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Archives of Microbiology (v.184, #6)
Effect of SsrA (tmRNA) tagging system on translational regulation in Streptomyces by Sandrine Braud; Celine Lavire; Audrey Bellier; Philippe Mazodier (pp. 343-352).
ssrA genes encoding tmRNA with transfer and messenger RNA functions are ubiquitous in bacteria. In a process called trans-translation, tmRNA enters a stalled ribosome and allows release of the original mRNA, then tmRNA becomes the template for translation of a short tag that signals for proteolytic degradation. We provide here the first evidences that the tmRNA tagging system (ssrA and cohort smpB) is active in Streptomyces. Transcription of the genes was shown and construction of a genetic probe allowed detection of a tmRNA-tagged peptide. Obtention of ssrA and smpB mutants of Streptomyces lividans showed that the ssrA system is dispensable in Streptomyces. Morphologies of the mutants colonies were similar to the wild type, thus tmRNA-mediated tagging does not seem to have, under conditions used, a significant effect in the Streptomyces differentiation.
Keywords: Streptomyces ; Differentiation; ssrA ; tmRNA; Rare codon; bldA ; Protease
Genotype versus phenotype in the circumscription of bacterial species: the case of Pseudomonas stutzeri and Pseudomonas chloritidismutans by Aina Maria Cladera; Elena García-Valdés; Jorge Lalucat (pp. 353-361).
The phenotypic characteristic of strain AW-1T of Pseudomonas chloritidismutans that is most relevant from the taxonomic point of view appears to be the capacity of growth under anaerobic conditions using chlorate as electron acceptor. This property is not restricted to this species only within the genus Pseudomonas, since it is also present in strains of genomovars 1 or 5, and 3 of Pseudomonas stutzeri. P. chloritidismutans has been described as a non-denitrifying species, but the isolation of variants that are able to grow anaerobically in the presence of nitrate is possible after subcultivation under selective conditions. The subdivision of P. stutzeri into a number of species on the basis of these characteristics does not help to clarify the phylogenetic relationships among the members of an otherwise coherent group of strains, and the considerations presented in this communication support the reclassification of the new species name P. chloritidismutans, which in our opinion, should be considered as a Junior name of P. stutzeri. A multilocus sequence analysis, together with a phenotypic analysis of the anaerobic oxidative metabolism, gives new insights into the phylogeny and evolution of the species.
Keywords: P . stutzeri ; P. chloritidismutans ; Denitrification; Multilocus sequence analysis (MLSA)
Analysing the outer membrane subproteome of Methylococcus capsulatus (Bath) using proteomics and novel biocomputing tools by Frode S. Berven; Odd André Karlsen; Anne Hege Straume; Kristian Flikka; J. Colin Murrell; Anne Fjellbirkeland; Johan R. Lillehaug; Ingvar Eidhammer; Harald B. Jensen (pp. 362-377).
High-resolution two-dimensional gel electrophoresis and mass spectrometry has been used to identify the outer membrane (OM) subproteome of the Gram-negative bacterium Methylococcus capsulatus (Bath). Twenty-eight unique polypeptide sequences were identified from protein samples enriched in OMs. Only six of these polypeptides had previously been identified. The predictions from novel bioinformatic methods predicting β-barrel outer membrane proteins (OMPs) and OM lipoproteins were compared to proteins identified experimentally. BOMP ( http://www.bioinfo.no/tools/bomp ) predicted 43 β-barrel OMPs (1.45%) from the 2,959 annotated open reading frames. This was a lower percentage than predicted from other Gram-negative proteomes (1.8–3%). More than half of the predicted BOMPs in M. capsulatus were annotated as (conserved) hypothetical proteins with significant similarity to very few sequences in Swiss-Prot or TrEMBL. The experimental data and the computer predictions indicated that the protein composition of the M. capsulatus OM subproteome was different from that of other Gram-negative bacteria studied in a similar manner. A new program, Lipo, was developed that can analyse entire predicted proteomes and give a list of recognised lipoproteins categorised according to their lipo-box similarity to known Gram-negative lipoproteins ( http://www.bioinfo.no/tools/lipo ). This report is the first using a proteomics and bioinformatics approach to identify the OM subproteome of an obligate methanotroph.
Keywords: Outer membrane proteins; Subproteomics; Methylococcus capsulatus (Bath); Genomic mining; Two-dimensional gel electrophoresis
Analysis of a non-canonical NtcA-dependent promoter in Synechococcus elongatus and its regulation by NtcA and PII by M. Fadi Aldehni; Karl Forchhammer (pp. 378-386).
In this communication, we present a genetic analysis of the glnN promoter region of Synechococcus elongatus PCC 7942. luxAB reporter fusions were used to characterize the glnN promoter by deletion and site-directed mutational analysis. Reporter gene expression analysis was performed in S. elongatus wild-type and mutant strains to reveal the role of the global nitrogen responsive transcription factor NtcA and of the PII signalling protein on regulation of glnN gene expression. A non-canonical NtcA-binding motif is responsible for NtcA-dependent, nitrogen-responsive regulation of glnN. The PII signalling protein has opposing effects on NtcA-dependent glnN expression. Under conditions of nitrate-growth, it depresses expression, whereas under conditions of combined nitrogen starvation, it is required for full induction. Furthermore, sequences upstream of the NtcA-binding site have repressive effect on glnN promoter activity.
Keywords: Cyanobacteria; GlnB; GlnN; Glutamine synthetase; Nitrogen regulation; NtcA; 2-oxoglutarate
Myxococcus xanthus twin-arginine translocation system is important for growth and development by Yoshio Kimura; Hiroyuki Saiga; Hiroko Hamanaka; Hideki Matoba (pp. 387-396).
The twin-arginine translocation (Tat) system serves to export fully folded proteins across the cytoplasmic membrane. In many bacteria, three major components, TatA, TatB and TatC, are the functionally essential constituents of the Tat system. A Myxococcus xanthus tatB–tatC deletion mutant could aggregate and form mounds, but was unable to form fruiting bodies under nutritionally limiting conditions. When tatB–tatC mutant vegetative cells were cultured with 0.5 M glycerol, the cell morphology changed to spore-like spherical cells, but the spores were not resistant to heat and sonication treatments. In contrast to the wild-type strain, the tatB–tatC mutant also showed a decreased cell growth rate and a lower maximum cell concentration. These results suggest possibility that the Tat system may contribute to export of various important proteins for development and growth for M. xanthus.
Structural modeling and environmental regulation of arginine decarboxylase in Synechocystis sp. PCC 6803 by Saowarath Jantaro; Heidi Kidron; Delphine Chesnel; Aran Incharoensakdi; Paula Mulo; Tiina Salminen; Pirkko Mäenpää (pp. 397-406).
Arginine decarboxylase (ADC) is the first enzyme in the alternative route to putrescine in the polyamine biosynthesis pathway in bacteria and plants. In this study, we have focused on the effects of various types of short-term stresses on the transcript amount and specific activity of Synechocystis sp. PCC 6803 ADC. Our results reveal that the steady-state transcript accumulation and enzyme activity are not connected in a simple manner, since only photoheterotrophy and synergistic salt and high-light stress affected both parameters similarly. Changes in the steady-state ADC mRNA accumulation under the other short-term stress conditions studied had only a small impact on enzyme activity, suggesting post-translational regulation. Based on structural modeling, Synechocystis ADCs have a putative extra domain, which might be involved in the post-translational regulation of ADC activity in Synechocystis. In addition, two symmetric inter-subunit disulfide bonds seem to stabilize the dimeric structure of ADCs. There are two genes coding for ADC and agmatinase, another polyamine pathway enzyme, in Synechocystis genome, while the genes coding for ornithine decarboxylase and for some other enzymes in the polyamine pathway were not identified with homology searches.
Keywords: Arginine decarboxylase; Gene expression; Pyridoxal-5′-phosphate; Polyamines; Protein structure; Synechocystis
Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida by Anirban Banerjee; Praveen Kaul; U. C. Banerjee (pp. 407-418).
The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH4)2SO4 fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40°C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (k cat/K m) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of (R)-(−)-mandelic acid from readily available mandelonitrile.
Keywords: Arylacetonitrilase; Purification; Pseudomonas putida ; Organic solvent; Stability; Specificity constant
A site-directed integration system for the nonuniversal CUGSer codon usage species Pichia farinosa by electroporation by Xiaoxing Wang; Gang LI; Yuntao Deng; Xuanwei Yu; Fang Chen (pp. 419-424).
Halotolerant yeast, Pichia farinosa, is a valuable yeast strain in fermentation industry because it produces high yield of glycerol and xylitol, and can tolerate both contamination and high-density growth during fermentation. However, the lack of genetic manipulation tools makes it less popular as a gene engineering strain. Expression systems commonly used in other yeast systems, such as Saccharomyces cerevisiae and Pichia pastoris cannot be used in P. farinosa because it translates universal Leu codon CUG as Ser. Here we reported a modified expression vector and a transformation system with enhanced efficiency in P. farinosa. The results showed that cells of OD600 0.8–1.0 with DTT treatment can obtain high transformation efficiency. The optimized electroporation condition was 900 V, 25 μF, and 200 Ω. The DNA concentration did not influence the transformation. Our system provides the potential not only for applying P. farinosa as an industrial strain of gene engineering, but also for studying gene function in its native host.
Keywords: Pichia farinosa ; Electroporation; Integration system; Codon usage; Ura3 gene; Competent cells
Identification of a lipoarabinomannan-like lipoglycan in the actinomycete Gordonia bronchialis by Natalie J. Garton; Iain C. Sutcliffe (pp. 425-427).
The cell envelopes of actinomycetes contain lipidated macroamphiphiles, of which the most extensively characterised are the lipoarabinomannans of mycobacteria and related bacteria. We have investigated the mycolic acid-containing actinomycete Gordonia bronchialis and identified the presence of a lipoarabinomannan-like lipoglycan. The extraction and purification procedures recovered a second amphiphilic fraction with properties suggesting a phosphatidylinositol mannoside, consistent with studies of other Gordonia species.
Keywords: Actinomycete; Glycolipid; Lipoglycan; Lipoteichoic acid; Mycobacterium ; Phosphatidylinositol mannoside; Rhodococcus