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Archives of Microbiology (v.192, #1)
The PQQ biosynthetic operons and their transcriptional regulation in Pseudomonas aeruginosa by Nicole Gliese; Viola Khodaverdi; Helmut Görisch (pp. 1-14).
Gene PA1990 of Pseudomonas aeruginosa, located downstream of pqqE and encoding a putative peptidase, was shown to be involved in excretion of PQQ into the culture supernatant. This gene is cotranscribed with the pqqABCDE cluster and was named pqqH. A PA1990::Kmr mutant (VK3) did not show any effect in growth behaviour; however, in contrast to the wild-type, no excretion of PQQ into the culture supernatant was observed. The putative pqqF gene of P. aeruginosa was shown to be essential for PQQ biosynthesis. A pqqF::Kmr mutant did not grow aerobically on ethanol, because of its inability to produce PQQ. Transcription of the pqqABCDEH operon was induced upon aerobic growth on ethanol, 1-propanol, 1,2-propanediol and 1-butanol, while on glycerol, succinate and acetate, transcription was low. Transcription of the pqqABCDEH operon was also found upon anoxic growth on ethanol with nitrate as electron acceptor, but no PQQ was produced. Expression of the pqqABCDEH operon is regulated at the transcriptional level. In contrast, the pqqF operon appeared to be transcribed constitutively at a very low level under all growth conditions studied.
Keywords: Pseudomonas aeruginosa ; Ethanol oxidation; Transcriptional regulation; pqqABCDEH ; pqqF
Characterization of an O-desmethylangolensin-producing bacterium isolated from human feces by Shin-ichiro Yokoyama; Toshio Niwa; Toshihiko Osawa; Tohru Suzuki (pp. 15-22).
A bacterium that converted daidzein to O-desmethylangolensin was isolated from the feces of healthy humans. It was an obligately anaerobic, nonsporeforming, nonmotile and Gram-positive rod. The isolate used glucose, sucrose, raffinose, maltose, and fructose as carbon sources. It did not hydrolyze gelatin, esculin, or starch. The strain was urease, acid phosphatase, and arginine dihydrolase positive. It was catalase, oxidase, H2S, and indole negative. The major products of glucose fermentation were butyrate and lactate. Its mol% G+C was 51.2. The major cellular fatty acids were C16:0 DMA, C16:0, and C16:0 aldehyde. The structural type of cell wall peptidoglycan was suggested to be A1γ. The isolate was susceptible to β-lactam, cefem, and macrolide antibiotics and resistant to aminoglycoside and quinolone antibiotics. The bacterium was related to Eubacterium ramulus ATCC29099T, Eubacterium rectale ATCC33656T, and species of the genus Roseburia, but the highest 16S rRNA gene similarity to these described species was only 94.4%, consistent with its being classified as a novel genus. Based on the above, the isolate, named strain SY8519, was identified as belonging to a novel genus in the Clostridium rRNA cluster XIVa.
Keywords: O-desmethylangolensin; Strain SY8519; Isolate from human feces; Taxonomical property; Clostridium rRNA cluster XIVa; Antimicrobial susceptibility
Early candidacy for differentiation into heterocysts in the filamentous cyanobacterium Anabaena sp. PCC 7120 by Masakazu Toyoshima; Naobumi V. Sasaki; Makoto Fujiwara; Shigeki Ehira; Masayuki Ohmori; Naoki Sato (pp. 23-31).
The filamentous cyanobacterium Anabaena sp. PCC 7120 fixes dinitrogen facultatively. Upon depletion of combined nitrogen, about 10% of vegetative cells within the filaments differentiate terminally into nitrogen-fixing cells. The heterocyst has been studied as a model system of prokaryotic cell differentiation, with major focus on signal transduction and pattern formation. The fate of heterocyst differentiation is determined at about the eighth hour of induction (point of no return), well before conspicuous morphological or metabolic changes occur. However, little is known about how the initial heterocysts are selected after the induction by nitrogen deprivation. To address this question, we followed the fate of every cells on agar plates after nitrogen deprivation with an interval of 4 h. About 10% of heterocysts were formed without prior division after the start of nitrogen deprivation. The intensity of fluorescence of GFP in the transformants of hetR-gfp increased markedly in the future heterocysts at the fourth hour with respect to other cells. We also noted that the growing filaments consisted of clusters of four consecutive cells that we call quartets. About 75% of initial heterocysts originated from either of the two outer cells of quartets at the start of nitrogen deprivation. These results suggest that the future heterocysts are loosely selected at early times after the start of nitrogen deprivation, before the commitment. Such early candidacy could be explained by different properties of the outer and inner cells of a quartet, but the molecular nature of candidacy remains to be uncovered.
Keywords: Filamentous cyanobacteria; Anabaena sp. PCC 7120; Heterocyst candidacy; Differentiation; Cell lineage; Cell division; Pattern formation; Frequently iterated observation; Spatial periodicity; Quartet
Activation of the transcription of Gal4-regulated genes by Physarum 14-3-3 in yeast is related to dimer-binding motif-2 and three phosphorylation sites by Shide Liu; Minghua Li; Jianhua Zhang; Kang Kang; Shengli Tian; Yisi Wang; Miao Xing (pp. 33-40).
The roles of 14-3-3 proteins in the lower eukaryotes are still elusive. We isolated a cDNA encoding the 14-3-3 protein (P14-3-3) from the lower eukaryote Physarum polycephalum. This P14-3-3 gene was then inserted downstream of the Gal4 DNA-binding domain in the yeast expression vector pGBKT7. The recombinant vector was transformed into auxotrophic AH109 and Y187 yeast cells to detect the activation of Gal4-regulated gene expression mediated by P14-3-3. The results showed that three reporter genes (ADE2, HIS3, and lacZ) could be normally expressed, indicating that the transcriptional activation function of P14-3-3 was retained. We subsequently used a truncated P14-3-3 peptides and mutant peptides to study the activation of the Gal4-regulated genes ADE2, HIS3, and lacZ. We found that deletion of the N-terminal second dimer-binding motif (DBM2) sequence or the C-terminal coil sequence led to the loss of P14-3-3’s transcriptional activation function. Specifically, any mutation at the potential phosphorylation sites (Ser62 and Ser67) on DBM2 or at the C-terminal potential phosphorylation site (235ThrSer236) led to the loss of the transcriptional activation function of P14-3-3. Taken together, these observations suggest that the transcriptional activation function of P14-3-3 in lower eukaryotes is related to DBM2 and the C-terminal coil structures.
Keywords: Physarum polycephalum ; 14-3-3; Transcriptional activation; Dimer-binding motif; Phosphorylation sites
On the photosynthetic properties of marine bacterium COL2P belonging to Roseobacter clade by Michal Koblížek; Jarmila Mlčoušková; Zbigniew Kolber; Jiří Kopecký (pp. 41-49).
Aerobic anoxygenic phototrophs (AAPs) are prokaryotic microorganisms capable of harvesting light using bacteriochlorophyll-based reaction centres. Marine AAP communities are generally dominated by species belonging to the Roseobacter clade. For this reason, we used marine Roseobacter-related strain COL2P as a model organism to characterize its photosynthetic apparatus, level of pigmentation and expression of photosynthetic complexes. This strain contained functional photosynthetic reaction centres with bacteriochlorophyll a and spheroidenone as the main light-harvesting pigments, but the expression of the photosynthetic apparatus was significantly reduced when compared to truly photoautotrophic species. Moreover, the absence of peripheral light-harvesting complexes largely reduced its light-harvesting capacity. The size of the photosynthetic unit was limited to 35.4 ± 1.0 BChl a molecules supplemented by the same number of spheroidenone molecules. The contribution of oxidative phosphorylation and photophosphorylation was analysed by respiration and fluorometric measurements. Our results indicate that even with a such reduced photosynthetic apparatus, photophosphorylation provides up to three times higher electron fluxes than aerobic respiration. These results suggest that light-derived energy can provide a substantial fraction of COL2P metabolic needs.
Keywords: Aerobic anoxygenic phototrophs; Aerobic photosynthetic bacteria; Bacteriochlorophyll a ; Roseobacter ; Photoheterotrophy
Functional analysis of a putative holin-like peptide-coding gene in the genome of Bacillus licheniformis AnBa9 by Thangamani Anthony; Gunasekaran Stalin Chellappa; Thangamani Rajesh; Paramasamy Gunasekaran (pp. 51-56).
BhlA, a putative holin-like protein of Bacillus licheniformis AnBa9 expressed in Escherichia coli BL21(DE3) showed antibacterial activity against several gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and Micrococcus luteus. Deletion analysis of bhlA suggests that a hydrophobic transmembrane domain, BhlATM is essential for antibacterial activity. Though the minimum inhibitory concentration (MIC) of BhlA was sevenfold lower than BhlATM, transmembrane domain deleted construct (BhlA∆TM) had no antibacterial activity. The expression of BhlA in E. coli was found to be toxic to cells. Therefore, the bhlA was cloned in yeast surface display vector pYD1 and expressed in Saccharomyces cerevisiae. The surface displayed yeast showed inhibition of several gram-positive bacteria. This recombinant yeast expressing BhlA may be used as biodrug for efficient control of multiple drug-resistant bacterial infections.
Keywords: Bacillus licheniformis ; BhlA; Holin-like protein; Transmembrane domain; Antibacterial activity; Yeast surface display
The putative Bacillus subtilis l,d-transpeptidase YciB is a lipoprotein that localizes to the cell poles in a divisome-dependent manner by Marc Bramkamp (pp. 57-68).
Cell wall synthesis in bacteria is spatially organized by cytoskeletal structures. Common to all cell wall-bearing bacteria, the cytokinetic machinery localizes the cell wall synthesis to the site of septation. Recently, MinJ, a new component of the cytokinetic machinery, or divisome, of Bacillus subtilis has been described. MinJ is part of the division site selection system but also essential for correct assembly of the divisome. Here, I used the isolated PDZ domain of MinJ for co-elution experiments. One of the proteins that co-eluted was the so far uncharacterized, putative l,d-transpeptidase protein YciB. Evidence is shown that YciB localizes to the cell poles. YciB localization depends on the existence of a mature divisome, suggesting that l,d-transpeptidases are, like penicillin-binding proteins, part of the divisome.
Keywords: l,d-transpeptidase; YciB; Cell division; Protein localization; Peptidoglycan synthesis; Lipoprotein
Mutation in the lysA gene impairs the symbiotic properties of Mesorhizobium ciceri by Subrata K. Das; Uma Shankar Gautam; Kiran V. Sandhu; Saumya Bandyopadhyay; Pran K. Chakrabartty; Aqbal Singh (pp. 69-77).
A Tn5-induced mutant of Mesorhizobium ciceri, TL28, requiring the amino acid lysine for growth on minimal medium was isolated and characterized. The Tn5 insertion in the mutant strain TL28 was located on a 6.8-kb EcoRI fragment of the chromosomal DNA. Complementation analysis with cloned DNA indicated that 1.269 kb of DNA of the 6.8-kb EcoRI fragment restored the wild-type phenotype of the lysine-requiring mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to lysA gene of different bacteria. The lys − mutant TL28 was unable to elicit development of effective nodules on the roots of Cicer arietinum L. There was no detectable level of lysine in the root exudates of chickpea. However, addition of lysine to the plant growth medium restored the ability of the mutant to produce effective nodules with nitrogen fixation ability on the roots of C. arietinum.
Keywords: Mesorhizobium ciceri ; Transposon mutagenesis; lysA gene; Symbiotic nitrogen fixation; Cicer arietinum (L); Root exudates
Reduced expression of virulence factors in multidrug-resistant Pseudomonas aeruginosa strains by Aleksander Deptuła; Eugenia Gospodarek (pp. 79-84).
MDR Pseudomonas aeruginosa strains are isolated from clinical specimens with increasing frequency. It seems that acquiring genes which determine antibiotic resistance usually comes at a biological cost of impaired bacterial physiology. There is no information on investigations comparing phenotypic differences in MDR and MDS P. aeruginosa strains in literature. The study included 150 clinical P. aeruginosa isolates (75 classified as MDS and 75 as MDR). PFGE analysis revealed five pairs of identical isolates in the group of MDR strains and the results obtained for these strains were not included in the statistical analyses. MDR strains adhered to polystyrene to a lesser extent than MDS strains. The growth rate in the liquid medium was significantly lower for MDR strains. Detectable amounts of alginate were present in the culture supernatants of seven MDS and six MDR strains. The MDR P. aeruginosa strains which were investigated produced significantly lower amounts of extracellular material binding Congo Red, lower lipolytic, elastase, LasA protease, phospholipase C activity and pyocyanin quantity in culture supernatants when compared with MDS strains. No significant differences were observed between MDR and MDS strains in proteolytic activity. In conclusion, the MDR P. aeruginosa strains have impaired virulence when compared to MDS strains.
Keywords: Pseudomonas aeruginosa ; Multidrug resistant; Multidrug sensitive; Reduced virulence