- Volume 153, Issue 1, 2007
Volume 153, Issue 1, 2007
- Mini-Review
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The first filamentous fungal genome sequences: Aspergillus leads the way for essential everyday resources or dusty museum specimens?
More LessThe published Aspergillus genome sequences (A. nidulans, A. fumigatus, A. oryzae) and further sequence data from A. clavatus, Neosartorya fischeri, A. flavus, A. niger, A. parasiticus and A. terreus are the first from a group of related filamentous fungi. They indicate the gains possible from genomic approaches, but also problems that arise after the sequences are finished. Benefits include a greater understanding of genome structure and evolution, insights into gene regulation, predictions of new factors that may be relevant to pathogenicity and the discovery of novel enzymes with biotechnological value. Areas where further developments are needed include gene and structure–function predictions, methods for comparative genome analysis and the interfaces for access to genome information. In addition, strategies for continued maintenance and updating need to be developed at the start of the post-genomic era to increase the value of genome sequences into the future.
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The first fifty microarray studies in filamentous fungi
More LessMicroarray studies have examined global gene expression in over 20 species of filamentous fungi encompassing a wide variety of research areas. The majority have addressed aspects of metabolism or pathogenicity. Metabolic studies have revealed important differences in the transcriptional regulation of genes for primary metabolic pathways between filamentous fungi and yeast. Transcriptional profiles for genes involved in secondary metabolism have also been established. Genes required for the biosynthesis of both useful and detrimental secondary metabolites have been identified. Due to the economic, ecological and medical implications, it is not surprising that many studies have used microarray analysis to examine gene expression in pathogenic filamentous fungi. Genes involved in various stages of pathogenicity have been identified, including those thought to be important for adaptation to the host environment. While most of the studies have simulated pathogenic conditions in vitro, a small number have also reported fungal gene expression within their plant hosts. This review summarizes the first 50 microarray studies in filamentous fungi and highlights areas for future investigation.
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- Biochemistry And Molecular Biology
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An rhl-like quorum-sensing system negatively regulates pyoluteorin production in Pseudomonas sp. M18
More LessPseudomonas sp. M18, isolated from the watermelon rhizosphere, is antagonistic against a number of soil-borne pathogens. This strain produces an uncharacterized red pigment, pyoluteorin (Plt), and two N-acylhomoserine lactones (AHLs). A previously isolated red-pigment-defective mutant, M18-T510, contains an insert within a gene similar to rhlI in P. aeruginosa PAO1. The M18 rhlI gene product is responsible for the production of two AHL signals: N-butyryl-homoserine lactone and N-hexanoylhomoserine lactone. Mutants defective in either rhlI or rhlR showed enhanced Plt biosynthesis due to loss of transcriptional repression, which was mediated, at least in part, by suppressed expression of the activator PltR. A Plt-specific ABC transporter was also upregulated in the rhl mutants in a Plt-dependent manner. In comparison with the wild-type strain, the rhl mutants survived longer during stationary-phase growth.
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Expression of furA is modulated by NtcA and strongly enhanced in heterocysts of Anabaena sp. PCC 7120
More LessFur (ferric uptake regulator) proteins are principally responsible for maintaining iron homeostasis in prokaryotes. Iron is usually a scarce resource. Its limitation reduces photosynthetic rates and cell growth in cyanobacteria in general and especially in cyanobacteria that are fixing dinitrogen, a process that requires the synthesis of numerous proteins with a high content of iron. This paper shows that in the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120, levels of furA mRNA and FurA protein increase significantly in response to nitrogen deprivation, and that furA up-regulation takes place specifically in proheterocysts and mature heterocysts. Great differences in a Northern blot, probed with furA, of RNA from an ntcA mutant relative to wild-type Anabaena sp. were attributable to binding of NtcA, a global regulator of nitrogen metabolism, to the promoter of furA and to the promoter of the furA antisense transcript alr1690-α-furA.
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A conserved extended signal peptide region directs posttranslational protein translocation via a novel mechanism
Members of the type V secretion family are among the most prevalent secreted proteins in Gram-negative bacteria. A subset of this family, including Pet, the prototypical member of the Enterobacteriaceae serine proteases, possess unusual signal peptides which can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions, which the authors have named the extended signal peptide region (ESPR), demonstrate remarkable conservation. In contrast, the N2, H2 and C regions show significant variability, and are reminiscent of typical Sec-dependent signal sequences. Despite several investigations, the function of the ESPR remains obscure. Here, it is shown that proteins possessing the ESPR are translocated in a posttranslational fashion. The presence of the ESPR severely impairs inner membrane translocation. Mutational analysis suggests that the ESPR delays inner membrane translocation by adopting a particular conformation, or by interacting with a cytoplasmic or inner membrane co-factor, prior to inner membrane translocation.
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Functional characterization of IScs605, an insertion element carried by tetracycline-resistant Chlamydia suis
More LessStable tetracycline resistance in Chlamydia suis is mediated by a family of genomic islands [the tet(C) islands] that are integrated into the chlamydial chromosome. The tet(C) islands contain several plasmid-specific genes, the tet(C) resistance gene and, in most cases, a novel insertion element (IScs605) encoding two predicted transposases. The hypothesis that IScs605 mediated the integration of the tet(C) resistance islands into the C. suis genome was tested using a plasmid-based transposition system in Escherichia coli. Both high- and medium-copy-number plasmids were used as carriers of IScs605 in these experiments. IScs605 integrated into a target plasmid (pOX38) when delivered by either donor plasmid, and integration of the entire donor plasmid was common. IScs605-mediated integration occurred at many positions within pOX38, with 36 of 38 events adjacent to a 5′-TTCAA-3′ sequence. Deletions in each of the candidate transposase genes within IScs605 demonstrated that only one of the two ORFs was necessary for the observed transposition activity and target specificity. Analysis of progeny from the mating assays also indicated that IScs605 can excise following integration into a target DNA, and, in each tested case, the sequence 5′-AATTCAA-3′ remained at the site of excision. Collectively, these results are consistent with the nucleotide sequence data collected for the tet(C) islands, and strongly suggest that a transposase within IScs605 is responsible for integration of these genomic islands into the C. suis chromosome.
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The dimeric repressor SoxR binds cooperatively to the promoter(s) regulating expression of the sulfur oxidation (sox) operon of Pseudaminobacter salicylatoxidans KCT001
More LessSulfur oxidation in Pseudaminobacter salicylatoxidans KCT001 is rendered by the combined action of several enzymes encoded by a thiosulfate-inducible sox operon. In this study it has been conclusively demonstrated by insertional mutagenesis that the regulatory gene of this operon is soxR, which encodes a DNA-binding protein belonging to the ArsR-SmtB family. SoxR was found to bind to two promoter-operator segments within the sox cluster, of which the one (wx) located between soxW and soxX controls the expression of sulfur-oxidation genes soxX through soxD while the other, a bi-directional element (sv) located between soxS and soxV, controls the expression of soxVW in one direction and the putative regulatory cluster soxSRT in the other. In the case of the wx promoter the repressor was found to bind in a cooperative manner to two distinct binding sites having different affinities, while in the case of the sv promoter binding occurred at a symmetric dimeric site and involved a higher degree of cooperativity. The high degree of cooperativity observed in the binding of SoxR to its target sites seemed to be due to the propensity of SoxR monomers to form dimers. The apparent dissociation constants of the SoxR–operator complexes were in the nanomolar range, indicating relatively strong interactions. It was demonstrated using a reporter system in Escherichia coli that this high-affinity binding of SoxR led to efficient repression in trans. Thus the role of SoxR as a repressor of the sox operon has not only been conclusively established but it has also been shown that this repression is brought about through cooperative interactions of SoxR with dimeric binding sites that occlude the operon promoters.
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Regulatory role of RsgI in sigI expression in Bacillus subtilis
More LessThe sigma gene, sigI, of Bacillus subtilis belongs to the group IV heat-shock response genes and has many orthologues in the bacterial phylum Firmicutes. The B. subtilis sigI gene is considered to constitute an operon with rsgI (regulation of sigI , formerly ykrI). As little is known about either the structure and function of the sigI-rsgI operon or the SigI regulons, the role of RsgI in heat-inducible transcription of the sigI-rsgI operon was investigated, using Northern analysis and a heat-stable β-galactosidase reporter assay. Heat-inducible, SigI-dependent transcription of the sigI-rsgI operon was stimulated greatly by disrupting rsgI. Yeast two-hybrid analysis showed direct interaction between the N-terminal portion of the presumed RsgI protein and SigI. Without RsgI function, induction of transcription of the sigI-rsgI operon upon transient heat stress depended on dnaK activity. However, transcription of the operon was induced during growth at prolonged higher temperature even without DnaK function. Without RsgI function, sigI-rsgI operon transcription was induced after the end of growth independent of any temperature shift in a sporulation medium and toward the end of growth in a rich complex medium. Furthermore, glucose addition resulted in a strong suppression of sigI-rsgI transcription. Therefore it is hypothesized that transcription of the sigI-rsgI operon of B. subtilis is negatively regulated by the putative transmembrane protein RsgI, which moderates SigI's sensitivity to heat shock or nutritional stress.
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Differential expression of two bc 1 complexes in the strict acidophilic chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans suggests a model for their respective roles in iron or sulfur oxidation
Three strains of the strict acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans, including the type strain ATCC 23270, contain a petIIABC gene cluster that encodes the three proteins, cytochrome c 1, cytochrome b and a Rieske protein, that constitute a bc 1 electron-transfer complex. RT-PCR and Northern blotting show that the petIIABC cluster is co-transcribed with cycA, encoding a cytochrome c belonging to the c 4 family, sdrA, encoding a putative short-chain dehydrogenase, and hip, encoding a high potential iron–sulfur protein, suggesting that the six genes constitute an operon, termed the petII operon. Previous results indicated that A. ferrooxidans contains a second pet operon, termed the petI operon, which contains a gene cluster that is similarly organized except that it lacks hip. Real-time PCR and Northern blot experiments demonstrate that petI is transcribed mainly in cells grown in medium containing iron, whereas petII is transcribed in cells grown in media containing sulfur or iron. Primer extension experiments revealed possible transcription initiation sites for the petI and petII operons. A model is presented in which petI is proposed to encode the bc 1 complex, functioning in the uphill flow of electrons from iron to NAD(P), whereas petII is suggested to be involved in electron transfer from sulfur (or formate) to oxygen (or ferric iron). A. ferrooxidans is the only organism, to date, to exhibit two functional bc 1 complexes.
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A σ 54-dependent promoter in the regulatory region of the Escherichia coli rpoH gene
The Escherichia coli rpoH gene is transcribed from four known and differently regulated promoters: P1, P3, P4 and P5. This study demonstrates that the conserved consensus sequence of the σ 54 promoter in the regulatory region of the rpoH gene, described previously, is a functional promoter, P6. The evidence for this conclusion is: (i) the specific binding of the σ 54–RNAP holoenzyme to P6, (ii) the location of the transcription start site at the predicted position (C, 30 nt upstream of ATG) and (iii) the dependence of transcription on σ 54 and on an ATP-dependent activator. Nitrogen starvation, heat shock, ethanol and CCCP treatment did not activate transcription from P6 under the conditions examined. Two activators of σ 54 promoters, PspF and NtrC, were tested but neither of them acted specifically. Therefore, PspFΔHTH, a derivative of PspF, devoid of DNA binding capability but retaining its ATPase activity, was used for transcription in vitro, taking advantage of the relaxed specificity of ATP-dependent activators acting in solution. In experiments in vivo overexpression of PspFΔHTH from a plasmid was employed. Thus, the σ 54-dependent transcription capability of the P6 promoter was demonstrated both in vivo and in vitro, although the specific conditions inducing initiation of the transcription remain to be elucidated. The results clearly indicate that the closed σ 54–RNAP–promoter initiation complex was formed in vitro and in vivo and needed only an ATP-dependent activator to start transcription.
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Three functional subdomains of the Escherichia coli FtsQ protein are involved in its interaction with the other division proteins
More LessFtsQ, an essential protein for the Escherichia coli divisome assembly, is able to interact with various division proteins, namely FtsI, FtsL, FtsN, FtsB and FtsW. In this paper, the FtsQ domains involved in these interactions were identified by two-hybrid assays and co-immunoprecipitations. Progressive deletions of the ftsQ gene suggested that the FtsQ self-interaction and its interactions with the other proteins are localized in three periplasmic subdomains: (i) residues 50–135 constitute one of the sites involved in FtsQ, FtsI and FtsN interaction, and this site is also responsible for FtsW interaction; (ii) the FtsB interaction is localized between residues 136 and 202; and (iii) the FtsL interaction is localized at the very C-terminal extremity. In this third region, the interaction site for FtsK and also the second site for FtsQ, FtsI, FtsN interactions are located. As far as FtsW is concerned, this protein interacts with the fragment of the FtsQ periplasmic domain that spans residues 67–75. In addition, two protein subdomains, one constituted by residues 1–135 and the other from 136 to the end, are both able to complement an ftsQ null mutant. Finally, the unexpected finding that an E. coli ftsQ null mutant can be complemented, at least transiently, by the Streptococcus pneumoniae divIB/ftsQ gene product suggests a new strategy for investigating the biological significance of protein–protein interactions.
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Structural and genetic evidence that the Escherichia coli O148 O antigen is the precursor of the Shigella dysenteriae type 1 O antigen and identification of a glucosyltransferase gene
Shigella dysenteriae type 1 is the most virulent serotype of Shigella. Enterotoxigenic Escherichia coli O148 is pathogenic and can cause diarrhoea. The following structure was established for the tetrasaccharide repeating unit of the E. coli O148 O antigen: →3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→2)-α-D-Glcp-(1→3)-α-D-GlcpNAc-(1→. This differs from the structure reported earlier for S. dysenteriae type 1 by having a glucose (Glc) residue in place of a galactose (Gal) residue. The two bacteria also have the same genes for O antigen synthesis, with the same organization and high level of DNA identity, except that in S. dysenteriae type 1 wbbG is interrupted by a deletion, and a galactosyltransferase gene wbbP located on a plasmid is responsible for the transfer of galactose to make a novel antigenic epitope of the O antigen. The S. dysenteriae type 1 O antigen was reconstructed by replacing the E. coli O148 wbbG gene with the wbbP gene, and it had the LPS structure and antigenic properties of S. dysenteriae type 1, indicating that the S. dysenteriae type 1 O antigen evolved from that of E. coli O148. It was also confirmed that wbbG of E. coli O148 is a glucosyltransferase gene, and two serotype-specific genes of E. coli O148 and S. dysenteriae type 1 were identified.
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The proteomic profile of Fusobacterium nucleatum is regulated by growth pH
More LessFusobacterium nucleatum is a saccharolytic Gram-negative anaerobic organism believed to play an important role in the microbial succession associated with the development of periodontal disease. Its genome contains niche-specific genes shared with the other inhabitants of dental plaque, which may help to explain its ability to survive and grow in the changing environmental conditions experienced in the gingival sulcus during the transition from health to disease. The pH of the gingival sulcus increases during the development of periodontitis and this is thought to occur by the metabolism of nutrients supplied by gingival crevicular fluid. In comparison with other plaque inhabitants, F. nucleatum has the greatest ability to neutralize acidic environments. The differential expression of soluble cytoplasmic proteins induced by acidic (pH 6.4) or basic (pH 7.4 and 7.8) conditions, during long-term anaerobic growth in a chemostat, was identified by two-dimensional gel electrophoresis and image analysis software. Twenty-two proteins, found to have altered expression in response to external pH, were identified by tryptic digestion and mass spectrometry. Eight differentially expressed proteins associated with increased energy (ATP) production via the 2-oxoglutarate and Embden–Meyerhof pathways appeared to be directed towards either cellular biosynthesis or the maintenance of internal homeostasis. Overall, these results represent the first proteomic investigation of F. nucleatum and the identification of gene products which may be important in the organism's persistence during the transition from health to disease in vivo.
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- Biodiversity And Evolution
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Rickettsiae phylogeny: a multigenic approach
More LessThe development of molecular taxonomic methods has provided a large amount of data in the reorganization of Rickettsiae taxonomy. Nevertheless, phylogenetic relationships among some groups and species delimitation remain unclear. To clarify rickettsial phylogeny, a multigenic approach was used for the first time for the genus Rickettsia, based on simultaneous analyses of eight loci: atpA, recA, virB4, dnaA, dnaK, rrl-rrf internal transcribed spacer, ompA and gltA. Concatenation of different nucleotide sequences resulted in an improvement in phylogenetic resolution when compared to single gene data. This multigenic approach has enabled the differentiation of many groups, including the spotted fever group which includes a great number of closely related species. The reliability of some previously recognized groups was evaluated.
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Phenotypic and molecular characterizations of Yersinia pestis isolates from Kazakhstan and adjacent regions
Recent interest in characterizing infectious agents associated with bioterrorism has resulted in the development of effective pathogen genotyping systems, but this information is rarely combined with phenotypic data. Yersinia pestis, the aetiological agent of plague, has been well defined genotypically on local and worldwide scales using multi-locus variable number tandem repeat analysis (MLVA), with emphasis on evolutionary patterns using old isolate collections from countries where Y. pestis has existed the longest. Worldwide MLVA studies are largely based on isolates that have been in long-term laboratory culture and storage, or on field material from parts of the world where Y. pestis has potentially circulated in nature for thousands of years. Diversity in these isolates suggests that they may no longer represent the wild-type organism phenotypically, including the possibility of altered pathogenicity. This study focused on the phenotypic and genotypic properties of 48 Y. pestis isolates collected from 10 plague foci in and bordering Kazakhstan. Phenotypic characterization was based on diagnostic tests typically performed in reference laboratories working with Y. pestis. MLVA was used to define the genotypic relationships between the central-Asian isolates and a group of North American isolates, and to examine Kazakh Y. pestis diversity according to predefined plague foci and on an intermediate geographical scale. Phenotypic properties revealed that a large portion of this collection lacks one or more plasmids necessary to complete the blocked flea/mammal transmission cycle, has lost Congo red binding capabilities (Pgm−), or both. MLVA analysis classified isolates into previously identified biovars, and in some cases groups of isolates collected within the same plague focus formed a clade. Overall, MLVA did not distinguish unique phylogeographical groups of Y. pestis isolates as defined by plague foci and indicated higher genetic diversity among older biovars.
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- Environmental Microbiology
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Abundance and ecophysiology of Defluviicoccus spp., glycogen-accumulating organisms in full-scale wastewater treatment processes
More LessThe activity of glycogen-accumulating organisms (GAOs) in enhanced biological phosphorus removal (EBPR) wastewater treatment plants has been proposed as one cause of deterioration of EBPR. Putative GAOs from the Alphaproteobacteria, Defluviicoccus spp. (including D. vanus), were studied in full-scale EBPR plants to determine their distribution, abundance and ecophysiology. Fluorescence in situ hybridization (FISH) demonstrated that Defluviicoccus spp. were generally low in abundance; however, in one plant surveyed, Cluster 2 Defluviicoccus constituted 9 % of all Bacteria. FISH combined with microautoradiography revealed that both Cluster 1 and Cluster 2 Defluviicoccus were capable of taking up a narrow range of substrates including acetate, propionate, pyruvate and glucose under anaerobic and aerobic conditions. Formate, butyrate, ethanol and several other substrates were not taken up. Cluster 2 Defluviicoccus demonstrated a phenotype consistent with the current metabolic model for GAOs – anaerobic assimilation of acetate and reduction to polyhydroxyalkanoates (PHA) using the glycolytic pathway, and aerobic consumption of PHA. Polyphosphate-accumulating organisms (PAOs, ‘Candidatus Accumulibacter phosphatis’) and other putative GAOs (‘Candidatus Competibacter phosphatis’) co-existed in two plants with Cluster 2 Defluviicoccus, but in both plants, the latter organisms were more abundant. Thus Cluster 2 Defluviicoccus can be relatively abundant and could be carbon competitors of PAOs and other GAOs in EBPR plants.
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Growth of the genetically engineered strain Cupriavidus necator RW112 with chlorobenzoates and technical chlorobiphenyls
More LessCupriavidus necator (formerly Ralstonia eutropha) strain H850 is known to grow on biphenyl, and to co-oxidize congeners of polychlorinated biphenyls (PCBs). Using a Tn5-based minitransposon shuttle system and the TOL plasmid, the rational construction of hybrids of H850 was achieved by subsequent introduction of three distinct elements carrying 11 catabolic loci from three other biodegrading bacteria into the parent strain, finally yielding C. necator RW112. The new genetic elements introduced into H850 and its derivatives were tcbRCDEF, which encode the catabolic enzymes needed for chlorocatechol biodegradation under the control of a transcriptional regulator, followed by cbdABC, encoding a 2-halobenzoate dioxygenase, and xylXYZ, encoding a broad-spectrum toluate dioxygenase. The expression of the introduced genes was demonstrated by measuring the corresponding enzymic activities. The engineered strain RW112 gained the ability to grow on all isomeric monochlorobenzoates and 3,5-dichlorobenzoate, all monochlorobiphenyls, and 3,5-dichloro-, 2,3′-dichloro- and 2,4′-dichlorobiphenyl, without accumulation of chlorobenzoates. It also grew and utilized two commercial PCB formulations, Aroclor 1221 and Aroclor 1232, as sole carbon and energy sources for growth. This is the first report on the aerobic growth of a genetically improved bacterial strain at the expense of technical Aroclor mixtures.
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- Pathogens And Pathogenicity
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The iron- and cAMP-regulated gene SIT1 influences ferrioxamine B utilization, melanization and cell wall structure in Cryptococcus neoformans
More LessThe mechanisms by which pathogens sense and transport iron are important during infection, because of the low availability of free iron in the mammalian host. Iron is a key nutritional cue for the pathogen Cryptococcus neoformans, because it influences expression of the polysaccharide capsule that is the major virulence factor of the fungus. In this study, C. neoformans mutants were constructed with a defect in the iron-regulated gene SIT1 that encodes a putative siderophore iron transporter. Analysis of mutants in serotype A and D strains demonstrated that SIT1 is required for the use of siderophore-bound iron, and for growth in a low-iron environment. The sit1 mutants also showed changes in melanin formation and cell wall density, and it was found that mutants defective in protein kinase A, which is known to influence melanization and capsule formation, showed elevated SIT1 transcripts in both the serotype A and the serotype D backgrounds. Finally, the mutants were tested for virulence in a murine model of cryptococcosis, and it was found that SIT1 was not required for virulence. Overall, these studies establish links between iron acquisition, melanin formation and cAMP signalling in C. neoformans.
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Autophagy in the pathogen Candida albicans
More LessAutophagy is a major cellular process that facilitates the bulk degradation of eukaryotic macromolecules and organelles, through degradation within the lysosomal/vacuole compartment. This has been demonstrated to influence a diverse array of eukaryotic cell functions including adaptation, differentiation and developmental programmes. For example, in Saccharomyces cerevisiae autophagy is required for sporulation and survival of nitrogen starvation. The opportunistic pathogen Candida albicans has the ability to colonize and cause disease within a diverse range of mammalian host sites. The ability to adapt and differentiate within the host is liable to be critical for host colonization and infection. Previous results indicated that the vacuole plays an important role in C. albicans adaptation to stress, differentiation, and survival within and injury of host cells. In this study the importance of vacuole-mediated degradation through the process of autophagy was investigated. This involved identification and deletion of ATG9, a C. albicans gene required for autophagy. The deletion strain was blocked in autophagy and the closely related cytoplasm to vacuole (cvt) trafficking pathway. This resulted in sensitivity to nitrogen starvation, but no defects in growth rate, vacuole morphology or resistance to other stresses. This indicates that the mutant has specific defects in autophagy/cvt trafficking. Given the importance of autophagy in the development and differentiation of other eukaryotes, it was surprising to find that the atg9Δ mutant was unaffected in either yeast–hypha or chlamydospore differentiation. Furthermore, the atg9Δ mutant survived within and killed a mouse macrophage-like cell line as efficiently as control strains. The data suggest that autophagy plays little or no role in C. albicans differentiation or during interaction with host cells.
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Proteomic comparison of Mycobacterium avium subspecies paratuberculosis grown in vitro and isolated from clinical cases of ovine paratuberculosis
More LessParatuberculosis (Johne's disease) poses a significant economic problem to beef, dairy and sheep industries worldwide, and is caused by Mycobacterium avium subspecies paratuberculosis. In this study, 2D PAGE was used as a tool to investigate the virulent state of M. avium subsp. paratuberculosis, incorporating the technique of beating the organism with zirconium/silica beads to provide a comprehensive representation of its proteome. A direct comparison of the proteomes of M. avium subsp. paratuberculosis scraped from the terminal ileum of ovine paratuberculosis cases, and the identical strain grown in vitro, is presented. These analyses identified a set of 10 proteins whose expression is upregulated during natural infection: 1-pyrroline-5-carboxylate dehydrogenase (RocA), a putative acyl-CoA dehydrogenase (FadE14), 2-methylcitrate dehydratase (2-mcd), arginosuccinate synthase (ArgG), universal stress protein (usp), 30S ribosomal protein S2 (RpsB), peptidyl-prolyl cis-trans isomerase (PpiA), luciferase-like monooxygenase (lmo), thiosulfate sulfurtransferase (SseA) and ATP-dependent Clp protease (ClpB). Most of the proteins identified do not have obviously related functions; however, ArgG and RocA function in the same pathway, and may have a concerted action for energy production in vivo.
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Proteomic identification and characterization of bacterial factors associated with Burkholderia cenocepacia survival in a murine host
More LessBurkholderia cenocepacia is a member of the Burkholderia cepacia complex, a diverse family of Gram-negative bacteria that are serious respiratory pathogens in immunocompromised patients and individuals with cystic fibrosis. To identify putative bacterial virulence determinants, proteomic profiles were compared between two B. cenocepacia isolates that demonstrated differential persistence in a mouse model of pulmonary infection; clinical isolate C1394 is rapidly cleared from the murine lung whereas the strain variant, C1394mp2, persists. Two-dimensional (2D) gel electrophoresis was used to identify candidate proteins involved in B. cenocepacia survival in a susceptible host. The 2D proteome of the persistent isolate (C1394mp2) revealed loss of an alkyl hydroperoxide reductase subunit C (AhpC) protein spot and increased production of flagellin proteins. Loss of AhpC expression in C1394mp2 correlated with enhanced susceptibility to oxidative stress. C1394mp2 expressed increased flagellin production and enhanced swimming motility, traits that were subject to regulation by heat and low pH. Together, these results revealed differential expression and stress regulation of putative virulence determinants associated with B. cenocepacia persistence in a susceptible host.
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The haem–haemopexin utilization gene cluster (hxuCBA) as a virulence factor of Haemophilus influenzae
Haemophilus influenzae has an absolute growth requirement for a porphyrin source, which can be supplied in vitro by haem, haemoglobin, or the haemoglobin–haptoglobin, haem–haemopexin and haem–albumin complexes. Utilization of the haem–haemopexin complex is known to be mediated by the products of the hxuCBA gene cluster. It was demonstrated that hxuC, but not hxuA or hxuB, is also essential for the utilization of haem from haem–albumin complexes. Mutants of the type b strain E1a lacking genes in the hxuCBA gene cluster were examined for their ability to cause bacteraemia in rat models of invasive disease. In 5-day-old rats, mutants in the hxuCBA genes yielded a significantly reduced bacteraemic titre compared to the wild-type strain. In addition, 5-day-old rats infected with the hxuCBA mutant strains exhibited significantly improved survival rates compared to those infected with the wild-type strain. Mutations in the haemoglobin/haemoglobin–haptoglobin-binding protein genes (hgps), either alone or in combination with the hxuCBA mutations, had no impact on virulence in 5-day-old rats. In 30-day-old rats infected with either the hxuCBA mutants or the wild-type strains, there was no significant difference in the ability to establish bacteraemia although bacterial titres were lower in rats infected with the hxuCBA mutants than in those infected with the wild-type strain. These age-related differences in the impact of mutations in the hxuCBA gene cluster may be related to changes in levels of host haem-binding proteins during development of the rat.
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Quorum-sensing-deficient (lasR) mutants emerge at high frequency from a Pseudomonas aeruginosa mutS strain
More LessIn Pseudomonas aeruginosa, quorum sensing constitutes a highly complex cell-to-cell communication system that, along with the cognate acylhomoserine lactone signals and regulators LasR and RhlR, modulates the production of virulence factors and a wide range of metabolic functions. In a previous paper, the authors reported that mismatch repair disruption in P. aeruginosa results in the spontaneous and reproducible emergence of defined morphological colony variants after a relatively short period of cultivation in an aerated rich medium, in contrast to the non-mutator parental strain, which does not display any kind of diversification under identical incubation conditions. One of the morphotypical variants, mS2, emerges at a high frequency and displays differences in virulence traits that could be regulated by major quorum-sensing regulators. The present study shows that mutS mS2 variants had defective LasR function due to simple but different point mutations along the lasR gene sequence, indicating that LasR inactivation is the main cause of mS2 phenotypic diversification. Moreover, it was determined that a non-functional LasR would confer a selective advantage in the late stationary phase, since viability was notably higher for mS2. Interestingly, in all mS2 variants analysed, no sequence alterations were found in the gacA and rhlR genes, suggesting that the selective pressures for GacA/RhlR and LasR were not the same and differed from those in other Pseudomonas species, which, when incubated in nutrient-rich liquid stationary-phase cultures, show specific high instability in the gacA–gacS genes.
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- Physiology
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The glutamate-dependent acid resistance system in Escherichia coli: essential and dual role of the His–Asp phosphorelay RcsCDB/AF
The RcsCDB signal transduction system is an atypical His–Asp phosphorelay. Notably, the response regulator RcsB can be activated either by phosphorylation through the RcsCD pathway or by an accessory cofactor RcsA. Although conserved in Enterobacteriaceae, the role of this system in adaptation to environmental stress conditions is largely unknown. This study reveals that the response regulator RcsB is essential to glutamate-dependent acid resistance, a condition pertinent to the lifestyle of Escherichia coli. The requirement for RcsB is independent of its activation by either the RcsCD or the RcsA pathway. The basal activity of RcsB appears to be necessary and sufficient for acid resistance. The sensitivity of the rcsB strain to low pH is correlated to a strong reduction of the expression of the glutamate decarboxylase genes, gadA and gadB, during the stationary phase of growth. This effect on gadA/B expression is not mediated by the general stress sigma factor RpoS, but does require a functional gadE allele and the previously identified GadE box. Therefore activation of gadAB expression and acid resistance absolutely requires both GadE and RcsB. In contrast, an increase in RcsB activity through the activation of the RcsCD phosphorelay or the RcsA pathway or through overproduction of the protein leads to general repression of the expression of the gad genes and a corresponding reduction in acid resistance.
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The influence of ribosome modulation factor on the survival of stationary-phase Escherichia coli during acid stress
More LessRibosome modulation factor (RMF) was shown to have an influence on the survival of Escherichia coli under acid stress during stationary phase, since the viability of cultures of a mutant strain lacking functional RMF decreased more rapidly than that of the parent strain at pH 3. Loss of ribosomes was observed in both strains when exposed to low pH, although this occurred at a higher rate in the RMF-deficient mutant strain, which also suffered from higher levels of rRNA degradation. It was concluded that the action of RMF in limiting the damage to rRNA contributed to the protection of E. coli under acid stress. Expression of the rmf gene was lower during stationary phase after growth in acidified media compared to media containing no added acid, and the increased rmf expression associated with transition from exponential phase to stationary phase was much reduced in acidified media. It was demonstrated that RMF was not involved in the stationary-phase acid-tolerance response in E. coli by which growth under acidic conditions confers protection against subsequent acid shock. This response was sufficient to overcome the increased vulnerability of the RMF-deficient mutant strain to acid stress at pH values between 6.5 and 5.5.
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Expression of the htrB gene is essential for responsiveness of Salmonella typhimurium and Campylobacter jejuni to harsh environments
More LessIn Campylobacter jejuni, an htrB homologous gene is located in the lipo-oligosaccharide synthesis gene cluster. This study examined the effects of htrB expression on the responsiveness of Salmonella typhimurium and C. jejuni to harsh environments. Complementation experiments showed that the C. jejuni htrB gene could restore the normal morphology of the Salmonella htrB mutant, and its ability to grow without inhibition under heat, acid and osmotic stresses, but not bile stress. This indicated that the htrB genes in C. jejuni and S. typhimurium exhibit similar pleiotropic effects. Moreover, quantitative real-time RT-PCR showed that expression of the C. jejuni htrB gene was upregulated under acid, heat, oxidative and osmotic stresses, but did not change under bile stress. This indicated that the C. jejuni htrB gene plays a role in regulating cell responses to various environmental changes. Furthermore, deletion mutation of the htrB gene in C. jejuni was lethal, indicating that the htrB gene is essential for C. jejuni survival. Therefore, these results showed that expression of the htrB gene is essential for the response of S. typhimurium and C. jejuni to environmental stresses.
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Role of the alternative sigma factors σ E and σ S in survival of Salmonella enterica serovar Typhimurium during starvation, refrigeration and osmotic shock
The ability of Salmonella enterica serovar Typhimurium to survive environmental stress requires specific, coordinated, responses, which induce resistance to the stress condition. This study investigated the relative contribution of σ E and σ S, the sigma factors regulating extracytoplasmic and general stress response functions, respectively, to survival at low temperature and also in media of differing osmotic strength, conditions relevant to food preservation. To determine if low-temperature storage is a signal for σ E- and σ S-mediated survival, the ability of S. Typhimurium rpoE, rpoS and rpoE/rpoS mutants to survive in a saline starvation-survival model at a refrigeration temperature (4.5 °C) was examined. Under these conditions, the rpoE mutant was significantly (P<0.05) compromised compared to the parent and to an rpoS mutant. The double mutant in rpoE and rpoS displayed a cumulative defect in survival. In hyperosmotic environments (low a w) containing 6 % NaCl and at refrigeration temperature, both sigma factors were important for maximum survival but σ S played the dominant role. Analysis of the metabolic activity of starved populations at 4.5 and 37 °C revealed significantly (P<0.001) elevated electron-transport system activity in mutants in rpoE and rpoS, indicating a role for σ E- and σ S-regulated genes in maintaining energy homeostasis. Together these data demonstrate that σ E and σ S are important for survival of S. Typhimurium in conditions encountered during food processing and that the relative contribution of σ E and σ S is critically dependent on the precise nature of the stress.
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Biochemical and genetic characterization of the pathways for trehalose metabolism in Propionibacterium freudenreichii, and their role in stress response
More LessPropionibacterium freudenreichii accumulates high levels of trehalose, especially in response to stress. The pathways for trehalose metabolism were characterized, and their roles in response to osmotic, oxidative and acid stress were studied. Two pathways were identified: the trehalose-6-phosphate synthase/phosphatase (OtsA–OtsB) pathway, and the trehalose synthase (TreS) pathway. The former was used for trehalose synthesis, whereas the latter is proposed to operate in trehalose degradation. The activities of OtsA, OtsB and TreS were detected in cell extracts; the corresponding genes were identified, and the recombinant proteins were characterized in detail. In crude extracts of P. freudenreichii, OtsA was specific for ADP-glucose, in contrast to the pure recombinant OtsA, which used UDP-, GDP- and TDP-glucose, in addition to ADP-glucose. Moreover, the substrate specificity of OtsA in cell extracts was lost during purification, and the recombinant OtsA became specific to ADP-glucose upon incubation with a dialysed cell extract. The level of OtsA was enhanced (approximately twofold) by osmotic, oxidative and acid stress, whereas the level of TreS remained constant, or it decreased, under identical stress conditions. Therefore, the OtsA–OtsB pathway plays an important role in the synthesis of trehalose in response to stress. It is most likely that trehalose degradation proceeds via TreS to yield maltose, which is subsequently catabolized via amylomaltase activity. Hydrolytic activities that are potentially involved in trehalose degradation (trehalase, trehalose phosphorylase, trehalose-6-phosphate phosphorylase and trehalose-6-phosphate hydrolase) were not present. The role of trehalose as a common response to three distinct stresses is discussed.
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Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates
The metabolic pathways associated with the tricarboxylic acid cycle intermediates of Pichia pastoris were studied using biosynthetically directed fractional 13C labelling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (1.39×10−5 s−1 and 4.44×10−5 s−1) with varying mixtures of glycerol and methanol as sole carbon sources. The results show that, with co-assimilation of methanol, the common amino acids are synthesized as in P. pastoris cells grown on glycerol only. During growth at the lower dilution rate, when both substrates are entirely consumed, the incorporation of methanol into the biomass increases as the methanol fraction in the feed is increased. Moreover, the co-assimilation of methanol impacts on how key intermediates of the pentose phosphate pathway (PPP) are synthesized. In contrast, such an impact on the PPP is not observed at the higher dilution rate, where methanol is only partially consumed. This finding possibly indicates that the distribution of methanol carbon into assimilatory and dissimilatory (direct oxidation to CO2) pathways are different at the two dilution rates. Remarkably, distinct flux ratios were registered at each of the two growth rates, while the dependency of the flux ratios on the varying fraction of methanol in the medium was much less pronounced. This study brings new insights into the complex regulation of P. pastoris methanol metabolism in the presence of a second carbon source, revealing important implications for biotechnological applications.
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Growth of probiotic lactobacilli in the presence of oleic acid enhances subsequent survival in gastric juice
More LessThe effect of inclusion of various C18 fatty acids with 0–2 double bonds in either cis or trans configuration on Lactobacillus rhamnosus GG survival was analysed in simulated gastric juice at pH 2.5. The incorporation of Tween 80 (1 g l−1) in the growth media enhanced subsequent survival of stationary-phase cultures up to 1000-fold following 90 min acid exposure compared with controls grown without Tween 80. There was a significant (P<0.05) increase in bacterial content of oleic acid [C18 : 1 (9c), up to 55-fold] after growth of bacteria in MRS supplemented with Tween 80. The inclusion of various C18 fatty acids in the growth media revealed that only oleic and vaccenic acids [C18 : 1 (11t)] had protective effects on the survival of Lb. rhamnosus GG when exposed to the acidic environment. Comparative analysis with other lactobacilli indicated that all strains exhibited increased survival when grown in the presence of Tween 80. Further work with a neomycin-resistant mutant with 48 % of the F0F1-ATPase activity of the parent indicated that the Tween 80 effect was independent of the complex. The mechanisms behind the effect of fatty acid protection were investigated and proton permeability assays showed that cultures grown in the presence of Tween 80 had higher extracellular pH than controls. Furthermore, there was a significant reduction of oleic acid and a significant increase in stearic acid (C18 : 0) (P<0.05) content of bacterial cells following exposure of Tween 80-supplemented cultures to simulated gastric juice. Overall, the data suggest that probiotic lactobacilli can use an exogenous oleic acid source to increase their acid survival and the underlying mechanism most likely involves the ability of increased membrane oleic acid to be reduced by H+ to stearic acid.
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Volumes and issues
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Volume 170 (2024)
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