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Volume 156,
Issue 5,
2010
Volume 156, Issue 5, 2010
- Microbial Pathogenicity
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Pseudomonas aeruginosa secreted factors impair biofilm development in Candida albicans
Signal-mediated interactions between the human opportunistic pathogens Pseudomonas aeruginosa and Candida albicans affect virulence traits in both organisms. Phenotypic studies revealed that bacterial supernatant from four P. aeruginosa strains strongly reduced the ability of C. albicans to form biofilms on silicone. This was largely a consequence of inhibition of biofilm maturation, a phenomenon also observed with supernatant prepared from non-clinical bacterial species. The effects of supernatant on biofilm formation were not mediated via interference with the yeast–hyphal morphological switch and occurred regardless of the level of homoserine lactone (HSL) produced, indicating that the effect is HSL-independent. A transcriptome analysis to dissect the effects of the P. aeruginosa supernatants on gene expression in the early stages of C. albicans biofilm formation identified 238 genes that exhibited reproducible changes in expression in response to all four supernatants. In particular, there was a strong increase in the expression of genes related to drug or toxin efflux and a decrease in expression of genes associated with adhesion and biofilm formation. Furthermore, expression of YWP1, which encodes a protein known to inhibit biofilm formation, was significantly increased. Biofilm formation is a key aspect of C. albicans infections, therefore the capacity of P. aeruginosa to antagonize this has clear biomedical implications.
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- Physiology And Biochemistry
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Molecular characterization of FinR, a novel redox-sensing transcriptional regulator in Pseudomonas putida KT2440
More LessFinR is required for the induction of fpr (ferredoxin-NADP+ reductase) under superoxide stress conditions in Pseudomonas putida. Many proteobacteria harbour FinR homologues in their genome as a putative LysR-type protein. Three cysteine residues (at positions 150, 239 and 289 in P. putida FinR) are conserved in all FinR homologues. When these conserved cysteines, along with two other cysteine residues present in FinR, were individually mutated to serines, the FinR remained active, unlike SoxR and OxyR in Escherichia coli. The results of our in vitro DNA-binding assay with cellular extracts showed that FinR binds directly to the fpr promoter region. In order to identify the FinR functional domain for sensing superoxide stress, we employed random and site-directed mutagenesis of FinR. Among 18 single amino acid mutants, three mutants (T39A, R194A and E225A) abolished fpr induction without any alteration of their DNA-binding ability, whereas other mutants also abrogated their DNA-binding abilities. Interestingly, two mutants (L215P and D51A) appeared to be constitutively active, regardless of superoxide stress conditions. Ferrous iron depletion, ferric iron addition and fdxA (ferredoxin) gene deletion also participate in the regulation of fpr. These data indicate that FinR has unusual residues for redox sensing and that the redox-sensing mechanism of FinR differs from the well-known mechanisms of OxyR and SoxR.
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Mycobacteriophage Ms6 LysB specifically targets the outer membrane of Mycobacterium smegmatis
LysB, a mycobacteriophage Ms6-encoded protein, was previously identified as a lipolytic enzyme able to hydrolyse the ester bond in lipase and esterase substrates. In the present work, we show that LysB can hydrolyse lipids containing mycolic acids from the outer membrane of the mycobacterial cell wall. LysB was shown to hydrolyse the mycolic acids from the mycolyl-arabinogalactan–peptidoglycan complex where the mycolates of the inner leaflet of the outer membrane are covalently attached to an arabinosyl head group. In addition, treatment of the extractable lipids from Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with LysB showed that trehalose 6,6′-dimycolate (TDM), a trehalose diester of two mycolic acid molecules, was hydrolysed by the enzyme. We have also determined the structures of the mycolic acid molecules that form the M. smegmatis TDM. The identification of a phage-encoded enzyme that targets the outer membrane of the mycobacterial cell wall enhances our understanding of the mechanism of mycobacteriophage lysis.
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A complex regulatory network controls aerobic ethanol oxidation in Pseudomonas aeruginosa: indication of four levels of sensor kinases and response regulators
More LessIn addition to the known response regulator ErbR (former AgmR) and the two-component regulatory system EraSR (former ExaDE), three additional regulatory proteins have been identified as being involved in controlling transcription of the aerobic ethanol oxidation system in Pseudomonas aeruginosa. Two putative sensor kinases, ErcS and ErcS′, and a response regulator, ErdR, were found, all of which show significant similarity to the two-component flhSR system that controls methanol and formaldehyde metabolism in Paracoccus denitrificans. All three identified response regulators, EraR (formerly ExaE), ErbR (formerly AgmR) and ErdR, are members of the luxR family. The three sensor kinases EraS (formerly ExaD), ErcS and ErcS′ do not contain a membrane domain. Apparently, they are localized in the cytoplasm and recognize cytoplasmic signals. Inactivation of gene ercS caused an extended lag phase on ethanol. Inactivation of both genes, ercS and ercS′, resulted in no growth at all on ethanol, as did inactivation of erdR. Of the three sensor kinases and three response regulators identified thus far, only the EraSR (formerly ExaDE) system forms a corresponding kinase/regulator pair. Using reporter gene constructs of all identified regulatory genes in different mutants allowed the hierarchy of a hypothetical complex regulatory network to be established. Probably, two additional sensor kinases and two additional response regulators, which are hidden among the numerous regulatory genes annotated in the genome of P. aeruginosa, remain to be identified.
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Effects of spontaneous mutations in PipX functions and regulatory complexes on the cyanobacterium Synechococcus elongatus strain PCC 7942
In Synechococcus elongatus sp. PCC 7942, PipX forms complexes with PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, and with the cyanobacterial nitrogen regulator NtcA. We recently showed that previous inactivation of pipX facilitates subsequent inactivation of the glnB gene. Here, we show that the three spontaneous pipX point mutations pipX-92delT, pipX160C>T and pipX194T>A, initially found in different glnB strains, are indeed suppressor mutations. When these mutations were reconstructed in the wild-type background, the glnB gene could be efficiently inactivated. Furthermore, the point mutations have different effects on PipX levels, coactivation of NtcA-dependent genes and protein–protein interactions. Further support for an in vivo role of PipX–PII complexes is provided by interaction analysis with the in vivo-generated PII T-loop+7 protein, a PII derivative unable to interact with its regulatory target N-acetyl-l-glutamate kinase, but which retains the ability to bind to PipX. The implications of these results are discussed.
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The enigmatic lack of glucose utilization in Streptomyces clavuligerus is due to inefficient expression of the glucose permease gene
More LessStreptomyces clavuligerus ATCC 27064 is unable to use glucose but has genes for a glucose permease (glcP) and a glucose kinase (glkA). Transformation of S. clavuligerus 27064 with the Streptomyces coelicolor glcP1 gene with its own promoter results in a strain able to grow on glucose. The glcP gene of S. clavuligerus encodes a 475 amino acid glucose permease with 12 transmembrane segments. GlcP is a functional protein when expressed from the S. coelicolor glcP1 promoter and complements two different glucose transport-negative Escherichia coli mutants. Transcription studies indicate that the glcP promoter is very weak and does not allow growth on glucose. These results suggest that S. clavuligerus initially contained a functional glucose permease gene, like most other Streptomyces species, and lost the expression of this gene by adaptation to glucose-poor habitats.
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Identification of two scyllo-inositol dehydrogenases in Bacillus subtilis
More Lessscyllo-Inositol (SI) is a stereoisomer of inositol whose catabolism has not been characterized in bacteria. We found that Bacillus subtilis 168 was able to grow using SI as its sole carbon source and that this growth was dependent on a functional iol operon for catabolism of myo-inositol (MI; another inositol isomer, which is abundant in nature). Previous studies elucidated the MI catabolic pathway in B. subtilis as comprising multiple stepwise reactions catalysed by a series of Iol enzymes. The first step of the pathway converts MI to scyllo-inosose (SIS) and involves the MI dehydrogenase IolG. Since IolG does not act on SI, we suspected that there could be another enzyme converting SI into SIS, namely an SI dehydrogenase. Within the whole genome, seven genes paralogous to iolG have been identified and two of these, iolX and iolW (formerly known as yisS and yvaA, respectively), were selected as candidate genes for the putative SI dehydrogenase since they were both prominently expressed when B. subtilis was grown on medium containing SI. iolX and iolW were cloned in Escherichia coli and both were shown to encode a functional enzyme, revealing the two distinct SI dehydrogenases in B. subtilis. Since inactivation of iolX impaired growth with SI as the carbon source, IolX was identified as a catabolic enzyme required for SI catabolism and it was shown to be NAD+ dependent. The physiological role of IolW remains unclear, but it may be capable of producing SI from SIS with NADPH oxidation.
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Isethionate formation from taurine in Chromohalobacter salexigens: purification of sulfoacetaldehyde reductase
More LessBacterial generation of isethionate (2-hydroxyethanesulfonate) from taurine (2-aminoethanesulfonate) by anaerobic gut bacteria was established in 1980. That phenomenon in pure culture was recognized as a pathway of assimilation of taurine-nitrogen. Based on the latter work, we predicted from genome-sequence data that the marine gammaproteobacterium Chromohalobacter salexigens DSM 3043 would exhibit this trait. Quantitative conversion of taurine to isethionate, identified by mass spectrometry, was confirmed, and the taurine-nitrogen was recovered as cell material. An eight-gene cluster was predicted to encode the inducible vectorial, scalar and regulatory enzymes involved, some of which were known from other taurine pathways. The genes (Csal_0153–Csal_0156) encoding a putative ATP-binding-cassette (ABC) transporter for taurine (TauAB1B2C) were shown to be inducibly transcribed by reverse transcription (RT-) PCR. An inducible taurine : 2-oxoglutarate aminotransferase [EC 2.6.1.55] was found (Csal_0158); the reaction yielded glutamate and sulfoacetaldehyde. The sulfoacetaldehyde was reduced to isethionate by NADPH-dependent sulfoacetaldehyde reductase (IsfD), a member of the short-chain alcohol dehydrogenase superfamily. The 27 kDa protein (SDS-PAGE) was identified by peptide-mass fingerprinting as the gene product of Csal_0161. The putative exporter of isethionate (IsfE) is encoded by Csal_0160; isfE was inducibly transcribed (RT-PCR). The presumed transcriptional regulator, TauR (Csal_0157), may autoregulate its own expression, typical of GntR-type regulators. Similar gene clusters were found in several marine and terrestrial gammaproteobacteria, which, in the gut canal, could be the source of not only mammalian, but also arachnid and cephalopod isethionate.
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2,3-Dihydroxypropane-1-sulfonate degraded by Cupriavidus pinatubonensis JMP134: purification of dihydroxypropanesulfonate 3-dehydrogenase
More Less2,3-Dihydroxypropane-1-sulfonate (DHPS) is a widespread intermediate in plant and algal transformations of sulfoquinovose (SQ) from the plant sulfolipid sulfoquinovosyl diacylglycerol. Further, DHPS is recovered quantitatively during bacterial degradation of SQ by Klebsiella sp. strain ABR11. DHPS is also a putative precursor of sulfolactate in e.g. Ruegeria pomeroyi DSS-3. A bioinformatic approach indicated that some 28 organisms with sequenced genomes might degrade DHPS inducibly via sulfolactate, with three different desulfonative enzymes involved in its degradation in different organisms. The hypothesis for Cupriavidus pinatubonensis JMP134 (formerly Ralstonia eutropha) involved a seven-gene cluster (Reut_C6093–C6087) comprising a LacI-type transcriptional regulator, HpsR, a major facilitator superfamily uptake system, HpsU, three NAD(P)+-coupled DHPS dehydrogenases, HpsNOP, and (R)-sulfolactate sulfo-lyase (SuyAB) [EC 4.4.1.24]. HpsOP effected a DHPS-racemase activity, and HpsN oxidized (R)-DHPS to (R)-sulfolactate. The hypothesis for Roseovarius nubinhibens ISM was similar, but involved a tripartite ATP-independent transport system for DHPS, HpsKLM, and two different desulfonative enzymes, (S)-cysteate sulfo-lyase [EC 4.4.1.25] and sulfoacetaldehyde acetyltransferase (Xsc) [EC 2.3.3.15]. Representative organisms were found to grow with DHPS and release sulfate. C. pinatubonensis JMP134 was found to express at least one NAD(P)+-coupled DHPS dehydrogenase inducibly, and three different peaks of activity were separated by anion-exchange chromatography. Protein bands (SDS-PAGE) were subjected to peptide-mass fingerprinting, which identified the corresponding genes (hpsNOP). Purified HpsN converted DHPS to sulfolactate. Reverse-transcription PCR confirmed that hpsNOUP were transcribed inducibly in strain JMP134, and that hpsKLM and hpsNOP were transcribed in strain ISM. DHPS degradation is widespread and diverse, implying that DHPS is common in marine and terrestrial environments.
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Novel insertion and deletion mutants of RpoB that render Mycobacterium smegmatis RNA polymerase resistant to rifampicin-mediated inhibition of transcription
The startling increase in the occurrence of rifampicin (Rif) resistance in the clinical isolates of Mycobacterium tuberculosis worldwide is posing a serious concern to tuberculosis management. The majority of Rif resistance in bacteria arises from mutations in the RpoB subunit of the RNA polymerase. We isolated M. smegmatis strains harbouring either an insertion (6 aa) or a deletion (10 aa) in their RpoB proteins. Although these strains showed a compromised fitness for growth in 7H9 Middlebrook medium, their resistance to Rif was remarkably high. The attenuated growth of the strains correlated with decreased specific activities of the RNA polymerases from the mutants. While the RNA polymerases from the parent or a mutant strain (harbouring a frequently occurring mutation, H442Y, in RpoB) were susceptible to Rif-mediated inhibition of transcription from calf thymus DNA, those from the insertion and deletion mutants were essentially refractory to such inhibition. Three-dimensional structure modelling revealed that the RpoB amino acids that interact with Rif are either deleted or unable to interact with Rif due to their unsuitable spatial positioning in these mutants. We discuss possible uses of the RpoB mutants in studying transcriptional regulation in mycobacteria and as potential targets for drug design.
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- Corrigendum
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