FEMS Microbiology Letters

Genest, Olivier; Méjean, Vincent; Iobbi-Nivol, Chantal

doi: 10.1111/j.1574-6968.2009.01660.xpmid: 19519768

AbstractBiogenesis of prokaryote molybdoenzymes is a complex process leading to the insertion of the molybdenum cofactor in the cytoplasm into a folded apoenzyme before transport through the cell membrane. Usually, specific chaperones belonging to the TorD family are required for the maturation of the molybdoenzymes of the dimethyl sulfoxide reductase family. These chaperones play a crucial role during the biogenesis and the incorporation of the molybdenum cofactor by interacting with the core of the apoprotein. Moreover, they are also involved in the protection of the apoproteins by binding to their N-terminal extremity in an early stage of synthesis. Finally, the TorD-like proteins may possess a proofreading activity and they could target their partners to the twin arginine translocation machinery system for cross-membrane transport of prefolded proteins. The roles of these chaperones during the different steps of molybdoenzyme biogenesis are described.

Gould, Simon W.J.; Chadwick, Maureen; Cuschieri, Paul; Easmon, Susan; Richardson, Anthony C.; Price, Robert G.; Fielder, Mark D.

doi: 10.1111/j.1574-6968.2009.01654.xpmid: 19558587

AbstractEight novel chromogenic substrates were evaluated for their efficacy in detecting lipase activity in clinical isolates of Staphylococcus aureus from the United Kingdom and Malta. All isolates metabolized the chromogenic lipase substrates 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothia-zolidin-4-one-3-ethanoic acid (SRA)-propionate, SRA-butyrate, SRA-octanoate and 2-[2-(4-hydroxy-3,5-dimethoxyphenyl)-vinyl]-3-methy-benzothiazolium salt (SBZTM)-acetate. Over 90% of the isolates metabolized the lipase substrates SRA-decanoate and SRA-laurate. However, only 0.6% of UK isolates and 2% of Maltese isolates metabolized the lipase substrate SRA-myristate; none of the isolates tested metabolized SBZTM-butyrate. Traditional Tween 80 assays showed that over 73% of the UK methicillin-resistant Staphylococcus aureus (MRSA) isolates and 83% of the UK methicillin-sensitive Staphylococcus aureus (MSSA) isolates demonstrated lipolytic activity. In contrast, Maltese isolates showed lipase activity in 94% and 88% of the MRSA and MSSA strains, respectively. Lipases in MRSA and MSSA demonstrated substrate specificity whose activity appeared dependent upon hydrocarbon chain length of the chromogen. These novel chromogens can be used for lipase enzyme detection and have application for full characterization of numerous S. aureus lipases.

Liu, Juan; Chen, Wen-Li

doi: 10.1111/j.1574-6968.2009.01644.xpmid: 19558588

AbstractHetN, a putative ketoacyl reductase, is required for heterocyst pattern maintenance in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. The hetN gene, when present on a multicopy plasmid, is able to suppress heterocyst differentiation. Little is known about the biochemical properties of HetN. In the present study, we found that HetN could hydrolyze ATP or GTP in vitro, and that this activity was dependent on the presence of magnesium in the reaction mixture. Mutations of the conserved active Ser142–Tyr155–Lys159 triad, predicted as necessary for the reductase activity of HetN, had only weak effect on the hydrolysis of ATP. The residue Lys159 is shown to be necessary for the heterocyst-suppressing activity of HetN, as the corresponding mutant allele present on a replicative plasmid failed to block heterocyst differentiation in contrast to the wild type. This result suggests that the reductase activity of HetN is involved in the HetN-mediated inhibition of heterocyst formation.

Hartley, Amanda J.; De Mattos-Shipley, Kate; Collins, Catherine M.; Kilaru, Sreedhar; Foster, Gary D.; Bailey, Andy M.

doi: 10.1111/j.1574-6968.2009.01656.xpmid: 19527297

AbstractPleuromutilin is a broad-spectrum antibiotic that has been used in veterinary medicine for over 20 years, but is now gaining interest as a human therapeutic. The compound is a fungal secondary metabolite, but there is some degree of confusion within the literature concerning which species may produce pleuromutilin, with several differently named fungi reported to make the compound. Here, we describe a taxonomic survey of publicly available cultures known to produce pleuromutilin, and other similar species. The pleuromutilin production of these strains was assessed and a phylogenetic assessment was carried out based on the sequence of the nuclear rRNA internal transcribed spacer region. Eleven strains were confirmed as being pleuromutilin producers and all of these isolates appear to fall within a discrete clade of the genus Clitopilus. The phylogenetic analysis also highlights the need for a revision of the taxonomic status of these fungi.

Miyashita, Ai; Mochimaru, Hanako; Kazama, Hiromi; Ohashi, Akiyoshi; Yamaguchi, Takashi; Nunoura, Takuro; Horikoshi, Koki; Takai, Ken; Imachi, Hiroyuki

doi: 10.1111/j.1574-6968.2009.01648.xpmid: 19486160

AbstractUncultured archaeal anaerobic methanotrophs (ANMEs) are known to operate the anaerobic oxidation of methane process, an important sink for the greenhouse gas methane in natural environments. In this study, we designed 16S rRNA gene-specific primers for each of the phylogenetic groups of ANMEs (ANME-1, Guaymas Basin hydrothermal sediment clones group within the ANME-1, ANME-2a, ANME-2b, ANME-2c and ANME-3) based on previously reported sequences. The newly designed primers were used for the detection of the various groups of ANMEs in the sulphate-limited anaerobic environmental samples, i.e. methanogenic sludges, rice field soils, lotus field sediments and natural gas fields. The ANME 16S rRNA gene sequences were detected only in a natural gas field sample among the environments examined in this study and were of the ANME-1 and -2c groups. In addition, the quantitative real-time PCR analysis using the designed primers showed that abundances of ANME-1 and -2c were estimated to be <0.02% of the total prokaryotic 16S rRNA gene community. The newly designed ANME group-specific primers in this study may be useful to survey the distribution and quantitative determination of ANMEs.

Lee, Yunho; Oh, Sejong; Park, Woojun

doi: 10.1111/j.1574-6968.2009.01650.xpmid: 19500143

AbstractTo identify genes essential to biofilm formation in Pseudomonas putida KT2440, 12 mutants defective in oxidative stress-related or metabolic pathway-related genes were evaluated. Of them, only the dsbA mutant lacking the disulfide bond isomerase exhibited significantly increased attachment to the polystyrene surface. Visual evaluation by extracellular matrix staining and scanning electron microscopy indicated that the KT2440-ΔdsbA strain displays enhanced extracellular matrix production, rugose colony morphology on agar plates and floating pellicles in static culture. Accordingly, we propose that deletion of the dsbA gene may stimulate production of the extracellular matrix, resulting in those phenotypes. In addition, the lack of detectable fluorescence in the KT2440-ΔdsbA under UV light as well as in both the wild type and the KT2440-ΔdsbA when grown on Luria–Bertani plates containing ferrous iron suggests that the fluorescent molecule may be a fluorescent siderophore with its synthesis/secretion controlled by DsbA in KT2440. These phenotypic defects observed in the dsbA mutant were complemented by the full-length KT2440 and Escherichia coli dsbA genes. In contrast to the role of DsbA in other bacteria, our results provide the first evidence that disruption of P. putida KT2440 dsbA gene overproduces the extracellular matrix and thus promotes biofilm formation.

Sukhi, Shibani S.; Shashidhar, Ravindranath; Kumar, Sanjukta A.; Bandekar, Jayant R.

doi: 10.1111/j.1574-6968.2009.01652.xpmid: 19490129

AbstractDeinococcus species exhibit an extraordinary ability to withstand ionizing radiation (IR). Most of the studies on radiation resistance have been carried out with exponential phase cells. The studies on radiation resistance of Deinococcus radiodurans R1 with respect to different phases of growth showed that late stationary phase cells of D. radiodurans R1 were fourfold more sensitive to IR and heat as compared with exponential or early stationary phase cells. The increased sensitivity of D. radiodurans R1 to IR in the late stationary phase was not due to a decrease in the intracellular Mn/Fe ratio or an increase in the level of oxidative protein damage. The resistance to IR was restored when late stationary phase cells were incubated for 15 min in fresh medium before irradiation, indicating that replenishment of exhausted nutrients restored the metabolic capability of the cells to repair DNA damage. These observations suggest that stress tolerance mechanisms in D. radiodurans R1 differ from established paradigms.

Gu, Ganyu; Wang, Nian; Chaney, Noel; Smith, Leif; Lu, Shi-En

doi: 10.1111/j.1574-6968.2009.01653.xpmid: 19500142

AbstractBurkholderia contaminans strain MS14 has a broad range of antifungal activities to plant and human pathogens. In previous studies, a 22.7-kb genomic fragment harboring six genes was shown to be involved in the production of an antifungal oligopeptide in B. contaminans strain MS14. In this study, another LuxR-type regulatory gene, named ambR1, was identified downstream of the ambR2 gene, and three new ORFs were found upstream of ORF6 of the 22.7-kb fragment. Site-directed mutagenesis revealed that ambR1 was required for expression of the antifungal activity against the indicator fungus Geotrichum candidum. Transcription of all the putative genes (ORFs 2–9) identified in the region except ORF1 was regulated by both ambR1 and ambR2. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44(ambR1∷nptII). Two of the three ORFs identified upstream from the ORF6 were predicted to encode two nonribosomal peptide synthetases (ORF7 and ORF9), and an insertion mutation in ORF9 resulted in the loss of antifungal activity against G. candidum. These results suggest that ambR1 is the key regulatory gene controlling the production of the antifungal activity of strain MS14.

Beck, Hans Christian; Madsen, Søren M.; Glenting, Jacob; Petersen, Jørgen; Israelsen, Hans; Nørrelykke, Mette Rindom; Antonsson, Martin; Hansen, Anne Maria

doi: 10.1111/j.1574-6968.2009.01662.xpmid: 19527296

AbstractIn the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Gel bands were excised and in-gel digested with trypsin. The resulting peptides were analysed by capillary-LC-ESI-MS/MS. The peptide sequences were used for a database search and allowed identification of a total of 29 proteins, many of which could potentially be involved in the action of probiotics in the gastrointestinal tract. The results provide the basis for future studies on the molecular mechanisms of probiotics.

Zampieri, Elisa; Mello, Antonietta; Bonfante, Paola; Murat, Claude

doi: 10.1111/j.1574-6968.2009.01655.xpmid: 19519770

AbstractTruffles are hypogeous Ascomycete fungi belonging to the genus Tuber and forming fruiting bodies highly prized for their taste and aroma. The identification of the genus Tuber and its species is important to investigate their ecology and avoid fraud in the food market. As genus-specific primers are not available, the aims of this work were (1) to assess the usefulness of the β-tubulin gene as a DNA barcoding region for designing Tuber genus-specific primers, (2) to test the primers on a range of fruiting bodies, representing a large part of truffle biodiversity and (3) to check their ecological usefulness, applying them to truffle-ground soil. The new primers designed on the β-tubulin gene were specific to the Tuber genus in nested PCR. When applied to DNA from soils, they gave a positive signal for 23 of 32 soils. Phylogenetic analysis confirmed that the bands corresponded to Tuber and that at least five Tuber species were present in the truffle-ground. β-tubulin was found to be a good barcoding region for designing Tuber genus-specific primers, detecting a high Tuber diversity in a natural environment. These primers will be useful for understanding truffle ecology and for practical needs in plantation management.