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Archives of Microbiology (v.194, #9)
Insights into the 1.59-Mbp largest plasmid of Azospirillum brasilense CBG497 by Erika Acosta-Cruz; Florence Wisniewski-Dyé; Zoé Rouy; Valérie Barbe; María Valdés; Patrick Mavingui (pp. 725-736).
The plant growth-promoting proteobacterium Azospirillum brasilense enhances growth of many economically important crops, such as wheat, maize, and rice. The sequencing and annotation of the 1.59-Mbp replicon of A. brasilense CBG497, a strain isolated from a maize rhizosphere grown on an alkaline soil in the northeast of Mexico, revealed a GC content of 68.7 % and the presence of 1,430 potential protein-encoding genes, 1,147 of them classified into clusters of orthologous groups categories, and 16 tRNA genes representing 11 tRNA species. The presence of sixty-two genes representatives of the minimal gene set and chromid core genes suggests its importance in bacterial survival. The phaAB → G operon, reported as involved in the bacterial adaptation to alkaline pH in the presence of K+, was also found on this replicon and detected in several Azospirillum strains. Phylogenetic analysis suggests that it was laterally acquired. We were not able to show its inference on the adaptation to basic pH, giving a hint about the presence of an alternative system for adaptation to alkaline pH.
Keywords: Azospirillum ; Proteobacteria; Plasmid; Essential genes; Phylogenetic analysis
Interactions of sulfur oxidation repressor with its promoters involve different binding geometries by Sukhendu Mandal; Sujoy K. Das Gupta (pp. 737-747).
The divergently transcribed sulfur oxidation (sox) operon of a sulfur chemolithotrophs, Pseudaminobacter salicylatoxidans KCT001, comprising sox TRS-VW-XYZABCD, is regulated by a repressor (SoxR). SoxR binds to two disparate operators, sv (present in between soxS and soxV) and wx (present in between soxW and soxX). Here we report details of the interaction between SoxR and these two operator regions of the sox operon, using methylation interference and hydroxyl radical footprinting. We propose that the sv operator is symmetric and compact, while the wx operator is asymmetric and extended. We report an interesting difference between the SoxR–sv interaction and the SoxR–wx interaction through a competition assay involving groove-specific ligands. SoxR binds in the major groove of the sv operator, but binds in the minor groove of the wx operator. The structural flexibility of the SoxR helps it to act differentially in its interactions with these two operators. Mutational analysis shows that SoxR uses different amino acid residues when binding to the sv operator versus the wx operator. Taken together, the results indicate that interaction between SoxR and the two operator sites involves different binding geometries. This makes SoxR the only known example of a ArsR-family protein that binds differentially to different operators.
Keywords: Sulfur chemolithotrophy; SoxR repressor; Operator–repressor interaction; Differential groove specificity
Species-specific real-time PCR cell number quantification of the bloom-forming cyanobacterium Planktothrix agardhii by Catarina Churro; Paulo Pereira; Vitor Vasconcelos; Elisabete Valério (pp. 749-757).
A species-specific method to detect and quantify Planktothrix agardhii was developed by combining the SYBR Green I real-time polymerase chain reaction technique with a simplified DNA extraction procedure for standard curve preparation. Newly designed PCR primers were used to amplify a specific fragment within the rpoC1 gene. Since this gene exists in single copy in the genome, it allows the direct achievement of cell concentrations. The cell concentration determined by real-time PCR showed a linear correlation with the cell concentration determined from direct microscopic counts. The detection limit for cell quantification of the method was 8 cells μL−1, corresponding to 32 cells per reaction. Furthermore, the real-time qPCR method described in this study allowed a successful quantification of P. agardhii from environmental water samples, showing that this protocol is an accurate and economic tool for a rapid absolute quantification of the potentially toxic cyanobacterium P. agardhii.
Keywords: Cyanobacteria; Planktothrix agardhii ; Real-time qPCR; rpoC1 gene
Role of altered rpoB alleles in Bacillus subtilis sporulation and spore resistance to heat, hydrogen peroxide, formaldehyde, and glutaraldehyde by Ralf Moeller; Ignacija Vlašić; Günther Reitz; Wayne L. Nicholson (pp. 759-767).
Mutations in the RNA polymerase β-subunit gene rpoB causing resistance to rifampicin (RifR) in Bacillus subtilis were previously shown to lead to alterations in the expression of a number of global phenotypes known to be under transcriptional control. To better understand the influence of rpoB mutations on sporulation and spore resistance to heat and chemicals, cells and spores of the wild-type and twelve distinct congenic RifR mutant strains of B. subtilis were tested. Different levels of glucose catabolite repression during sporulation and spore resistance to heat and chemicals were observed in the RifR mutants, indicating the important role played by the RNA polymerase β-subunit, not only in the catalytic aspect of transcription, but also in the initiation of sporulation and in the spore resistance properties of B. subtilis.
Keywords: Bacillus subtilis ; Sporulation; Spore resistance; Heat; Chemicals
Activity and selectivity of histidine-containing lytic peptides to antibiotic-resistant bacteria by Riddhi Kharidia; Zhigang Tu; Long Chen; Jun F. Liang (pp. 769-778).
Lytic peptides are a group of membrane-acting peptides that are active to antibiotic-resistant bacteria but demonstrate high toxicity to tissue cells. Here, we reported the construction of new lytic peptide derivatives through the replacement of corresponding lysine/arginine residues in lytic peptide templates with histidines. Resulting lytic peptides had the same lethality to antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus, but showed greatly improved selectivity to bacteria. When incubated with co-cultured bacteria and tissue cells, these histidine-containing lytic peptide derivatives killed bacteria selectively but spared co-cultured human cells. Membrane insertion and peptide-quenching studies revealed that histidine protonation controlled peptide interactions with cell membranes determined the bacterial selectivity of lytic peptide derivatives. Compared with parent peptides, lytic peptide derivatives bound to bacteria strongly and inserted deeply into the bacterial cell membrane. Therefore, histidine-containing lytic peptides represent a new group of antimicrobial peptides for bacterial infections in which the antibiotic resistance has developed.
Keywords: Antimicrobial peptides; Lytic activity; Antibiotic resistance; MRSA; Selectivity
The biosynthetic pathway to a novel derivative of 4,4′-diapolycopene-4,4′-oate in a red strain of Sporosarcina aquimarina by Sabine Steiger; Laura Perez-Fons; Paul D. Fraser; Gerhard Sandmann (pp. 779-784).
In a red bacterial strain SF238 belonging to Sporosarcina aquimarina, a C30 carotenoid biosynthetic pathway was identified. It has been reconstructed by analysis of intermediates that accumulate in two different pigment mutants. It starts with the synthesis of 4,4′-diapophytoene and proceeds with its desaturation to 4,4′-diapolycopene, which is then oxidized to 4,4′-diapolycopene-4,4′-dioate. Using a combination of HPLC–PDA and LC–MS/MS analyses, the final product of this pathway was identified as acetyl-4,4′-diapolycopene-4,4′-dioate. This is a novel carotenoid not reported in any organisms to date. It could be demonstrated that this carotenoid has excellent antioxidative properties to protect from photosensitized peroxidation reactions like other related 4,4′-diapolycopene-4,4′-dioate derivatives.
Keywords: Acetyl-4; 4′-Diapolycopene-4; 4′-Dioate; Antioxidative property; C30 biosynthesis pathway; Chemical modification
Thiofractor thiocaminus gen. nov., sp. nov., a novel hydrogen-oxidizing, sulfur-reducing epsilonproteobacterium isolated from a deep-sea hydrothermal vent chimney in the Nikko Seamount field of the northern Mariana Arc by Hiroko Makita; Satoshi Nakagawa; Masayuki Miyazaki; Ko-ichi Nakamura; Fumio Inagaki; Ken Takai (pp. 785-794).
A novel chemolithoautotrophic hydrogen-oxidizing and sulfur-reducing bacterium, strain 496ChimT, was isolated from a deep-sea hydrothermal vent chimney collected from the hydrothermal field at the summit of Nikko Seamount field, in the Mariana Arc. Cells were rods or curved rods, motile by means of a single polar flagellum. Growth was observed between 15 and 45 °C (optimum 37 °C; doubling time, 2.1 h) and between pH 5.3 and 8.0 (optimum pH 6.0). The isolate was a strictly anaerobic, obligate chemolithoautotroph capable of growth using molecular hydrogen as the sole energy source, carbon dioxide as the sole carbon source, ammonium or nitrate as the sole nitrogen source, and elemental sulfur as the electron acceptor. The G+C content of genomic DNA was 35 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the new isolate belonged to the class Epsilonproteobacteria, but the isolate was distantly related to the previously described Epsilonproteobacteria species potentially at the genus level (<90 %). On the basis of its physiological and molecular characteristics, strain 496ChimT (=DSM 22050Τ = JCM 15747Τ = NBRC 105224Τ) represents the sole species of a new genus, Thiofractor, for which the name Thiofractor thiocaminus is proposed.
Keywords: Epsilonproteobacteria ; Hydrogen-oxidizing; Sulfur-reducing; Chemolithoautotroph; Hydrothermal field; Mariana
A TonB-dependent outer membrane receptor of Pseudomonas fluorescens: virulence and vaccine potential by Yong-hua Hu; Wei Dang; Li Sun (pp. 795-802).
Pseudomonas fluorescens is a Gram-negative bacterium and a common aquaculture pathogen. In this study, we identified from a pathogenic P. fluorescens strain a TonB-dependent outer membrane receptor, TdrA, as a secreted protein and examined its function and vaccine potential. TdrA is composed of 746 residues and possesses conserved structural domains of TonB-dependent outer membrane receptors. Quantitative real-time reverse transcriptase-PCR analysis showed that expression of tdrA was upregulated under conditions of iron starvation and during infection of host cells. Consistently, iron depletion induced increased production of TdrA protein in the outer membrane. Compared to the wild type, a tdrA-knock out mutant (1) was unable to grow in the absence of iron, (2) exhibited drastically attenuated overall bacterial virulence, and (3) was impaired in the ability to establish lethal infection in host tissues. Purified recombinant TdrA (rTdrA), when used as a subunit vaccine to immunize flounder, was able to induce strong protective immunity, including production of serum-specific antibodies that resulted in effective protection against lethal-dose P. fluorescens challenge. Together, these results indicate that TdrA is an outer membrane receptor and a protective immunogen that is likely to be involved in iron acquisition and, as a result, required for optimal bacterial virulence.
Keywords: Pseudomonas fluorescens ; TonB-dependent receptor; Virulence; Vaccine
Importance of RNA stabilization: evaluation of ansB, ggt, and rpoA transcripts in microaerophilic Campylobacter jejuni 81-176 by Heidi Hyytiäinen; Pekka Juntunen; Nina Akrenius; Marja-Liisa Hänninen (pp. 803-808).
A subset of food-borne Campylobacter jejuni strains utilizes amino acids asparagine and glutamine as carbon sources that may enhance the ability of this microaerophilic pathogen to colonize specific tissues. In this study, we analyzed the transcript sizes of the ansB and ggt genes encoding the periplasmic asparaginase and γ-glutamyltranspeptidase in C. jejuni 81-176, respectively, and compared the expression level of mRNAs at different time points during the growth in vitro. In addition, we included the housekeeping rpoA gene, encoding the α-subunit of DNA-directed RNA polymerase, to monitor sample processing as it has been described as a stable reference gene in gene expression studies in C. jejuni. Our results revealed that both the ansB and ggt genes were expressed in the end of the logarithmic growth phase and their corresponding monocistronic mRNAs were not affected by sample processing steps. In contrast, the mRNAs of the polycistronic operon containing rpoA gene were highly induced at earlier stage of the logarithmic growth and were clearly differentially responding to external factors during cell harvesting step.
Keywords: Microaerophilic bacteria; RNA isolation; Northern blot analysis; RT-qPCR