- Volume 155, Issue 4, 2009
Volume 155, Issue 4, 2009
- Sgm Special Lecture
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Bacterial variation, virulence and vaccines
More LessResearch into Haemophilus influenzae, a commensal and pathogen of humans, has resulted in major scientific contributions to biology. The first endonucleases (restriction enzymes), which paved the way for the new genetics, and the DNA used to obtain the first complete genome sequence of a free-living organism were obtained from H. influenzae. Prevention of invasive bacterial infections of infants, such as meningitis, has been achieved using a novel class of vaccines, of which the glycoconjugates of H. influenzae were the first to be licensed. Originally fallaciously proposed to be the aetiological agent of epidemic influenza, now known to be caused by a virus, H. influenzae is a pathogen of global public health importance. Research into the pathogenesis of the infections it causes (for example, meningitis, septicaemia, pneumonia and otitis media) are case studies in understanding the molecular basis of the variation in gene expression and gene sequences that are critical to its commensal and virulence behaviour and for the strategies that can be pursued to prevent H. influenzae diseases through vaccines.
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- Cell And Molecular Biology Of Microbes
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Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis
More LessThe obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.
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ParB deficiency in Pseudomonas aeruginosa destabilizes the partner protein ParA and affects a variety of physiological parameters
More LessDeletions leading to complete or partial removal of ParB were introduced into the Pseudomonas aeruginosa chromosome. Fluorescence microscopy of fixed cells showed that ParB mutants lacking the C-terminal domain or HTH motif formed multiple, less intense foci scattered irregularly, in contrast to the one to four ParB foci per cell symmetrically distributed in wild-type P. aeruginosa. All parB mutations affected both bacterial growth and swarming and swimming motilities, and increased the production of anucleate cells. Similar effects were observed after inactivation of parA of P. aeruginosa. As complete loss of ParA destabilized its partner ParB it was unclear deficiency of which protein is responsible for the mutant phenotypes. Analysis of four parB mutants showed that complete loss of ParB destabilized ParA whereas three mutants that retained the N-terminal 90 aa of ParB did not. As all four parB mutants demonstrate the same defects it can be concluded that either ParB, or ParA and ParB in combination, plays an important role in nucleoid distribution, growth and motility in P. aeruginosa.
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M1.MboII and M2.MboII type IIS methyltransferases: different specificities, the same target
More LessMethylation of a base in a specific DNA sequence protects the DNA from nucleolytic cleavage by restriction enzymes recognizing the same sequence. The MboII restriction–modification (R–M) system of Moraxella bovis ATCC 10900 consists of a restriction endonuclease gene and two methyltransferase genes. The enzymes encoded by this system recognize an asymmetrical sequence 5′-GAAGA-3′/3′-CTTCT-5′. M1.MboII modifies the last adenine in the recognition sequence 5′-GAAGA-3′ to N 6-methyladenine. A second methylase, M2.MboII, was cloned and purified to electrophoretic homogeneity using a four-step chromatographic procedure. It was demonstrated that M2.MboII modifies the internal cytosine in the recognition sequence 3′-CTTCT-5′, yielding N 4-methylcytosine, and moreover is able to methylate single-stranded DNA. The protein exists in solution as a monomer of molecular mass 30 000±1000 Da under denaturing conditions. Divalent cations (Ca2+, Mg2+, Mn2+ and Zn2+) inhibit M2.MboII methylation activity. It was found that the isomethylomer M2.NcuI from Neisseria cuniculi ATCC 14688 behaves in the same manner. Functional analysis showed that the complete MboII R–M system, consisting of two methyltransferases genes and the mboIIR gene, is the most stable and the least harmful to bacterial cells.
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The Spo0E phosphatase of Bacillus subtilis is a substrate of the FtsH metalloprotease
More LessIn the absence of the ATP-dependent metalloprotease FtsH, the sporulation frequency of Bacillus subtilis cells is reduced by several orders of magnitude. This indicates that FtsH has to degrade or to regulate the steady-state level of one or more proteins that interfere with successful sporulation. Here, we show that the amount of the master regulator protein Spo0A is reduced in an ftsH knockout and the small amounts of Spo0A protein present are inactive. Phosphorylation of Spo0A occurs through a phosphorelay. Four negative regulators have been identified here which directly interfere with the phosphorelay through ftsH, namely the phosphatases RapA, RapB, RapE and Spo0E. If a null allele in any one of them was combined with an ftsH knockout, the sporulation frequency was increased by two to three orders of magnitude, but remained below 1 %. When purified Spo0E was incubated with FtsH, partial degradation of the phosphatase was observed. In contrast, two mutant versions of Spo0E with truncated C-termini remained stable. Transfer of the C-terminal 25 aa of Spo0E to a shorter homologue of Spo0E, YnzD, which is not a substrate of FtsH, conferred instability. When a mutant Spo0A was produced that was active in the absence of phosphorylation, spores were formed at a normal rate in an ftsH knockout, indicating that ftsH is needed only during phase 0.
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Localization and assembly of proteins comprising the outer structures of the Bacillus anthracis spore
Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. In Bacillus anthracis, however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue of exsFA/bxpB) and IunH (similar to an inosine–uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, an exsFA/bxpB mutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB–GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB–GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB–GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. IunH–GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB–GFP and IunH–GFP resides in the region between the coat and the exosporium, called the interspace.
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Localization of the GerD spore germination protein in the Bacillus subtilis spore
More LessThe GerD protein of Bacillus subtilis is required for efficient spore germination in l-alanine, and for germination in the alternative germinant combination of amino acids plus sugars. Only germination via nutrient receptors is affected in the mutant. The GerD protein is predicted to be a lipoprotein that is produced in the forespore compartment of the sporulating cell. Using antibody raised against the GerD protein, Western blots of proteins from spore fractions revealed that, as might be expected, the protein was detected in the inner membrane of spores, but it was also present at a high level in spore integuments (comprising coat, cortex and germ cell wall layers), and to some extent in the soluble fraction. It is likely that the GerD protein in the outer layers of dormant spores is located in the germ cell wall, as it was detected in coat-defective spores, and in the cell wall fraction of cells that were outgrowing from spores. Which of the multiple locations of GerD is important for its function is not known, but the inner membrane association would be appropriate for any interaction with germinant receptor proteins or SleB cortex lytic enzyme. Substitution of alanine for cysteine in the conserved cleavage site of the predicted prelipoprotein signal sequence of GerD resulted in mutant spores that lacked the GerD protein entirely.
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Single-copy chromosomal integration systems for Francisella tularensis
More LessFrancisella tularensis is a fastidious Gram-negative bacterium responsible for the zoonotic disease tularemia. Investigation of the biology and molecular pathogenesis of F. tularensis has been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of genetic tools. However, recent advances have substantially improved the ability of researchers to genetically manipulate this organism. To expand the molecular toolbox we have developed two systems to stably integrate genetic elements in single-copy into the F. tularensis genome. The first system is based upon the ability of transposon Tn7 to insert in both a site- and orientation-specific manner at high frequency into the attTn7 site located downstream of the highly conserved glmS gene. The second system consists of a sacB-based suicide plasmid used for allelic exchange of unmarked elements with the blaB gene, encoding a β-lactamase, resulting in the replacement of blaB with the element and the loss of ampicillin resistance. To test these new tools we used them to complement a novel d-glutamate auxotroph of F. tularensis LVS, created using an improved sacB-based allelic exchange plasmid. These new systems will be helpful for the genetic manipulation of F. tularensis in studies of tularemia biology, especially where the use of multi-copy plasmids or antibiotic markers may not be suitable.
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Characterization and recombinant expression of a divergent ice nucleation protein from ‘Pseudomonas borealis’
More LessIsolates of ‘Pseudomonas borealis’ were recovered after ice-affinity selection of summer-collected soils. ‘P. borealis’ DL7 was further characterized and shown to have ice nucleation activity (INA), a property that allows the crystallization of ice at temperatures close to the melting point, effectively preventing the supercooling of water. INA was optimally detected after culturing at temperatures consistent with psychrophilic growth. The sequence encoding the ‘P. borealis’ ice nucleation protein (INP) was obtained using both PCR and chromosome walking. When expressed in Escherichia coli, the resulting inaPb recombinants had INA. The ‘P. borealis’ sequence, dubbed inaPb, is clearly related to previously cloned INP genes, but it shows greater divergence. Sequence analysis suggests that there are two opposite flat surfaces, one relatively hydrophobic that likely serves as an ice template, and the other that could function as a complementary face to facilitate interprotein interaction for ice-step formation.
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A fixed-time diffusion analysis method determines that the three cheV genes of Helicobacter pylori differentially affect motility
Helicobacter pylori is a chemotactic bacterium that has three CheV proteins in its predicted chemotaxis signal transduction system. CheV proteins contain both CheW- and response-regulator-like domains. To determine the function of these proteins, we developed a fixed-time diffusion method that would quantify bacterial direction change without needing to define particular behaviours, to deal with the many behaviours that swimming H. pylori exhibit. We then analysed mutants that had each cheV gene deleted individually and found that the behaviour of each mutant differed substantially from wild-type and the other mutants. cheV1 and cheV2 mutants displayed smooth swimming behaviour, consistent with decreased cellular CheY-P, similar to a cheW mutant. In contrast, the cheV3 mutation had the opposite effect and the mutant cells appeared to change direction frequently. Additional analysis showed that the cheV mutants displayed aberrant behaviour as compared to the wild-type in the soft-agar chemotaxis assay. The soft-agar assay phenotype was less extreme compared to that seen in the fixed-time diffusion model, suggesting that the cheV mutants are able to partially compensate for their defects under some conditions. Each cheV mutant furthermore had defects in mouse colonization that ranged from severe to modest, consistent with a role in chemotaxis. These studies thus show that the H. pylori CheV proteins each differently affect swimming behaviour.
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Alkyl hydroperoxide reductase has a role in oxidative stress resistance and in modulating changes in cell-surface properties in Azospirillum brasilense Sp245
More LessAn ahpC mutant derivative of Azospirillum brasilense Sp245 (strain SK586) that encodes an alkyl hydroperoxide reductase was found to be more sensitive to oxidative stress caused by organic hydroperoxides compared with the wild-type. In addition, the ahpC mutant strain had multiple defects in a large array of cellular functions that were consistent with alteration of cell-surface properties, such as cell morphology in stationary phase, Calcofluor White-, Congo Red- and lectin-binding abilities, as well as cell-to-cell aggregation and flocculation. All phenotypes of the ahpC mutant were complemented by in trans expression of AhpC, and overexpression of AhpC in the wild-type strain was found to affect the same set of phenotypes, suggesting that the pleiotropic effects were caused by the ahpC mutation. SK586 was also found to be fully motile, but it lost motility at a higher rate than the wild-type during growth, such that most SK586 cells were non-motile in stationary phase. Despite these defects, the mutant did not differ from the wild-type in short-term colonization of sterile wheat roots when inoculated alone, and in competition with the wild-type strain; this implied that AhpC activity may not endow the cells with a competitive advantage in colonization under these conditions. Although the exact function of AhpC in affecting these phenotypes remains to be determined, changes in cell morphology, surface properties, cell-to-cell aggregation and flocculation are common adaptive responses to various stresses in bacteria, and the data obtained here suggest that AhpC contributes to modulating such stress responses in A. brasilense.
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- Genes And Genomes
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Genomic analysis of the PAI ICL3 locus in pathogenic LEE-negative Shiga toxin-producing Escherichia coli and Citrobacter rodentium
More LessShiga toxin-producing Escherichia coli (STEC) causes a spectrum of human illnesses such as haemorrhagic colitis and haemolytic–uraemic syndrome. Although the locus of enterocyte effacement (LEE) seems to confer enhanced virulence, LEE-negative STEC strains are also associated with severe human disease, suggesting that other unknown factors enhance the virulence potential of STEC strains. A novel hybrid pathogenicity island, termed PAI ICL3, has been previously characterized in the LEE-negative O113 : H21 STEC strain CL3. Screening for the presence of PAI ICL3 elements in 469 strains of E. coli, including attaching and effacing (A/E) pathogens [enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC)], non-A/E pathogens [LEE-negative STEC, extra-intestinal pathogenic E. coli (ExPEC), enterotoxigenic E. coli (ETEC) and enteroaggregative E. coli (EAEC)] and commensal E. coli isolates, showed that PAI ICL3 is unique to LEE-negative STEC strains linked to disease, providing a new marker for these strains. We also showed that a PAI ICL3-equivalent gene cluster is present in the genome of Citrobacter rodentium, on a 53 kb genomic island inserted into the pheV tRNA locus. While the C. rodentium PAI ICL3 shows high similarities at the nucleotide level and in organization with the E. coli PAI ICL3, the genetic context of the integration differs completely. In addition, blast searches revealed that other E. coli pathotypes (O157 : H7 EHEC, ExPEC, EPEC and EAEC) possess incomplete PAI ICL3 elements that contain only the genes located at the extremities of the island. Six of the 16 sequenced E. coli genomes showed deleted PAI ICL3 gene clusters which are carried on mobile genetic elements inserted into pheV, selC or serW tRNA loci, which is compatible with the idea that the PAI ICL3 gene cluster entered E. coli and C. rodentium at multiple times through independent events. The phylogenetic distribution of the PAI ICL3 variants suggests that a B1 genetic background is necessary for the maintenance of the full complement of PAI ICL3 genes in E. coli.
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Global transcriptional response to vancomycin in Mycobacterium tuberculosis
More LessIn order to gain additional understanding of the physiological mechanisms used by bacteria to maintain surface homeostasis and to identify potential targets for new antibacterial drugs, we analysed the variation of the Mycobacterium tuberculosis transcriptional profile in response to inhibitory and subinhibitory concentrations of vancomycin. Our analysis identified 153 genes differentially regulated after exposing bacteria to a concentration of the drug ten times higher than the MIC, and 141 genes differentially expressed when bacteria were growing in a concentration of the drug eightfold lower than the MIC. Hierarchical clustering analysis indicated that the response to these different conditions is different, although with some overlap. This approach allowed us to identify several genes whose products could be involved in the protection from antibiotic stress targeting the envelope and help to confer the basal level of M. tuberculosis resistance to antibacterial drugs, such as Rv2623 (UspA-like), Rv0116c, PE20-PPE31, PspA and proteins related to toxin–antitoxin systems. Moreover, we also demonstrated that the alternative sigma factor σ E confers basal resistance to vancomycin, once again underlining its importance in the physiology of the mycobacterial surface stress response.
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Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression
More LessRegulation of the Helicobacter pylori flagellar gene cascade involves the transcription factors σ 54 (RpoN), employed for expression of genes required midway through flagellar assembly, and σ 28 (FliA), required for expression of late genes. Previous studies revealed that mutations in genes encoding components of the flagellar protein export apparatus block expression of the H. pylori RpoN and FliA regulons. FlhB is a membrane-bound component of the export apparatus that possesses a large cytoplasmic domain (FlhBC). The hook length control protein FliK interacts with FlhBC to modulate the substrate specificity of the export apparatus. FlhBC undergoes autocleavage as part of the switch in substrate specificity. Consistent with previous reports, deletion of flhB in H. pylori interfered with expression of RpoN-dependent reporter genes, while deletion of fliK stimulated expression of these reporter genes. In the ΔflhB mutant, disrupting fliK did not restore expression of RpoN-dependent reporter genes, suggesting that the inhibitory effect of the ΔflhB mutation is not due to the inability to export FliK. Amino acid substitutions (N265A and P266G) at the putative autocleavage site of H. pylori FlhB prevented processing of FlhB and export of filament-type substrates. The FlhB variants supported wild-type expression of RpoN- and FliA-dependent reporter genes. In the strain producing FlhBN265A, expression of RpoN- and FliA-dependent reporter genes was inhibited when fliK was disrupted. In contrast, expression of these reporter genes was unaffected or slightly stimulated when fliK was disrupted in the strain producing FlhBP266G. H. pylori HP1575 (FlhX) shares homology with the C-terminal portion of FlhBC (FlhBCC) and can substitute for FlhBCC in flagellar assembly. Disrupting flhX inhibited expression of a flaB reporter gene in the wild-type but not in the ΔfliK mutant or strains producing FlhB variants, suggesting a role for FlhX or FlhBCC in normal expression of the RpoN regulon. Taken together, these data indicate that the mechanism by which the flagellar protein export apparatus exerts control over the H. pylori RpoN regulon is complex and involves more than simply switching substrate specificity of the flagellar protein export apparatus.
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Fis negatively affects binding of Tn4652 transposase by out-competing IHF from the left end of Tn4652
More LessTransposition activity in bacteria is generally maintained at a low level. The activity of mobile DNA elements can be controlled by bacterially encoded global regulators. Regulation of transposition of Tn4652 in Pseudomonas putida is one such example. Activation of transposition of Tn4652 in starving bacteria requires the stationary-phase sigma factor RpoS and integration host factor (IHF). IHF plays a dual role in Tn4652 translocation by activating transcription of the transposase gene tnpA of the transposon and facilitating TnpA binding to the inverted repeats of the transposon. Our previous results have indicated that besides IHF some other P. putida-encoded global regulator(s) might bind to the ends of Tn4652 and regulate transposition activity. In this study, employing a DNase I footprint assay we have identified a binding site of P. putida Fis (factor for inversion stimulation) centred 135 bp inside the left end of Tn4652. Our results of gel mobility shift and DNase I footprint studies revealed that Fis out-competes IHF from the left end of Tn4652, thereby abolishing the binding of TnpA. Thus, the results obtained in this study indicate that the transposition of Tn4652 is regulated by the cellular amount of P. putida global regulators Fis and IHF.
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Comparative analysis of Caulobacter chromosome replication origins
More LessCaulobacter crescentus (CB15) initiates chromosome replication only in stalked cells and not in swarmers. To better understand this dimorphic control of chromosome replication, we isolated replication origins (oris) from freshwater Caulobacter (FWC) and marine Caulobacter (MCS) species. Previous studies implicated integration host factor (IHF) and CcrM DNA methylation sites in replication control. However, ori IHF and CcrM sites identified in the model FWC CB15 were only conserved among closely related FWCs. DnaA boxes and CtrA binding sites are established CB15 ori components. CtrA is a two-component regulator that blocks chromosome replication selectively in CB15 swarmers. DnaA boxes and CtrA sites were found in five FWC and three MCS oris. Usually, a DnaA box and a CtrA site were paired, suggesting that CtrA binding regulates DnaA activity. We tested this hypothesis by site-directed mutagenesis of an MCS10 ori which contains only one CtrA binding site overlapping a critical DnaA box. This overlapping site is unique in the whole MCS10 genome. Selective DnaA box mutations decreased replication, while selective CtrA binding site mutations increased replication of MCS10 ori plasmids. Therefore, both FWC and MCS oris use CtrA to repress replication. Despite this similarity, phylogenetic analysis unexpectedly shows that CtrA usage evolved separately among these Caulobacter oris. We discuss consensus oris and convergent ori evolution in differentiating bacteria.
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Novel subfamily of mitochondrial HMG box-containing proteins: functional analysis of Gcf1p from Candida albicans
Mitochondria of eukaryotic organisms contain populations of DNA molecules that are packed into higher-order structures called mitochondrial nucleoids (mt-nucleoids). In Saccharomyces cerevisiae, the compaction of mitochondrial DNA (mtDNA) into mt-nucleoids is mediated primarily by the high-mobility group (HMG) box-containing protein Abf2, which is an important player in stabilization and metabolism of mtDNA. Although it is evident that analogous proteins must exist in other yeast species, an apparently fast divergence rate has precluded their identification, characterization and comparative analysis. Using in silico analysis of the complete genome sequence of the pathogenic yeast Candida albicans we predicted that the ORF 19.400/19.8030 assigned as GCF1 encodes a putative mitochondrial HMG box-containing protein. In contrast to Abf2p, which contains two HMG boxes, Gcf1p contains only one C-terminal HMG box. In addition, it contains one putative coiled-coil domain with a potential role in protein dimerization. Fluorescence microscopy analysis of a C-terminally tagged Gcf1p with green fluorescent protein (GFP) revealed its mitochondrial localization in both heterologous (S. cerevisiae) and native (C. albicans) hosts. Biochemical analyses of DNA-binding properties indicate that Gcf1p is, similarly to Abf2p, a non-specific DNA-binding protein. To analyse the role of Gcf1p in mtDNA metabolism, we constructed strains lacking one functional allele of the GCF1 gene and carrying one GCF1 allele under the control of the MET3 promoter. Under repressible conditions this strain exhibited a more than 3000-fold decrease in levels of GCF1 mRNA, which was correlated with a substantial decrease in the number of mtDNA copies as well as recombination intermediates. The dramatic effect of reduced levels of Gcf1p on mtDNA metabolism indicates that the protein is involved in essential molecular transactions that relate to the mitochondrial genome.
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Identification of a site-specific tyrosine recombinase that mediates promoter inversions of phase-variable mpl lipoprotein genes in Mycoplasma penetrans
More LessMycoplasma penetrans has the ability to change its surface lipoprotein profiles frequently. The P35 family lipoproteins encoded by the mpl genes are key players in this profile variation. The M. penetrans HF-2 genome has 38 mpl genes that form three gene clusters. Most of these mpl genes have an invertible promoter sequence that is responsible for the ON/OFF switching of individual mpl gene expression. Here, we identified the recombinase that catalyses inversions of the mpl gene promoters. We focused on two open reading frames of the M. penetrans HF-2 genome, namely MYPE2900 and MYPE8180, which show significant homology to the tyrosine site-specific recombinase (Tsr) family proteins. Since genetic tools for M. penetrans are still not developed, we cloned the MYPE2900 and MYPE8180 genes and expressed them in Mycoplasma pneumoniae and Escherichia coli. The promoter regions of the mpl genes [p35 (MYPE6810) or p42 (MYPE6630) genes] were also introduced into M. pneumoniae and E. coli cells expressing MYPE2900 or MYPE8180. Inversion of these promoters occurred in the presence of the MYPE2900 gene but not in the presence of the MYPE8180 gene, indicating that the MYPE2900 gene product is the recombinase that catalyses mpl gene promoter inversions. We used a PCR-based method to detect mpl promoter inversion. This method also enabled us to detect inversions of 10 mpl gene promoters in M. penetrans HF-2 cells. All these promoter inversions occurred at the 12 bp inverted repeat (IR) sequences flanking the promoter sequence. The consensus sequence of these IRs was proposed as TAAYNNNDATTA (Y=C or T; D=A, G or T).
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Hierarchical control of virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR
More LessTwo regulatory genes encoding a Streptomyces antibiotic regulatory protein (vmsS) and a response regulator (vmsT) of a bacterial two-component signal transduction system are present in the left-hand region of the biosynthetic gene cluster of the antibiotic virginiamycin, which is composed of virginiamycin M (VM) and virginiamycin S (VS), in Streptomyces virginiae. Disruption of vmsS abolished both VM and VS biosynthesis, with drastic alteration of the transcriptional profile for virginiamycin biosynthetic genes, whereas disruption of vmsT resulted in only a loss of VM biosynthesis, suggesting that vmsS is a pathway-specific regulator for both VM and VS biosynthesis, and that vmsT is a pathway-specific regulator for VM biosynthesis alone. Gene expression profiles determined by semiquantitative RT-PCR on the virginiamycin biosynthetic gene cluster demonstrated that vmsS controls the biosynthetic genes for VM and VS, and vmsT controls unidentified gene(s) of VM biosynthesis located outside the biosynthetic gene cluster. In addition, transcriptional analysis of a deletion mutant of vmsR located in the clustered regulatory region in the virginiamycin cluster (and which also acts as a SARP-family activator for both VM and VS biosynthesis) indicated that the expression of vmsS and vmsT is under the control of vmsR, and vmsR also contributes to the expression of VM and VS biosynthetic genes, independent of vmsS and vmsT. Therefore, coordinated virginiamycin biosynthesis is controlled by three pathway-specific regulators which hierarchically control the expression of the biosynthetic gene cluster.
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- Microbial Pathogenicity
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Delayed association of the NADPH oxidase complex with macrophage vacuoles containing the opportunistic pathogen Burkholderia cenocepacia
More LessBurkholderia cenocepacia causes chronic lung infections in patients suffering from cystic fibrosis and chronic granulomatous disease. We have previously shown that B. cenocepacia survives intracellularly in macrophages within a membrane vacuole (BcCV) that delays acidification. Here, we report that after macrophage infection with live B. cenocepacia there is a ∼6 h delay in the association of NADPH oxidase with BcCVs, while heat-inactivated bacteria are normally trafficked into NADPH oxidase-positive vacuoles. BcCVs in macrophages treated with a functional inhibitor of the cystic fibrosis transmembrane conductance regulator exhibited a further delay in the assembly of the NADPH oxidase complex at the BcCV membrane, but the inhibitor did not affect NADPH oxidase complex assembly onto vacuoles containing heat-inactivated B. cenocepacia or live Escherichia coli. Macrophages produced less superoxide following B. cenocepacia infection as compared to heat-inactivated B. cenocepacia and E. coli controls. Reduced superoxide production was associated with delayed deposition of cerium perhydroxide precipitates around BcCVs of macrophages infected with live B. cenocepacia, as visualized by transmission electron microscopy. Together, our results demonstrate that intracellular B. cenocepacia resides in macrophage vacuoles displaying an altered recruitment of the NADPH oxidase complex at the phagosomal membrane. This phenomenon may contribute to preventing the efficient clearance of this opportunistic pathogen from the infected airways of susceptible patients.
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Lipotoxin F of Pseudomonas aeruginosa is an AlgU-dependent and alginate-independent outer membrane protein involved in resistance to oxidative stress and adhesion to A549 human lung epithelia
More LessChronic lung infection with P. aeruginosa and excessive neutrophil-associated inflammation are major causes of morbidity and mortality in patients with cystic fibrosis (CF). Overproduction of an exopolysaccharide known as alginate leads to the formation of mucoid biofilms that are resistant to antibiotics and host defences. Alginate overproduction or mucoidy is controlled by a stress-related ECF sigma factor AlgU/T. Mutation in the anti-sigma factor MucA is a known mechanism for conversion to mucoidy. Recently, we showed that inactivation of a kinase (KinB) in nonmucoid strain PAO1 results in overproduction of alginate. Here, we report the initial characterization of lipotoxin F (LptF, PA3692), an OmpA-like outer membrane protein that exhibited increased expression in the mucoid PAO1kinB mutant. The lipotoxin family of proteins has been previously shown to induce inflammation in lung epithelia, which may play a role in CF disease progression. Expression of LptF was observed to be AlgU-dependent and upregulated in CF isolates. Deletion of lptF from the kinB mutant had no effect on alginate production. Deletion of lptF from PAO1 caused a differential susceptibility to oxidants that can be generated by phagocytes. The lptF and algU mutants were more sensitive to hypochlorite than PAO1. However, the lptF mutant displayed increased resistance to hydrogen peroxide. LptF also contributed to adhesion to A549 human lung epithelial cells. Our data suggest that LptF is an outer membrane protein that may be important for P. aeruginosa survival in harsh environments, including lung colonization in CF.
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Inactivation of the hmgA gene of Pseudomonas aeruginosa leads to pyomelanin hyperproduction, stress resistance and increased persistence in chronic lung infection
Clinical isolates of Pseudomonas aeruginosa that hyperproduce a dark-brown pigment are quite often found in the lungs of chronically infected patients, suggesting that they may have an adaptive advantage in chronic infections. We have screened a library of random transposon insertions in P. aeruginosa. Transposon insertions resulting in the hyperproduction of a dark-brown pigment were found to be located in the hmgA gene, which putatively encodes the enzyme homogentisate-1,2-dioxygenase. Complementation studies indicate that hmgA disruption is responsible for the hyperproduction of pyomelanin in both laboratory and clinical isolates. A relationship between hmgA disruption and adaptation to chronic infection was explored and our results show that the inactivation of hmgA produces a slight reduction of killing ability in an acute murine model of lung infection. On the other hand, it also confers decreased clearance and increased persistence in chronic lung infections. Whether pyomelanin production is the cause of the increased adaptation to chronicity or just a side effect of hmgA inactivation is a question to be studied in future; however, this adaptation is consistent with the higher resistance to oxidative stress conferred in vitro by the pyomelanin pigment. Our results clearly demonstrate that hmgA inactivation leads to a better adaptation to chronic infection, and strongly suggest that this mechanism may be exploited in naturally occurring P. aeruginosa strains.
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Loss of mannosylphosphate from Candida albicans cell wall proteins results in enhanced resistance to the inhibitory effect of a cationic antimicrobial peptide via reduced peptide binding to the cell surface
More LessThe outermost layer of the Candida albicans cell wall is enriched with mannosylated glycoproteins. We have used a range of isogenic glycosylation mutants of C. albicans, which are defective to varying degrees in cell wall protein mannosylation, to investigate the role of the outermost layer of the yeast cell wall in mediating the fungicidal action of the cationic, α-helical antimicrobial peptide dermaseptin S3(1-16) [DsS3(1-16)]. The degree of phosphomannan loss, and concomitant reduction in surface negative charge, from the series of glycosylation mutants correlated with reduced levels of peptide binding to the cells. In turn, the reduced peptide binding correlated with enhanced resistance to DsS3(1-16). To ascertain whether DsS3(1-16) binds to negatively charged phosphate, we studied the effect of exogenous glucosamine 6-phosphate, and glucosamine hydrochloride as a negative control, on the antifungal efficacy of DsS3(1-16). Glucosamine 6-phosphate retarded the efficacy of DsS3(1-16), and this was attributed to the presence of phosphate, because addition of identical concentrations of glucosamine hydrochloride had little detrimental effect on peptide efficacy. Fluorescence microscopy with DsS3(1-16) tagged with fluorescein revealed that the peptide binds to the outer surface of the yeast cell, supporting our previous conclusion that the presence of exterior phosphomannan is a major determinant of the antifungal potency of DsS3(1-16). The binding of the peptide to the cell surface was a transient event that was followed by apparent localization of DsS3(1-16) in the vacuole or dissemination throughout the entire cytosol. The presence of glucosamine 6-phosphate clearly reduced the proportion of cells in the population that showed complete cytosolic staining, implying that the binding and entry of the peptide into the cytosol is significantly reduced due to the exogenous phosphate sequestering the peptide and reducing the amount of peptide able to bind to the surface phosphomannan. In conclusion, we present evidence that an antimicrobial peptide, similar to those employed by cells of the human immune system, has evolved to recognize molecular patterns on the surface of pathogens in order to maximize efficacy.
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Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC
Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31–137 of EseC are required for EseC-EscA interaction. Mutation of EseC residues 31–137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31–137 of EseC increased the LD50 by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.
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Activation of the eis gene in a W-Beijing strain of Mycobacterium tuberculosis correlates with increased SigA levels and enhanced intracellular growth
There is growing evidence that strains of Mycobacterium tuberculosis differ in pathogenicity and transmissibility, but little is understood about the contributory factors. We have previously shown that increased expression of the principal sigma factor, SigA, mediates the capacity of M. tuberculosis strain 210 to grow more rapidly in human monocytes, compared with other strains. Strain 210 is part of the widespread W-Beijing family of M. tuberculosis strains and includes clinical isolate TB294. To identify genes that respond to changes in SigA levels and that might enhance intracellular growth, we examined RNA and protein expression patterns in TB294-pSigA, a recombinant strain of TB294 that overexpresses sigA from a multicopy plasmid. Lysates from broth-grown cultures of TB294-pSigA contained high levels of Eis, a protein known to modulate host–pathogen interactions. DNA microarray analysis indicated that the eis gene, Rv2416c, was expressed at levels in TB294-pSigA 40-fold higher than in the vector control strain TB294-pCV, during growth in the human monocyte cell line MonoMac6. Other genes with elevated expression in TB294-pSigA showed much smaller changes from TB294-pCV, and the majority of genes with expression differences between the two strains had reduced expression in TB294-pSigA, including an unexpected number of genes associated with the DNA-damage response. Real-time PCR analyses confirmed that eis was expressed at very high levels in TB294-pSigA in monocytes as well as in broth culture, and further revealed that, like sigA, eis was also more highly expressed in wild-type TB294 than in the laboratory strain H37Rv, during growth in monocytes. These findings suggested an association between increased SigA levels and eis activation, and results of chromatin immunoprecipitation confirmed that SigA binds the eis promoter in live TB294 cells. Deletion of eis reduced growth of TB294 in monocytes, and complementation of eis reversed this effect. We conclude that SigA regulates eis, that there is a direct correlation between upregulation of SigA and high expression levels of eis, and that eis contributes to the enhanced capacity of a clinical isolate of M. tuberculosis strain 210 to grow in monocytes.
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Lipopolysaccharide biosynthesis-related genes are required for colony pigmentation of Porphyromonas gingivalis
More LessThe periodontopathic bacterium Porphyromonas gingivalis forms pigmented colonies when incubated on blood agar plates as a result of accumulation of μ-oxo haem dimer on the cell surface. Gingipain–adhesin complexes are responsible for production of μ-oxo haem dimer from haemoglobin. Non-pigmented mutants (Tn6-5, Tn7-1, Tn7-3 and Tn10-4) were isolated from P. gingivalis by Tn4351 transposon mutagenesis [Hoover & Yoshimura (1994), FEMS Microbiol Lett 124, 43–48]. In this study, we found that the Tn6-5, Tn7-1 and Tn7-3 mutants carried Tn4351 DNA in a gene homologous to the ugdA gene encoding UDP-glucose 6-dehydrogenase, a gene encoding a putative group 1 family glycosyltransferase and a gene homologous to the rfa gene encoding ADP heptose-LPS heptosyltransferase, respectively. The Tn10-4 mutant carried Tn4351 DNA at the same position as that for Tn7-1. Gingipain activities associated with cells of the Tn7-3 mutant (rfa) were very weak, whereas gingipain activities were detected in the culture supernatants. Immunoblot and mass spectrometry analyses also revealed that gingipains, including their precursor forms, were present in the culture supernatants. A lipopolysaccharide (LPS) fraction of the rfa deletion mutant did not show the ladder pattern that was usually seen for the LPS of the wild-type P. gingivalis. A recombinant chimera gingipain was able to bind to an LPS fraction of the wild-type P. gingivalis in a dose-dependent manner. These results suggest that the rfa gene product is associated with biosynthesis of LPS and/or cell-surface polysaccharides that can function as an anchorage for gingipain–adhesin complexes.
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- Physiology And Biochemistry
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Dormant forms of Mycobacterium smegmatis with distinct morphology
Cultivation of Mycobacterium smegmatis cells in a nitrogen-limited minimal medium (SR-1) followed by prolonged storage at room temperature without shaking resulted in the gradual accumulation of morphologically distinct ovoid forms characterized by (i) low metabolic activity; (ii) elevated resistance to antibiotics and to heat treatment; and (iii) inability to produce colonies on standard agar plates (non-platable cells). Detailed microscopic examination confirmed that ovoid cells possessed an intact cell envelope, specific fine structure and large electron-transparent bodies in the cytoplasm. Cell staining with Nile red and analysis of the lipid content by TLC revealed the presence of significant amounts of apolar lipids in these bodies. The ovoid forms could be stored for significant periods (up to 5 months) and resuscitated afterwards in a modified Sauton's medium. Importantly, resuscitation of ovoid cells was accompanied by their transformation into the typical rod-shaped cells. We suggest that the observed ovoid cells represent dormant forms, resembling morphologically distinct cells of Mycobacterium tuberculosis previously isolated from tuberculosis patients and infected animals.
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Insights into the physiology of Methylotenera mobilis as revealed by metagenome-based shotgun proteomic analysis
While the shotgun proteomics approach is gaining momentum in understanding microbial physiology, it remains limited by the paucity of high-quality genomic data, especially when it comes to poorly characterized newly identified phyla. At the same time, large-scale metagenomic sequencing projects produce datasets representing genomes of a variety of environmental microbes, although with lower sequence coverage and sequence quality. In this work we tested the utility of a metagenomic dataset enriched in sequences of environmental strains of Methylotenera mobilis, to assess the protein profile of a laboratory-cultivated strain, M. mobilis JLW8, as a proof of principle. We demonstrate that a large portion of the proteome predicted from the metagenomic sequence (approx. 20 %) could be identified with high confidence (three or more peptide sequences), thus gaining insights into the physiology of this bacterium, which represents a new genus within the family Methylophilaceae.
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Ferric reductase A is essential for effective iron acquisition in Paracoccus denitrificans
More LessBased on N-terminal sequences obtained from the purified cytoplasmic ferric reductases FerA and FerB, their corresponding genes were identified in the published genome sequence of Paracoccus denitrificans Pd1222. The ferA and ferB genes were cloned and individually inactivated by insertion of a kanamycin resistance marker, and then returned to P. denitrificans for exchange with their wild-type copies. The resulting ferA and ferB mutant strains showed normal growth in brain heart infusion broth. Unlike the ferB mutant, the strain lacking FerA did not grow on succinate minimal medium with ferric 2,3-dihydroxybenzoate as the iron source, and grew only poorly in the presence of ferric sulfate, chloride, citrate, NTA, EDTA and EGTA. Moreover, the ferA mutant strain was unable to produce catechols, which are normally detectable in supernatants from iron-limited wild-type cultures. Complementation of the ferA mutation using a derivative of the conjugative broad-host-range plasmid pEG400 that contained the whole ferA gene and its putative promoter region largely restored the wild-type phenotype. Partial, though significant, restoration could also be achieved with 1 mM chorismate added to the growth medium. The purified FerA protein acted as an NADH : FMN oxidoreductase and catalysed the FMN-mediated reductive release of iron from the ferric complex of parabactin, the major catecholate siderophore of P. denitrificans. The deduced amino acid sequence of the FerA protein has closest similarity to flavin reductases that form part of the flavin-dependent two-component monooxygenases. Taken together, our results demonstrate an essential role of reduced flavins in the utilization of exogenous ferric iron. These flavins not only provide the electrons for Fe(III) reduction but most probably also affect the rate of siderophore production.
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Ferrous iron oxidation and rusticyanin in halotolerant, acidophilic ‘Thiobacillus prosperus’
More LessThe halotolerant acidophile ‘Thiobacillus prosperus’ was shown to require chloride for growth. With ferrous iron as substrate, growth occurred at a rate similar to that of the well-studied acidophile Acidithiobacillus ferrooxidans. Previously, the salt (NaCl) requirement of ‘T. prosperus’ was not clear and its growth on ferrous iron was described as poor. A subtractive hybridization of cDNAs from ferrous-iron-grown and sulfur-grown ‘T. prosperus’ strain V6 led to identification of a cluster of genes similar to the rus operon reported to encode ferrous iron oxidation in A. ferrooxidans. However, the ‘T. prosperus’ gene cluster did not contain a homologue of cyc1, which is thought to encode a key cytochrome c in the pathway of electron transport from ferrous iron in A. ferrooxidans. Rusticyanin, another key protein in ferrous iron oxidation by A. ferrooxidans, was present in ‘T. prosperus’ at similar concentrations in cells grown on either ferrous iron or sulfur.
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Solar disinfection (SODIS) and subsequent dark storage of Salmonella typhimurium and Shigella flexneri monitored by flow cytometry
More LessPathogenic enteric bacteria are a major cause of drinking water related morbidity and mortality in developing countries. Solar disinfection (SODIS) is an effective means to fight this problem. In the present study, SODIS of two important enteric pathogens, Shigella flexneri and Salmonella typhimurium, was investigated with a variety of viability indicators including cellular ATP levels, efflux pump activity, glucose uptake ability, and polarization and integrity of the cytoplasmic membrane. The respiratory chain of enteric bacteria was identified to be a likely target of sunlight and UVA irradiation. Furthermore, during dark storage after irradiation, the physiological state of the bacterial cells continued to deteriorate even in the absence of irradiation: apparently the cells were unable to repair damage. This strongly suggests that for S. typhimurium and Sh. flexneri, a relatively small light dose is enough to irreversibly damage the cells and that storage of bottles after irradiation does not allow regrowth of inactivated bacterial cells. In addition, we show that light dose reciprocity is an important issue when using simulated sunlight. At high irradiation intensities (>700 W m−2) light dose reciprocity failed and resulted in an overestimation of the effect, whereas reciprocity applied well around natural sunlight intensity (<400 W m−2).
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Gene expression patterns and differential input into curli fimbriae regulation of all GGDEF/EAL domain proteins in Escherichia coli
Switching from the motile planktonic bacterial lifestyle to a biofilm existence is stimulated by the signalling molecule bis-(3′-5′)-cyclic-diguanosine monophosphate (cyclic-di-GMP), which is antagonistically controlled by diguanylate cyclases (DGCs; characterized by GGDEF domains) and specific phosphodiesterases (PDEs; mostly featuring EAL domains). Here, we present the expression patterns of all 28 genes that encode GGDEF/EAL domain proteins in Escherichia coli K-12. Twenty-one genes are expressed in Luria–Bertani medium, with 15 being under σ S control. While a small subset of GGDEF/EAL proteins (YeaJ and YhjH) is dominant and modulates motility in post-exponentially growing cells, a diverse battery of GGDEF/EAL proteins is deployed during entry into stationary phase, especially in cells grown at reduced temperature (28 °C). This suggests that multiple signal input into cyclic-di-GMP control is particularly important in growth-restricted cells in an extra-host environment. Six GGDEF/EAL genes differentially control the expression of adhesive curli fimbriae. Besides the previously described ydaM, yciR, yegE and yhjH genes, these are yhdA (csrD), which stimulates the expression of the DGC YdaM and the major curli regulator CsgD, and yeaP, which contributes to expression of the curli structural operon csgBAC. Finally, we discuss why other GGDEF/EAL domain-encoding genes, despite being expressed, do not influence motility and/or curli formation.
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The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis
Mycobacterium tuberculosis can utilize various nutrients including nitrate as a source of nitrogen. Assimilation of nitrate requires the reduction of nitrate via nitrite to ammonium, which is then incorporated into metabolic pathways. This study was undertaken to define the molecular mechanism of nitrate assimilation in M. tuberculosis. Homologues to a narGHJI-encoded nitrate reductase and a nirBD-encoded nitrite reductase have been found on the chromosome of M. tuberculosis. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a narG mutant of M. tuberculosis failed to grow on nitrate. A nirB mutant of M. tuberculosis failed to grow on both nitrate and nitrite. Mutant strains of Mycobacterium smegmatis mc2155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from M. tuberculosis, and a gene with homology to the response regulator gene glnR of Streptomyces coelicolor was identified. A ΔglnR mutant of M. tuberculosis was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when nirBD was expressed from a plasmid, suggesting a role of GlnR in regulating nirBD expression. A specific binding site for GlnR within the nirB promoter was identified and confirmed by electrophoretic mobility shift assay using purified recombinant GlnR. Semiquantitative reverse transcription PCR, as well as microarray analysis, demonstrated upregulation of nirBD expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of M. tuberculosis mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of nirBD.
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Energetic limits to metabolic flexibility: responses of Saccharomyces cerevisiae to glucose–galactose transitions
More LessGlucose is the favoured carbon source for Saccharomyces cerevisiae, and the Leloir pathway for galactose utilization is only induced in the presence of galactose during glucose-derepressed conditions. The goal of this study was to investigate the dynamics of glucose–galactose transitions. To this end, well-controlled, glucose-limited chemostat cultures were switched to galactose-excess conditions. Surprisingly, galactose was not consumed upon a switch to galactose excess under anaerobic conditions. However, the transcripts of the Leloir pathway were highly increased upon galactose excess under both aerobic and anaerobic conditions. Protein and enzyme-activity assays showed that impaired galactose consumption under anaerobiosis coincided with the absence of the Leloir-pathway proteins. Further results showed that absence of protein synthesis was not caused by glucose-mediated translation inhibition. Analysis of adenosine nucleotide pools revealed a fast decrease of the energy charge after the switch from glucose to galactose under anaerobic conditions. Similar results were obtained when glucose–galactose transitions were analysed under aerobic conditions with a respiratory-deficient strain. It is concluded that under fermentative conditions, the energy charge was too low to allow synthesis of the Leloir proteins. Hence, this study conclusively shows that the intracellular energy status is an important factor in the metabolic flexibility of S. cerevisiae upon changes in its environment.
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Relaxed control of sugar utilization in Lactobacillus brevis
More LessPrioritization of sugar consumption is a common theme in bacterial growth and a problem for complete utilization of five and six carbon sugars derived from lignocellulose. Growth studies show that Lactobacillus brevis simultaneously consumes numerous carbon sources and appears to lack normal hierarchical control of carbohydrate utilization. Analysis of several independent L. brevis isolates indicated that co-utilization of xylose and glucose is a common trait for this species. Moreover, carbohydrates that can be used as a single carbon source are simultaneously utilized with glucose. Analysis of the proteome of L. brevis cells grown on glucose, xylose or a glucose/xylose mixture revealed the constitutive expression of the enzymes of the heterofermentative pathway. In addition, fermentative mass balances between mixed sugar inputs and end-products indicated that both glucose and xylose are simultaneously metabolized through the heterofermentative pathway. Proteomic and mRNA analyses revealed that genes in the xyl operon were expressed in the cells grown on xylose or on glucose/xylose mixtures but not in those grown on glucose alone. However, the expression level of XylA and XylB proteins in cells grown on a glucose/xylose mixture was reduced 2.7-fold from that observed in cells grown solely on xylose. These results suggest that regulation of xylose utilization in L. brevis is not stringently controlled as seen in other lactic acid bacteria, where carbon catabolite repression operates to prioritize carbohydrate utilization more rigorously.
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Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum
More LessCorynebacterium glutamicum is a biotin-auxotrophic bacterium and some strains efficiently produce glutamic acid under biotin-limiting conditions. In an effort to understand C. glutamicum metabolism under biotin limitation, growth of the type strain ATCC 13032 was investigated in batch cultures and a time-course analysis was performed. A transient excretion of organic acids was observed and we focused our attention on lactate synthesis. Lactate synthesis was due to the ldh-encoded l-lactate dehydrogenase (Ldh). Features of Ldh activity and ldh transcription were analysed. The ldh gene was shown to be regulated at the transcriptional level by SugR, a pleiotropic transcriptional repressor also acting on most phosphotransferase system (PTS) genes. Electrophoretic mobility shift assays (EMSAs) and site-directed mutagenesis allowed the identification of the SugR-binding site. Effector studies using EMSAs and analysis of ldh expression in a ptsF mutant revealed fructose 1-phosphate as a highly efficient negative effector of SugR. Fructose 1,6-bisphosphate also affected SugR binding.
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