- Volume 151, Issue 7, 2005
Volume 151, Issue 7, 2005
- Genes And Genomes
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The ‘yeast cell wall chip’ – a tool to analyse the regulation of cell wall biogenesis in Saccharomyces cerevisiae
Within the field of Saccharomyces cerevisiae functional genomics, DNA microarrays have become a very useful tool to study genome-wide gene-expression changes under diverse experimental conditions. Here, the design and production of a gene microarray, called the ‘yeast cell wall chip’, specifically tailored to investigate cell wall functions, is described. This array has been validated and shown to be useful to address gene involvement in the regulation of the response to cell wall damage in yeast. The advantages of this tailored gene microarray, which contains 390 genes, in terms of reproducibility, accuracy, versatility and ease of use are reported. Importantly, the microarray design permits the performance of a double hybridization process (two experiments) on the same slide. Cell wall stress leads to the transcriptional activation of a set of genes involved in cell wall remodelling. This response has been shown to be strongly controlled by the MAP kinase (MAPK) Slt2p, but other signalling pathways have also been suggested to be involved in this process. Here, using the tailored microarray, the role of the HOG1 pathway in the regulation of the transcriptional compensatory response to cell wall damage was evaluated by comparing the transcriptional profiles of a hog1 mutant and a wild-type strain in the presence of Congo red. Two genes, YFL014W (HSP12) and YLR414C, were found to be dependent on the Hog1p MAPK for their induction, indicating that an additional level of regulation of cell wall functions is mediated by this MAPK.
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Functional analysis of the lysis genes of Staphylococcus aureus phage P68 in Escherichia coli
More LessDouble-stranded DNA phages of both Gram-positive and Gram-negative bacteria typically use a holin–endolysin system to achieve lysis of their host. In this study, the lysis genes of Staphylococcus aureus phage P68 were characterized. P68 gene lys16 was shown to encode a cell-wall-degrading enzyme, which causes cell lysis when externally added to clinical isolates of S. aureus. Another gene, hol15, was identified embedded in the −1 reading frame at the 3′ end of lys16. The deduced Hol15 protein has three putative transmembrane domains, and thus resembles class I holins. An additional candidate holin gene, hol12, was found downstream of the endolysin gene lys16 based on two predicted transmembrane domains of the encoded protein, which is a typical trait of class II holins. The synthesis of either Hol12 or Hol15 resulted in growth retardation of Escherichia coli, and both hol15 and hol12 were able to complement a phage λ Sam mutation. The hol15 gene has a dual start motif beginning with the codons Met1-Lys2-Met3…. Evidence is presented that the hol15 gene encodes a lysis inhibitor (anti-holin) and a lysis effector (actual holin). As depolarization of the membrane converted the anti-holin to a functional holin, these studies suggested that hol15 functions as a typical dual start motif class I holin. The unusual arrangement of the P68 lysis genes is discussed.
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Acd, a peptidoglycan hydrolase of Clostridium difficile with N-acetylglucosaminidase activity
A gene encoding a putative peptidoglycan hydrolase was identified by sequence similarity searching in the Clostridium difficile 630 genome sequence, and the corresponding protein, named Acd (autolysin of C. difficile) was expressed in Escherichia coli. The deduced amino acid sequence of Acd shows a modular structure with two main domains: an N-terminal domain exhibiting repeated sequences and a C-terminal catalytic domain. The C-terminal domain exhibits sequence similarity with the glucosaminidase domains of Staphylococcus aureus Atl and Bacillus subtilis LytD autolysins. Purified recombinant Acd produced in E. coli was confirmed to be a cell-wall hydrolase with lytic activity on the peptidoglycan of several Gram-positive bacteria, including C. difficile. The hydrolytic specificity of Acd was studied by RP-HPLC analysis and MALDI-TOF MS using B. subtilis cell-wall extracts. Muropeptides generated by Acd hydrolysis demonstrated that Acd hydrolyses peptidoglycan bonds between N-acetylglucosamine and N-acetylmuramic acid, confirming that Acd is an N-acetylglucosaminidase. The transcription of the acd gene increased during vegetative cellular growth of C. difficile 630. The sequence of the acd gene appears highly conserved in C. difficile strains. Regarding deduced amino acid sequences, the C-terminal domain with enzymic function appears to be the most conserved of the two main domains. Acd is the first known autolysin involved in peptidoglycan hydrolysis of C. difficile.
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- Pathogens And Pathogenicity
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Glutathione-dependent extracellular ferric reductase activities in dimorphic zoopathogenic fungi
More LessIn this study, extracellular glutathione-dependent ferric reductase (GSH-FeR) activities in different dimorphic zoopathogenic fungal species were characterized. Supernatants from Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis and Sporothrix schenckii strains grown in their yeast form were able to reduce iron enzymically with glutathione as a cofactor. Some variations in the level of reduction were noted amongst the strains. This activity was stable in acidic, neutral and slightly alkaline environments and was inhibited when trivalent aluminium and gallium ions were present. Using zymography, single bands of GSH-FeRs with apparent molecular masses varying from 430 to 460 kDa were identified in all strains. The same molecular mass range was determined by size exclusion chromatography. These data demonstrate that dimorphic zoopathogenic fungi produce and secrete a family of similar GSH-FeRs that may be involved in the acquisition and utilization of iron. Siderophore production by these and other fungi has sometimes been considered to provide a full explanation of iron acquisition in these organisms. Our work reveals an additional common mechanism that may be biologically and pathogenically important. Furthermore, while some characteristics of these enzymes such as extracellular location, cofactor utilization and large size are not individually unique, when considered together and shared across a range of fungi, they represent an important novel physiological feature.
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Prc protease promotes mucoidy in mucA mutants of Pseudomonas aeruginosa
More LessMucoid strains of Pseudomonas aeruginosa that overproduce the exopolysaccharide alginate are a frequent cause of chronic respiratory infections in cystic fibrosis (CF) patients. The overproduction of alginate by these strains is often caused by mutations within mucA of the algU mucABCD gene cluster. This gene cluster encodes an extreme stress response system composed of the ECF alternative sigma factor AlgU, the anti-sigma factor MucA located in the inner membrane and the negative regulator MucB located in the periplasm. Most of the mutations in mucA found in mucoid strains cause a truncation of the C-terminal, periplasmic domain of MucA. The most significant effect of these mutations appears to be to reduce the levels of MucA. PA3257 (prc) was identified as a regulator of alginate production in P. aeruginosa through the isolation and study of mutations that partially suppressed the mucoid phenotype of a mucA22 strain. The suppressor of mucoidy (som) mutants isolated produced very little alginate when grown on LB medium, but were still mucoid when grown on Pseudomonas isolation agar. These som mutations and another previously isolated suppressor mutation were complemented by cosmids or plasmids carrying PA3257. PA3257 is predicted to encode a periplasmic protease similar to Prc or Tsp of Escherichia coli. Sequencing of prc from three strains with som suppressor mutations confirmed that each had a mutation within the prc coding region. The authors propose that Prc acts to degrade mutant forms of MucA. Additional evidence in support of this hypothesis is: (1) transcription from the AlgU-regulated algD reporter was reduced in som mutants; (2) inactivation of prc affected alginate production in mucoid strains with other mucA mutations found in CF isolates; (3) inactivation or overexpression of prc did not affect alginate production in strains with wild-type MucA.
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Effect of static growth and different levels of environmental oxygen on toxA and ptxR expression in the Pseudomonas aeruginosa strain PAO1
More LessWithin certain infection sites, such as the lung of cystic fibrosis patients, Pseudomonas aeruginosa grows statically under either decreased oxygen tension or anaerobic conditions, a situation that is likely to influence the production of virulence factors. The goal of this study was to determine the effect of static growth under microaerobic (decreased oxygen) and anaerobic conditions on the expression of the P. aeruginosa exotoxin A (ETA) gene toxA and its positive regulator ptxR. Using toxA–lacZ and ptxR–lacZ fusion plasmids, the level of toxA and ptxR expression was measured throughout the growth cycle of strain PAO1, which was grown in either iron-deficient or iron-sufficient medium under four different conditions: 20 %-SH (aerobic, shaking), 20 %-ST (aerobic, static), 10 %-ST (microaerobic, static) and 0 %-ST (anaerobic, static). In iron-deficient medium, toxA expression was higher under 20 %-ST and 10 %-ST than under 20 %-SH. However, the highest level of toxA expression occurred under 0 %-ST. Analysis of ETA protein using sandwich ELISA revealed that at time points between 8 and 24 h of the growth curve, PAO1 produced higher levels of ETA under 0 %-ST than under 20 %-SH. In iron-sufficient medium, toxA expression was significantly repressed under all conditions. Additional analyses using PAO1 strains that carry lacZ fusions with the toxA regulatory genes regA and pvdS revealed that the expression of regA and pvdS is reduced rather than increased at 0 %-ST. ptxR expression under different conditions paralleled that of toxA expression, except that it was repressed by iron under 20 %-SH only. Between 6 and 24 h of growth, and under all conditions, the level of dissolved oxygen (DO) within the PAO1 cultures was sharply reduced. These results suggest that (1) the combined effect of static growth and anaerobic conditions produce a significant increase in toxA and ptxR expression in PAO1; (2) this effect appears to be unique to toxA and ptxR, since the level of regA and pvdS expression was reduced under the same conditions; (3) neither static growth nor anaerobic conditions interfere with the repression of toxA expression by iron, although static growth deregulates ptxR expression with respect to iron; and (4) the enhanced expression of toxA and ptxR is not related to the reduced levels of DO in PAO1 cultures.
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Multiple genotypes of Chlamydia pneumoniae identified in human carotid plaque
More LessChlamydia pneumoniae is an obligate intracellular respiratory pathogen that causes 10 % of community-acquired pneumonia and has been associated with cardiovascular disease. Both whole-genome sequencing and specific gene typing suggest that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. The genotypes of C. pneumoniae present in human atherosclerotic carotid plaque were analysed and several polymorphisms in the variable domain 4 (VD4) region of the outer-membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD–urk) genes were found. While one genotype was identified that was the same as one reported previously in humans (respiratory and cardiovascular), another genotype was found that was identical to a genotype from non-human sources (frog/koala).
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DNA methylation in Yersinia enterocolitica: role of the DNA adenine methyltransferase in mismatch repair and regulation of virulence factors
More LessDNA adenine methyltransferase (Dam) plays an important role in physiological processes of Gram-negative bacteria such as mismatch repair and replication. In addition, Dam regulates the expression of virulence genes in various species. The authors cloned the dam gene of Yersinia enterocolitica and showed that Dam is essential for viability. Dam overproduction in Y. enterocolitica resulted in an increased frequency of spontaneous mutation and decreased resistance to 2-aminopurine; however, these effects were only marginal compared to the effect of overproduction of Escherichia coli-derived Dam in Y. enterocolitica, implying different roles or activities of Dam in mismatch repair of the two species. These differences in Dam function are not the cause for the essentiality of Dam in Y. enterocolitica, as Dam of E. coli can complement a dam defect in Y. enterocolitica. Instead, Dam seems to interfere with expression of essential genes. Furthermore, Dam mediates virulence of Y. enterocolitica. Dam overproduction results in increased tissue culture invasion of Y. enterocolitica, while the expression of specifically in vivo-expressed genes is not altered.
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Role of type III secretion in Edwardsiella tarda virulence
More LessEdwardsiella tarda is a Gram-negative enteric bacterium affecting both animals and humans. Recently, a type III secretion system (TTSS) was found in Ed. tarda. Such systems are generally used by bacterial pathogens to deliver virulence factors into host cells to subvert normal cell functions. Genome-walking was performed from the eseB and esrB genes (homologues of Salmonella sseB and ssrB, respectively) identified in previous studies, to determine the sequences of the TTSS. Thirty-five ORFs were identified which encode the TTSS apparatus, chaperones, effectors and regulators. Mutants affected in genes representing each category were generated and found to have decreased survival and growth in fish phagocytes. LD50 values of the mutants were increased by at least 10-fold in comparison to those of the wild-type strain. The adherence and invasion rates of the esrA and esrB mutants were enhanced while those of the other mutants remained similar to the wild-type. The eseC and eseD mutants showed slight autoaggregation in Dulbecco's Modified Eagle Medium, whereas the rest of the mutants failed to autoaggregate. Regulation of the TTSS was found to involve the two-component regulatory system esrA–esrB. This study showed that the TTSS is important for Ed. tarda pathogenesis. An understanding of this system will provide greater insight into the virulence mechanisms of this bacterial pathogen.
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Porins limit the intracellular persistence of Mycobacterium smegmatis
The genus Mycobacterium comprises highly pathogenic as well as opportunistic or apathogenic species exhibiting a great variability with respect to their ability to persist or multiply within monocytic host cells. The impact of the permeability of the mycobacterial outer membrane on intracellular persistence was studied. For this purpose, a Mycobacterium smegmatis mutant with a deletion of the major porin gene mspA and a second mutant lacking mspA and the homologous porin gene mspC were used. Deletion of mspA together with mspC significantly enhanced intracellular persistence in murine bone marrow macrophages, the mouse macrophage cell line J774A.1 and Acanthamoeba castellanii. Complementation of mspA in the porin mutant strains resulted in restoration of the wild-type phenotype with respect to intracellular persistence. This is the first report to show that the deletion of porins of mycobacteria results in improved persistence in eukaryotic cells, demonstrating that the intracellular persistence of M. smegmatis depends upon the permeability of the outer membrane.
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Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics
Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73 % of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-γ production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses.
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Campylobacter jejuni inhibits the absorptive transport functions of Caco-2 cells and disrupts cellular tight junctions
More LessCaco-2 cells are models of absorptive enterocytes. The net transport of fluid from apical to basolateral surfaces results in ‘domes' forming in differentiated monolayers. Here, the effect of Campylobacter jejuni on this process has been examined. C. jejuni caused no changes in short-circuit current upon infection of Caco-2 cell monolayers in Ussing chambers. Thus, no active secretory events could be demonstrated using this model. It was therefore hypothesized that C. jejuni could inhibit the absorptive function of enterocytes and that this may contribute to diarrhoeal disease. C. jejuni infection of fluid-transporting (‘doming’) Caco-2 cells resulted in a significant reduction in dome number, which correlated with a decrease in tight junction integrity in infected monolayers, when measured as transepithelial electrical resistance. Defined mutants of C. jejuni also reduced dome numbers in infected monolayers. C. jejuni also altered the distribution of the tight junction protein occludin within cell monolayers. The addition to monolayers of extracellular gentamicin prevented these changes, indicating the contribution of extracellular bacteria to this process. Thus, tight junction integrity is required for fluid transport in Caco-2 cell monolayers as leaky tight junctions cannot maintain support of transported fluid at the basolateral surface of infected cell monolayers. Inhibition of absorptive cell function, changes in epithelial resistance and rearrangement of tight junctional proteins such as occludin represent a potential diarrhoeal mechanism of C. jejuni.
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Atomic force microscopy study on specificity and non-specificity of interaction forces between Enterococcus faecalis cells with and without aggregation substance
Enterococcus faecalis is one of the leading causes of hospital-acquired infections, and indwelling medical devices are especially prone to infection. E. faecalis expressing aggregation substance (Agg) adheres to biomaterial surfaces by means of positive cooperativity, i.e. the ability of one adhering organism to stimulate adhesion of other organisms in its immediate vicinity. In this study, atomic force microscopy (AFM) was used to measure the specificity and non-specificity of interaction forces between E. faecalis cells with and without Agg. Bacteria were attached to a substratum surface and a tip-less cantilever. Two E. faecalis strains expressing different forms of Agg showed nearly twofold higher interaction forces between bacterial cells than a strain lacking Agg [adhesive force (F adh), −1·3 nN]. The strong interaction forces between the strains with Agg were reduced after adsorption of antibodies against Agg from −2·6 and −2·3 nN to −1·2 and −1·3 nN, respectively. This suggests that the non-specific interaction force between the enterococci amounts to approximately 1·2 nN, while the specific force component is only twofold stronger. Comparison of the results of the AFM interaction forces with the positive cooperativity after adhesion to a biomaterial in a parallel-plate flow chamber showed that in the absence of strong interaction forces between the cells, positive cooperativity was also absent. In conclusion, this is believed to be the first time that the influence of specific antibodies on interaction forces between E. faecalis cells has been demonstrated by AFM, thereby experimentally distinguishing between specific and non-specific force components.
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Bap-dependent biofilm formation by pathogenic species of Staphylococcus: evidence of horizontal gene transfer?
More LessThe biofilm-associated protein (Bap) is a surface protein implicated in biofilm formation by Staphylococcus aureus isolated from chronic mastitis infections. The bap gene is carried in a putative composite transposon inserted in SaPIbov2, a mobile staphylococcal pathogenicity island. In this study, bap orthologue genes from several staphylococcal species, including Staphylococcus epidermidis, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus simulans and Staphylococcus hyicus, were identified, cloned and sequenced. Sequence analysis comparison of the bap gene from these species revealed a very high sequence similarity, suggesting the horizontal gene transfer of SaPIbov2 amongst them. However, sequence analyses of the flanking region revealed that the bap gene of these species was not contained in the SaPIbov2 pathogenicity island. Although they did not contain the icaADBC operon, all the coagulase-negative staphylococcal isolates harbouring bap were strong biofilm producers. Disruption of the bap gene in S. epidermidis abolished its capacity to form a biofilm, whereas heterologous complementation of a biofilm-negative strain of S. aureus with the Bap protein from S. epidermidis bestowed the capacity to form a biofilm on a polystyrene surface. Altogether, these results demonstrate that Bap orthologues from coagulase-negative staphylococci induce an alternative mechanism of biofilm formation that is independent of the PIA/PNAG exopolysaccharide.
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A surface-exposed DraD protein of uropathogenic Escherichia coli bearing Dr fimbriae may be expressed and secreted independently from DraC usher and DraE adhesin
The dra gene cluster, expressed by uropathogenic Escherichia coli strains, determines bacterial attachment and invasion. The Dr fimbrial structures formed at the bacterial cell surface are composed of DraE subunits. The Dr fimbriae-coding cluster contains six open reading frames – draA, draB, draC, draD, draP and draE – among which the draE gene encodes the structural fimbrial subunit DraE. Very little is known about E. coli surface expression of the draD gene product. The expression of DraD and its role in the biogenesis of Dr fimbriae were determined by constructing mutants in the dra operon and by immunoblot and immunofluorescence experiments. In this study, DraD was found to be a surface-exposed protein. The expression of DraD was independent of the DraC usher and DraE fimbrial subunits. Polymerization of DraE fimbrial subunits into fimbrial structures did not require expression of the DraD protein.
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- Physiology
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CreA influences the metabolic fluxes of Aspergillus nidulans during growth on glucose and xylose
More LessThe physiological phenotype of Aspergillus nidulans was investigated for different genetic and environmental conditions of glucose repression through the quantification of in vivo fluxes in the central carbon metabolism using 13C-metabolic-flux analysis. The particular focus was the role of the carbon repressor CreA, which is the major regulatory protein mediating carbon repression in many fungal species, in the primary metabolism of A. nidulans. Batch cultivations were performed with a reference strain and a deletion mutant strain (creAΔ4) using [1-13C]glucose as carbon source. The mutant strain was also grown on a mixture of [1-13C]glucose and unlabelled xylose. Fractional enrichment data were measured by gas chromatography-mass spectrometry. A model describing the central metabolism of A. nidulans was used in combination with fractional enrichment data, and measurements of extracellular rates and biomass composition for the estimation of the in vivo metabolic fluxes. The creA-mutant strain showed a lower maximum specific growth rate than the reference strain when grown on glucose (0·11 and 0·25 h−1, respectively), whereas the specific growth rate of the mutant strain grown on the glucose/xylose mixture was identical to that on glucose (0·11 h−1). Different patterns and increased levels of extracellular polyols were observed both upon deletion of the creA gene and upon addition of xylose to the growth medium of the mutant strain. Concerning metabolic fluxes, the major change observed in the flux distribution of A. nidulans upon deletion of the creA gene was a 20 % decrease in the flux through the oxidative part of the pentose-phosphate pathway. Addition of xylose to the growth medium of the mutant resulted in an increase of about 40 % in the activity of the oxidative part of the pentose-phosphate pathway, as well as decreases in the fluxes through the Embden–Meyerhof–Parnas pathway and the tricarboxylic acid cycle (in the range of 20–30 %). The derepression of key pathways leads to alterations in the demands for cofactors, thereby imposing changes in the central metabolism due to the coupling of the many different reactions via the redox and energy metabolism of the cells.
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Utilization of geraniol is dependent on molybdenum in Pseudomonas aeruginosa: evidence for different metabolic routes for oxidation of geraniol and citronellol
More LessMini-Tn5-induced mutants with defects in utilization of linear terpenes such as citronellol, geraniol, citronellate and/or geranylate were isolated from Pseudomonas aeruginosa. One mutant was unable to utilize geraniol but showed wild-type growth with the three other acyclic terpenes tested. The Tn5 insertion site of the mutant was determined by DNA sequencing. Comparison with the P. aeruginosa genome sequence revealed that PA3028, an ORF with high similarity on the amino acid level to molybdenum cofactor biosynthesis protein A2 (encoded by moeA2), was the target of mini-Tn5 in the mutant. Disruption of moeA2 in P. aeruginosa PAO1 wild-type by insertion mutagenesis resulted in the same geraniol-minus phenotype. The ability to utilize geraniol was restored to the mutant by conjugative transfer of PCR-cloned wild-type moeA2 on a broad-host-range plasmid. Growth of P. aeruginosa PAO1 on geraniol and geranial, but not on citronellol, citronellate or geranylate, was inhibited by the presence of 10 mM tungstate, a molybdenum-specific inhibitor. Inhibition by tungstate was prevented by addition of molybdate. The results indicate that at least one step in the oxidation of geraniol to geranic acid (geranial oxidation) is a molybdenum-dependent reaction in P. aeruginosa and is different from the molybdenum-independent oxidation of citronellol to citronellate.
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Shifts from glucose to certain secondary carbon-sources result in activation of the extracytoplasmic function sigma factor σ E in Salmonella enterica serovar Typhimurium
More LessSalmonella enterica serovar Typhimurium (S. Typhimurium) elicits the starvation-stress response (SSR) due to starvation for an essential nutrient, e.g. a carbon/energy source (C-source). As part of the SSR, the alternative sigma factor σ E is activated and induced. The authors suspect that this activation is, in part, triggered by changes in the S. Typhimurium cell envelope occurring during the adaptation from growth to carbon/energy starvation (C-starvation), and resulting in an increased need for σ E-regulated factors involved in the proper folding and assembly of newly synthesized proteins destined for this extracytoplasmic compartment. This led to the hypothesis that a σ E activation signal might arise during C-source shifts that cause the induction of proteins localized to the extracytoplasmic compartment, i.e. the outer membrane or periplasm, of the cell. To test this hypothesis, cultures were grown in minimal medium containing enough glucose to reach mid-exponential-phase, plus a non-limiting amount of a secondary ‘less-preferred’ but utilizable carbon/energy source. The σ E activity was then monitored using plasmids carrying rpoEP1– and rpoEP2–lacZ transcriptional fusions, which exhibit σ E-independent and -dependent lacZ expression, respectively. The secondary C-sources maltose, succinate and citrate, which have extracytoplasmic components involved in their utilization (e.g. LamB), resulted in a discernible diauxic lag period and a sustained increase in σ E activity. Growth transition from glucose to other utilizable phosphotransferase (PTS) and non-PTS C-sources, such as trehalose, mannose, mannitol, fructose, glycerol, d-galactose or l-arabinose, did not cause a discernible diauxic lag period or a sustained increase in σ E activity. Interestingly, a shift from glucose to melibiose, which does not use an extracytoplasmic-localized protein for uptake, did cause an observable diauxic lag period but did not result in a sustained increase in σ E activity. In addition, overexpression of LamB from an arabinose-inducible promoter leads to a significant increase in σ E activity in the absence of a glucose to maltose shift or C-starvation. Furthermore, a ΔlamB : : Ω-Kmr mutant, lacking the LamB maltoporin, exhibited an approximately twofold reduction in the sustained σ E activity observed during a glucose to maltose shift, again supporting the hypothesis. Interestingly, the LamB protein lacks the typical Y-X-F terminal tripeptide of the OmpC-like peptides that activate DegS protease activity leading to σ E activation. It does, however, possess a terminal pentapeptide (Q-M-E-I-W-W) that may function as a ligand for a putative class II PDZ-binding site. The authors therefore propose that the σ E regulon of S. Typhimurium not only is induced in response to deleterious environmental conditions, but also plays a role in the adaptation of cells to new growth conditions that necessitate changes in the extracytoplasmic compartment of the cell, which may involve alternative signal recognition and activation pathways that are independent of DegS.
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Two functional FAS-I type fatty acid synthases in Corynebacterium glutamicum
The lipid-rich Corynebacterianeae, to which Corynebacterium glutamicum and Mycobacterium species belong, produce both fatty acids and mycolic acids. Compared with most other bacteria, C. glutamicum possesses two fatty acid synthases, encoded by fasA (8907 kb; FAS-IA) and fasB (8988 kb; FAS-IB). Here, it was shown by mutational analyses that fasA is essential but fasB is not. However, in a fasA background, the fasB mutation results in a slightly reduced growth yield, l-glutamate production is increased, and comparative lipid analysis suggests that in vivo FAS-IB is active primarily to supply palmitate. Transcript quantifications revealed that the fasB transcript contributes 32 % to both fas transcripts during growth on glucose, affirmative for fasB expression, and that fasB is subordinate to fasA. The fasA transcript is downregulated by 8·3-fold during growth on acetate as compared with glucose. The lipid analyses also demonstrate that cells grown on propionate produce a number of uneven fatty acids (e.g. 15 : 0, 17 : 0, 17 : 1), which are not present in cells grown on glucose or acetate, suggesting that fatty acid synthase in vivo may also use propionyl-CoA as the priming unit in fatty acid synthesis. The fatty acid auxotrophic fasAB double mutant was used to determine the suggested incorporation of fatty acids into mycolic acids. Supplementation of this mutant with uniformly labelled [13C]oleate and analysis of isolated mycolic acids confirmed that mature mycolic acids in the mutant consist exclusively of two fused [13C]oleate molecules. In addition to an altered phospholipid profile, the fasB mutant also exhibits differences in its mycolic acid profile. Taken together, the results show that although FAS-IA is the most relevant fatty acid synthase of C. glutamicum and FAS-IB is supplementary, both synthases are necessary to produce the characteristic lipid environment of this organism.
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Volumes and issues
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Volume 84 (1974)
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Volume 83 (1974)
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Volume 82 (1974)
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Volume 81 (1974)
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Volume 80 (1974)
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Volume 79 (1973)
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Volume 78 (1973)
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Volume 77 (1973)
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Volume 76 (1973)
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Volume 75 (1973)
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Volume 74 (1973)
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Volume 73 (1972)
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Volume 72 (1972)
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Volume 71 (1972)
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Volume 70 (1972)
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Volume 69 (1971)
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Volume 68 (1971)
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Volume 67 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)