possibly co-transcribed with macA, and a presumed regulatory gene, macR, which is divergently transcribed from macBA. MacA was found to be most closely related to TftE, the maleylacetate reductase from Burkholderia cepacia AC1100 (62 % identical positions) and to a presumed maleylacetate reductase from a dinitrotoluene catabolic gene cluster from B. cepacia R34 (61 % identical positions). By expressing macA in Escherichia coli, it was confirmed that macA encodes a functional maleylacetate reductase. Purification of maleylacetate reductase from 4-fluorobenzoate-grown R. eutropha 335T cells allowed determination of the N-terminal sequence of the purified protein, which was shown to be identical to that predicted from the cloned macA gene, thus proving that the gene is, in fact, recruited for growth of R. eutropha 335T with this substrate.
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- Genes And Genomes
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Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum
Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections and grow exclusively in the stratum corneum, nails and hair. In a culture medium containing soy proteins as sole nitrogen source a substantial proteolytic activity was secreted by Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. This proteolytic activity was 55–75 % inhibited by o-phenanthroline, attesting that metalloproteases were secreted by all three species. Using a consensus probe constructed on previously characterized genes encoding metalloproteases (MEP) of the M36 fungalysin family in Aspergillus fumigatus, Aspergillus oryzae and M. canis, a five-member MEP family was isolated from genomic libraries of T. rubrum, T. mentagrophytes and M. canis. A phylogenetic analysis of genomic and protein sequences revealed a robust tree consisting of five main clades, each of them including a MEP sequence type from each dermatophyte species. Each MEP type was remarkably conserved across species (72–97 % amino acid sequence identity). The tree topology clearly indicated that the multiplication of MEP genes in dermatophytes occurred prior to species divergence. In culture medium containing soy proteins as a sole nitrogen source secreted Meps accounted for 19–36 % of total secreted protein extracts; characterization of protein bands by proteolysis and mass spectrometry revealed that the three dermatophyte species secreted two Meps (Mep3 and Mep4) encoded by orthologous genes.
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Regulation of ndh expression in Escherichia coli by Fis
More LessThe Escherichia coli ndh gene encodes NADH dehydrogenase II, a primary dehydrogenase used during aerobic and nitrate respiration. The anaerobic transcription factor FNR represses ndh expression by binding at two sites centred at −94·5 and −50·5. In vivo transcription studies using promoter fusions with 5′ deletions confirmed that both FNR sites are required for maximum repression under anaerobic conditions. The histone-like protein Fis binds to three sites [centred at −123 (Fis I), −72, (Fis II) and +51 (Fis III)] in the ndh promoter. Using ndh : : lacZ promoter fusions carrying 5′ deletions, or replacement mutations it is shown that Fis III is a repressing site and that Fis I and II are activating sites, with the greatest contribution from Fis II. Deletion of the C-terminal domain of the RNA polymerase α-subunit abolished Fis-mediated activation of ndh expression, suggesting that ndh has a Class I Fis-activated promoter. In accordance with the established pattern of Fis synthesis, ndh transcription was greatest during exponential growth. Thus, it is suggested that Fis enhances ndh expression during periods of rapid growth, by acting as a Class I activator, and that the binding of tandem FNR dimers represses ndh expression by preventing interaction of the RNA polymerase α-subunit with DNA and Fis.
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Macro-array and bioinformatic analyses reveal mycobacterial ‘core’ genes, variation in the ESAT-6 gene family and new phylogenetic markers for the Mycobacterium tuberculosis complex
To better understand the biology and the virulence determinants of the two major mycobacterial human pathogens Mycobacterium tuberculosis and Mycobacterium leprae, their genome sequences have been determined recently. In silico comparisons revealed that among the 1439 genes common to both M. tuberculosis and M. leprae, 219 genes code for proteins that show no similarity with proteins from other organisms. Therefore, the latter ‘core’ genes could be specific for mycobacteria or even for the intracellular mycobacterial pathogens. To obtain more information as to whether these genes really were mycobacteria-specific, they were included in a focused macro-array, which also contained genes from previously defined regions of difference (RD) known to be absent from Mycobacterium bovis BCG relative to M. tuberculosis. Hybridization of DNA from 40 strains of the M. tuberculosis complex and in silico comparison of these genes with the near-complete genome sequences from Mycobacterium avium, Mycobacterium marinum and Mycobacterium smegmatis were undertaken to answer this question. The results showed that among the 219 conserved genes, very few were not present in all the strains tested. Some of these missing genes code for proteins of the ESAT-6 family, a group of highly immunogenic small proteins whose presence and number is variable among the genomically highly conserved members of the M. tuberculosis complex. Indeed, the results suggest that, with few exceptions, the ‘core’ genes conserved among M. tuberculosis H37Rv and M. leprae are also highly conserved among other mycobacterial strains, which makes them interesting potential targets for developing new specific anti-mycobacterial drugs. In contrast, the genes from RD regions showed great variability among certain members of the M. tuberculosis complex, and some new specific deletions in Mycobacterium canettii, Mycobacterium microti and seal isolates were identified and further characterized during this study. Together with the distribution of a particular 6 or 7 bp micro-deletion in the gene encoding the polyketide synthase pks15/1, these results confirm and further extend the revised phylogenetic model for the M. tuberculosis complex recently presented.
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Transcriptome and proteome analysis of Bacillus subtilis gene expression in response to superoxide and peroxide stress
More LessThe Gram-positive soil bacterium Bacillus subtilis responds to oxidative stress by the activation of different cellular defence mechanisms. These are composed of scavenging enzymes as well as protection and repair systems organized in highly sophisticated networks. In this study, the peroxide and the superoxide stress stimulons of B. subtilis were characterized by means of transcriptomics and proteomics. The results demonstrate that oxidative-stress-responsive genes can be classified into two groups. One group encompasses genes which show similar expression patterns in the presence of both reactive oxygen species. Examples are members of the PerR and the Fur regulon which were induced by peroxide and superoxide stress. Similarly, both kinds of stress stimulated the activation of the stringent response. The second group is composed of genes primarily responding to one stimulus, like the members of the SOS regulon which were particularly upregulated in the presence of peroxide, and many genes involved in sulfate assimilation and methionine biosynthesis which were only induced by superoxide. Several genes encoding proteins of unknown function could be assigned to one of these groups.
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- Pathogens And Pathogenicity
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RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms
An RT-PCR assay was developed to analyse expression patterns of genes in the Candida albicans ALS (agglutinin-like sequence) family. Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by C. albicans and also formation of an upper fungal layer that had characteristics similar to a biofilm. RT-PCR analysis of total RNA samples extracted from C. albicans-inoculated buccal RHE showed that ALS1, ALS2, ALS3, ALS4, ALS5 and ALS9 were consistently detected over time as destruction of the RHE progressed. Detection of transcripts from ALS7, and particularly from ALS6, was more sporadic, but not associated with a strictly temporal pattern. The expression pattern of ALS genes in C. albicans cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of C. albicans with buccal RHE does little to alter ALS gene expression. RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens. Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various C. albicans strains during biofilm development and were comparable to gene expression patterns in planktonic cells. The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models. The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between C. albicans and its host.
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Accessibility of the peptide backbone of protein ligands is a key specificity determinant in Candida albicans SRS adherence
More LessCandida albicans displays a high degree of specificity in selecting and adhering to targets in vivo. The features of target recognition are poorly understood and likely to involve more than the mere chemical composition of the ligand. Using an adherence assay in which protein and peptide ligands are covalently coupled to magnetic beads, the authors have previously described a new adherence mechanism in C. albicans, henceforth referred to as SRS (stable, reversible, specific) adherence. It was previously demonstrated that C. albicans and Saccharomyces cerevisiae expressing agglutinin-like sequence 5 protein (Als5p, previously referred to as Ala1p or Ala1/Als5p) adhere to peptides containing patches of threonine, serine and alanine residues when these are located in the free end of immobilized peptides. The interaction with protein ligands in SRS adherence predominantly involves the formation of hydrogen bonds. Accordingly, this interaction may occur (1) to the peptide backbone of the protein ligand or (2) to the amino acid side chain with an appropriate functional group. Evidence is provided that the primary interaction occurs with the peptide backbone and the secondary interaction occurs with the side chain. The primary interaction with the peptide backbone is sufficient for adherence to occur, whereas the secondary interaction with a side chain possessing an appropriate functional group stabilizes the interaction. In agreement with these results, it is also demonstrated that proteins lacking secondary and tertiary structure, wherein the peptide backbone is sterically accessible, interact with C. albicans and S. cerevisiae expressing Als5p. C. albicans Als proteins are resistant to denaturation by harsh conditions that kill the yeast cells. The proposed interactions in SRS adherence have striking similarities with those of the molecular chaperone Hsp70, which specifically binds to non-native proteins and resists denaturation.
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Deregulation of Listeria monocytogenes virulence gene expression by two distinct and semi-independent pathways
More LessExpression of the major virulence cluster in Listeria monocytogenes is positively regulated by the transcription factor PrfA and is influenced by several environmental factors, including the presence of readily metabolized carbohydrates such as cellobiose and glucose. Although little is understood about the mechanisms through which environmental factors influence expression of the PrfA regulon, evidence for structural and functional similarities of PrfA to the CRP-FNR family of regulatory proteins suggests the possibility that PrfA activity could be modulated by a small molecule ligand. The identity of components of the PrfA-associated regulatory pathway was sought through the isolation of mutants that exhibit high levels of PrfA-controlled gene expression in the presence of cellobiose or glucose. Here are described the properties and preliminary genetic analysis in two different genetic loci, gcr and csr, both unlinked by general transduction to the major virulence cluster. A mutation in gcr deregulates the expression of PrfA-controlled genes in the presence of several repressing sugars and other environmental conditions, a phenotype similar to that of a G145S substitution in PrfA itself. A mutation in the csr locus, within csrA, results in a cellobiose-specific defect in virulence gene regulation. Gene products encoded by the csr locus share homology with proteins involved in the sensing and transport of β-glucosides in other bacteria. Mutations in both gcr and csr are required for full relief of cellobiose-mediated repression of the PrfA regulon. These results suggest the existence of two semi-independent pathways for cellobiose-mediated repression and further reconcile conflicting reports in previous literature concerning the repressive effects of carbohydrates on virulence gene expression in L. monocytogenes.
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Physiological implications of class IIa bacteriocin resistance in Listeria monocytogenes strains
More LessHigh-level resistance to class IIa bacteriocins has been directly associated with the absent EIIABMan (MptA) subunit of the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) () in Listeria monocytogenes strains. Class IIa bacteriocin-resistant strains used in this study were a spontaneous resistant, L. monocytogenes B73-MR1, and a defined mutant, L. monocytogenes EGDe-mptA. Both strains were previously reported to have the EIIABMan PTS component missing. This study shows that these class IIa bacteriocin-resistant strains have significantly decreased specific growth and glucose consumption rates, but they also have a significantly higher growth yield than their corresponding wild-type strains, L. monocytogenes B73 and L. monocytogenes EGDe, respectively. In the presence of glucose, the strains showed a shift from a predominantly lactic-acid to a mixed-acid fermentation. It is here proposed that elimination of the EIIABMan in the resistant strains has caused a reduced glucose consumption rate and a reduced specific growth rate. The lower glucose consumption rate can be correlated to a shift in metabolism to a more efficient pathway with respect to ATP production per glucose, leading to a higher biomass yield. Thus, the cost involved in obtaining bacteriocin resistance, i.e. losing substrate transport capacity leading to a lower growth rate, is compensated for by a higher biomass yield.
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Absence of Yersinia pestis-specific DNA in human teeth from five European excavations of putative plague victims
This study reports the results of a collaborative study undertaken by two independent research groups to (a) confirm recent PCR-based detection of Yersinia pestis DNA in human teeth from medieval plague victims in France, and (b) to extend these observations over five different European burial sites believed to contain plague victims dating from the late 13th to 17th centuries. Several different sets of primers were used, including those previously documented to yield positive results on ancient DNA extracts. No Y. pestis DNA could be amplified from DNA extracted from 108 teeth belonging to 61 individuals, despite the amplification of numerous other bacterial DNA sequences. Several methods of extracting dentine prior to the DNA extraction were also compared. PCR for bacterial 16S rDNA indicated the presence of multiple bacterial species in 23 out of 27 teeth DNA extracts where dentine was extracted using previously described methods. In comparison, positive results were obtained from only five out of 44 teeth DNA extracts for which a novel contamination-minimizing embedding technique was used. Therefore, high levels of environmental bacterial DNA are present in DNA extracts where previously described methods of tooth manipulation are used. To conclude, the absence of Y. pestis-specific DNA in an exhaustive search using specimens from multiple putative European plague burial sites does not allow us to confirm the identification of Y. pestis as the aetiological agent of the Black Death and subsequent plagues. In addition, the utility of the published tooth-based ancient DNA technique used to diagnose fatal bacteraemias in historical epidemics still awaits independent corroboration.
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Identification of proteins in the exosporium of Bacillus anthracis
More LessSpores of Bacillus anthracis, the causative agent of anthrax, possess an exosporium. As the outer surface layer of these mature spores, the exosporium represents the primary contact surface between the spore and environment/host and is a site of spore antigens. The exosporium was isolated from the endospores of the B. anthracis wild-type Ames strain, from a derivative of the Ames strain cured of plasmid pXO2−, and from a previously isolated pXO1−, pXO2− doubly cured strain, B. anthracis UM23Cl2. The protein profiles of SDS-PAGE-separated exosporium extracts were similar for all three. This suggests that avirulent variants lacking either or both plasmids are realistic models for studying the exosporium from spores of B. anthracis. A number of loosely adsorbed proteins were identified from amino acid sequences determined by either nanospray-MS/MS or N-terminal sequencing. Salt and detergent washing of the exosporium fragments removed these and revealed proteins that are likely to represent structural/integral exosporium proteins. Seven proteins were identified in washed exosporium: alanine racemase, inosine hydrolase, ExsF, CotY, ExsY, CotB and a novel protein, named ExsK. CotY, ExsY and CotB are homologues of Bacillus subtilis outer spore coat proteins, but ExsF and ExsK are specific to B. anthracis and other members of the Bacillus cereus group.
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Mouse skin passage of Streptococcus pyogenes results in increased streptokinase expression and activity
More LessThe plasminogen activator streptokinase has been proposed to be a key component of a complex mechanism that promotes skin invasion by Streptococcus pyogenes. This study was designed to compare ska gene message and protein levels in wild-type M1 serotype isolate 1881 and a more invasive variant recovered from the spleen of a lethally infected mouse. M1 isolates selected for invasiveness demonstrated enhanced levels of active plasminogen activator activity in culture. This effect was due to a combination of increased expression of the ska gene and decreased expression of the speB gene. The speB gene product, SpeB, was found to efficiently degrade streptokinase in vitro.
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- Physiology
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Role of the hprT–ftsH locus in Staphylococcus aureus
More LessThe roles of two adjacent genes in the Staphylococcus aureus chromosome with functions in starvation survival and the response to stressful conditions have been characterized. One of these, hprT, encoding a hypoxanthine–guanine phosphoribosyltransferase homologue, was initially identified in a transposon mutagenesis screen. Mutation of hprT affects starvation survival in amino-acid-limiting conditions and the ability of S. aureus to grow in high-salt concentrations. Downstream of hprT is ftsH, which encodes a membrane-bound, ATP- and Zn2+-dependent ‘AAA’-type protease. Mutation of ftsH in S. aureus leads to pleiotropic defects including slower growth, sensitivity to salt, acid, methyl viologen and potassium tellurite stresses, and reduced survival in amino-acid- or phosphate-limiting conditions. Both hprT–lacZ and ftsH–lacZ gene fusions are expressed maximally in the post-exponential phase of growth. Although secretion of exoproteins is not affected, an ftsH mutant is attenuated in a murine skin lesion model of pathogenicity.
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Characterization of cyst cell formation in the purple photosynthetic bacterium Rhodospirillum centenum
More LessRhodospirillum centenum is an anoxygenic photosynthetic bacterium that is capable of differentiating into several cell types. When grown phototrophically in liquid, cells exhibit a vibrioid shape and have a single polar flagellum. When grown on a solid surface, R. centenum will differentiate into rod-shaped swarm cells that display numerous lateral flagella. Upon starvation for nutrients, R. centenum also forms desiccation-resistant cysts. In this study, it was determined that R. centenum has heat- and desiccation-resistance properties similar to other cyst-forming species. In addition, microscopic analyses of the morphological changes that occur during cyst cell development were performed. It was observed that R. centenum typically forms multi-celled clusters of cysts that contain from four to more than 10 cells per cluster. It was also determined that cell density has a minor effect on the percentage of cyst cells formed, with cell densities of 105–107 cells per 5 μl spot yielding the highest percentage of cyst cells. The striking similarities between the life cycle of R. centenum and the life cycle exhibited by Azospirillum spp. are discussed.
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Regulation of RcsA by the ClpYQ (HslUV) protease in Escherichia coli
More LessEscherichia coli ClpYQ protease and Lon protease possess a redundant function for degradation of SulA, a cell division inhibitor. An experimental cue implied that the capsule synthesis activator RcsA, a known substrate of Lon, is probably a specific substrate for the ClpYQ protease. This paper shows that overexpression of ClpQ and ClpY suppresses the mucoid phenotype of a lon mutant. Since the cpsB (wcaB) gene, involved in capsule synthesis, is activated by RcsA, the reporter construct cpsB–lacZ was used to assay for β-galactosidase activity and thus follow RcsA stability. The expression of cpsB–lacZ was increased in double mutants of lon in combination with clpQ or/and clpY mutation(s) compared with the wild-type or lon single mutants. Overproduction of ClpYQ or ClpQ decreased cpsB–lacZ expression. Additionally, a PBAD–rcsA fusion construct showed quantitatively that an inducible RcsA activates cpsB–lacZ expression. The effect of RcsA on cpsB–lacZ expression was shown to be influenced by the ClpYQ activities. Moreover, a rcsA Red –lacZ translational fusion construct showed higher activity of RcsARed–LacZ in a clpQ clpY strain than in the wild-type. By contrast, overproduction of cellular ClpYQ resulted in decreased β-galactosidase levels of RcsARed–LacZ. Taken together, the data indicate that ClpYQ acts as a secondary protease in degrading the Lon substrate RcsA.
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Pseudomonas syringae pv. phaseolicola can be separated into two genetic lineages distinguished by the possession of the phaseolotoxin biosynthetic cluster
More LessThe bean (Phaseolus spp.) plant pathogen Pseudomonas syringae pv. phaseolicola is characterized by the ability to produce phaseolotoxin (Tox+). We recently reported that the majority of the Spanish P. syringae pv. phaseolicola population is unable to synthesize this toxin (Tox−). These Tox− isolates appear to lack the entire DNA region for the biosynthesis of phaseolotoxin (argK-tox gene cluster), as shown by PCR amplification and DNA hybridization using DNA sequences specific for separated genes of this cluster. Tox+ and Tox− isolates also showed genomic divergence that included differences in ERIC-PCR and arbitrarily primed-PCR profiles. Tox+ isolates showed distinct patterns of IS801 genomic insertions and contained a chromosomal IS801 insertion that was absent from Tox− isolates. Using a heteroduplex mobility assay, sequence differences were observed only among the intergenic transcribed spacer of the five rDNA operons of the Tox− isolates. The techniques used allowed the unequivocal differentiation of isolates of P. syringae pv. phaseolicola from the closely related soybean (Glycine max) pathogen, P. syringae pv. glycinea. Finally, a pathogenicity island that is essential for the pathogenicity of P. syringae pv. phaseolicola on beans appears to be conserved among Tox+, but not among Tox− isolates, which also lacked the characteristic large plasmid that carries this pathogenicity island. It is proposed that the results presented here justify the separation of the Tox+ and Tox− P. syringae pv. phaseolicola isolates into two distinct genetic lineages, designated Pph1 and Pph2, respectively, that show relevant genomic differences that include the pathogenicity gene complement.
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