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Volume 157,
Issue 9,
2011
Volume 157, Issue 9, 2011
- Microbial Pathogenicity
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A Mig-14-like protein (PA5003) affects antimicrobial peptide recognition in Pseudomonas aeruginosa
More LessThe evolution of antibiotic resistance in pathogenic bacteria is a growing global health problem which is gradually making the treatment of infectious diseases less efficient. Antimicrobial peptides are small charged molecules found in organisms from the complete phylogenetic spectrum. The peptides are attractive candidates for novel drug development due to their activity against bacteria that are resistant to conventional antibiotics, and reports of peptide resistance are rare in the clinical setting. Paradoxically, many clinically relevant bacteria have mechanisms that can recognize and respond to the presence of cationic antimicrobial peptides (CAMPs) in the environment by changing the properties of the microbial surface thereby increasing the tolerance of the microbes towards the peptides. In Pseudomonas aeruginosa an essential component of this inducible tolerance mechanism is the lipopolysaccharide modification operon arnBCADTEF–PA3559 which encodes enzymes required for LPS alterations leading to increased antimicrobial peptide tolerance. The expression of the operon is induced by the presence of CAMPs in the environment but the molecular mechanisms underlying the cellular recognition of the peptides are poorly elucidated. In this work, we investigate the factors influencing arnB expression by transposon mutagenesis and arnB promoter green fluorescent protein reporters. We have identified a novel gene encoding a Mig-14-like protein that is required for recognition of the CAMPs colistin and Novispirin G10 by P. aeruginosa. Moreover, we show that this gene is also required for the formation of CAMP-tolerant subpopulations in P. aeruginosa hydrodynamic flow chamber biofilms.
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HXK1 regulates carbon catabolism, sporulation, fumonisin B1 production and pathogenesis in Fusarium verticillioides
More LessIn Fusarium verticillioides, a ubiquitous pathogen of maize, virulence and mycotoxigenesis are regulated in response to the types and amounts of carbohydrates present in maize kernels. In this study, we investigated the role of a putative hexokinase-encoding gene (HXK1) in growth, development and pathogenesis. A deletion mutant (Δhxk1) of HXK1 was not able to grow when supplied with fructose as the sole carbon source, and growth was impaired when glucose, sucrose or maltotriose was provided. Additionally, the Δhxk1 mutant produced unusual swollen hyphae when provided with fructose, but not glucose, as the sole carbon source. Moreover, the Δhxk1 mutant was impaired in fructose uptake, although glucose uptake was unaffected. On maize kernels, the Δhxk1 mutant was substantially less virulent than the wild-type, but virulence on maize stalks was not impaired, possibly indicating a metabolic response to tissue-specific differences in plant carbohydrate content. Finally, disruption of HXK1 had a pronounced effect on fungal metabolites produced during colonization of maize kernels; the Δhxk1 mutant produced approximately 50 % less trehalose and 80 % less fumonisin B1 (FB1) than the wild-type. The reduction in trehalose biosynthesis likely explains observations of increased sensitivity to osmotic stress in the Δhxk1 mutant. In summary, this study links early events in carbohydrate sensing and glycolysis to virulence and secondary metabolism in F. verticillioides, and thus provides a new foothold from which the genetic regulatory networks that underlie pathogenesis and mycotoxigenesis can be unravelled and defined.
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- Physiology And Biochemistry
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Characterization of the KstR-dependent promoter of the gene for the first step of the cholesterol degradative pathway in Mycobacterium smegmatis
More LessThe KstR-dependent promoter of the MSMEG_5228 gene of Mycobacterium smegmatis, which encodes the 3-β-hydroxysteroid dehydrogenase (3-β HSDMS) responsible for the first step in the cholesterol degradative pathway, has been characterized. Primer extension analysis of the P5228 promoter showed that the transcription starts at the ATG codon, thus generating a leaderless mRNA lacking a 5′ untranslated region (5′UTR). Footprint analyses demonstrated experimentally that KstR specifically binds to an operator region of 31 nt containing the quasi-palindromic sequence AACTGGAACGTGTTTCAGTT, located between the −5 and −35 positions with respect to the transcription start site. This region overlaps with the −10 and −35 boxes of the P5228 promoter, suggesting that KstR represses MSMEG_5228 transcription by preventing the binding of RNA polymerase. Using a P5228 –β-galactosidase fusion we have demonstrated that KstR is able to work as a repressor in a heterologous system like Escherichia coli. A 3D model of the KstR protein revealed folding typical of TetR-type regulators, with two domains, i.e. a DNA-binding N-terminal domain and a regulator-binding C-terminal domain composed of six helices with a long tunnel-shaped hydrophobic pocket that might interact with a putative highly hydrophobic inducer. The finding that similar P5228 promoter regions have been found in all mycobacterial strains examined, with the sole exception of Mycobacterium tuberculosis, provides new clues about the role of cholesterol in the pathogenicity of this micro-organism.
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The plant pathogen Streptomyces scabies 87-22 has a functional pyochelin biosynthetic pathway that is regulated by TetR- and AfsR-family proteins
Siderophores are high-affinity iron-chelating compounds produced by bacteria for iron uptake that can act as important virulence determinants for both plant and animal pathogens. Genome sequencing of the plant pathogen Streptomyces scabies 87-22 revealed the presence of a putative pyochelin biosynthetic gene cluster (PBGC). Liquid chromatography (LC)-MS analyses of culture supernatants of S. scabies mutants, in which expression of the cluster is upregulated and which lack a key biosynthetic gene from the cluster, indicated that pyochelin is a product of the PBGC. LC-MS comparisons with authentic standards on a homochiral stationary phase confirmed that pyochelin and not enantio-pyochelin (ent-pyochelin) is produced by S. scabies. Transcription of the S. scabies PBGC occurs via ~19 kb and ~3 kb operons and transcription of the ~19 kb operon is regulated by TetR- and AfsR-family proteins encoded by the cluster. This is the first report, to our knowledge, of pyochelin production by a Gram-positive bacterium; interestingly regulation of pyochelin production is distinct from characterized PBGCs in Gram-negative bacteria. Though pyochelin-mediated iron acquisition by Pseudomonas aeruginosa is important for virulence, in planta bioassays failed to demonstrate that pyochelin production by S. scabies is required for development of disease symptoms on excised potato tuber tissue or radish seedlings.
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JmjN interacts with JmjC to ensure selective proteolysis of Gis1 by the proteasome
More LessA group of JmjC domain-containing proteins also harbour JmjN domains. Although the JmjC domain is known to possess histone demethylase activity, the function of the JmjN domain remains largely undetermined. Previously, we have demonstrated that the yeast Gis1 transcription factor, bearing both JmjN and JmjC domains at its N terminus, is subject to proteasome-mediated selective proteolysis to downregulate its transcription activation ability. Here, we reveal that the JmjN and JmjC domains interact with each other through two β-sheets, one in each domain. Removal of either or both β-strands or the entire JmjN domain leads to complete degradation of Gis1, mediated partially by the proteasome. Mutating the core residues essential for histone demethylase activity demonstrated for other JmjC-containing proteins or deleting both Jumonji domains enhances the transcription activity of Gis1, but has no impact on its selective proteolysis by the proteasome. Together, these data suggest that JmjN and JmjC interact physically to form a structural unit that ensures the stability and appropriate transcription activity of Gis1.
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PBP5, PBP6 and DacD play different roles in intrinsic β-lactam resistance of Escherichia coli
More LessEscherichia coli PBP5, PBP6 and DacD, encoded by dacA, dacC and dacD, respectively, share substantial amino acid identity and together constitute ~50 % of the total penicillin-binding proteins of E. coli. PBP5 helps maintain intrinsic β-lactam resistance within the cell. To test if PBP6 and DacD play simlar roles, we deleted dacC and dacD individually, and dacC in combination with dacA, from E. coli 2443 and compared β-lactam sensitivity of the mutants and the parent strain. β-Lactam resistance was complemented by wild-type, but not dd-carboxypeptidase-deficient PBP5, confirming that enzymic activity of PBP5 is essential for β-lactam resistance. Deletion of dacC and expression of PBP6 during exponential or stationary phase did not alter β-lactam resistance of a dacA mutant. Expression of DacD during mid-exponential phase partially restored β-lactam resistance of the dacA mutant. Therefore, PBP5 dd-carboxypeptidase activity is essential for intrinsic β-lactam resistance of E. coli and DacD can partially compensate for PBP5 in this capacity, whereas PBP6 cannot.
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Disruption of the alsSD operon of Enterococcus faecalis impairs growth on pyruvate at low pH
More LessDiacetyl and acetoin are pyruvate-derived metabolites excreted by many micro-organisms, and are important in their physiology. Although generation of these four-carbon (C4) compounds in Enterococcus faecalis is a well-documented phenotype, little is known about the gene regulation of their biosynthetic pathway and the physiological role of the pathway in this bacterium. In this work, we identified the genes involved in C4 compound biosynthesis in Ent. faecalis and report their transcriptional analysis. These genes are part of the alsSD bicistronic operon, which encodes α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD). Our studies showed that alsSD operon transcription levels are maximal during the exponential phase of growth, decreasing thereafter. Furthermore, we found that this transcription is enhanced upon addition of pyruvate to the growth medium. In order to study the functional role of the alsSD operon, an isogenic alsSD mutant strain was constructed. This strain lost its capacity to generate C4 compounds, confirming the role of alsSD genes in this metabolic pathway. In contrast to the wild-type strain, the alsSD-deficient strain was unable to grow in LB medium supplemented with pyruvate at an initial pH of 4.5. This dramatic reduction in growth parameters for the mutant strain was simultaneously accompanied by the inability to alkalinize the internal and external medium under these conditions. In sum, these results suggest that the decarboxylation reactions related to the C4 biosynthetic pathway give enterococcal cells a competitive advantage during pyruvate metabolism at low pH.
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Oxygen reduction in the strict anaerobe Desulfovibrio vulgaris Hildenborough: characterization of two membrane-bound oxygen reductases
More LessAlthough Desulfovibrio vulgaris Hildenborough (DvH) is a strictly anaerobic bacterium, it is able to consume oxygen in different cellular compartments, including extensive periplasmic O2 reduction with hydrogen as electron donor. The genome of DvH revealed the presence of cydAB and cox genes, encoding a quinol oxidase bd and a cytochrome c oxidase, respectively. In the membranes of DvH, we detected both quinol oxygen reductase [inhibited by heptyl-hydroxyquinoline-N-oxide (HQNO)] and cytochrome c oxidase activities. Spectral and HPLC data for the membrane fraction revealed the presence of o-, b- and d-type haems, in addition to a majority of c-type haems, but no a-type haem, in agreement with carbon monoxide-binding analysis. The cytochrome c oxidase is thus of the cc(o/b)o 3 type, a type not previously described. The monohaem cytochrome c 553 is an electron donor to the cytochrome c oxidase; its encoding gene is located upstream of the cox operon and is 50-fold more transcribed than coxI encoding the cytochrome c oxidase subunit I. Even when DvH is grown under anaerobic conditions in lactate/sulfate medium, the two terminal oxidase-encoding genes are expressed. Furthermore, the quinol oxidase bd-encoding genes are more highly expressed than the cox genes. The cox operon exhibits an atypical genomic organization, with the gene coxII located downstream of coxIV. The occurrence of these membrane-bound oxygen reductases in other strictly anaerobic Deltaproteobacteria is discussed.
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