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Operon
Kinds of Operon Terms modified by Operon Selected AbstractsLarge-scale distribution and activity patterns of an extremely low-light-adapted population of green sulfur bacteria in the Black SeaENVIRONMENTAL MICROBIOLOGY, Issue 5 2010Evelyn Marschall Summary The Black Sea chemocline represents the largest extant habitat of anoxygenic phototrophic bacteria and harbours a monospecific population of Chlorobium phylotype BS-1. High-sensitivity measurements of underwater irradiance and sulfide revealed that the optical properties of the overlying water column were similar across the Black Sea basin, whereas the vertical profiles of sulfide varied strongly between sampling sites and caused a dome-shaped three-dimensional distribution of the green sulfur bacteria. In the centres of the western and eastern basins the population of BS-1 reached upward to depths of 80 and 95 m, respectively, but were detected only at 145 m depth close to the shelf. Using highly concentrated chemocline samples from the centres of the western and eastern basins, the cells were found to be capable of anoxygenic photosynthesis under in situ light conditions and exhibited a photosynthesis,irradiance curve similar to low-light-adapted laboratory cultures of Chlorobium BS-1. Application of a highly specific RT-qPCR method which targets the internal transcribed spacer (ITS) region of the rrn operon of BS-1 demonstrated that only cells at the central station are physiologically active in contrast to those at the Black Sea periphery. Based on the detection of ITS-DNA sequences in the flocculent surface layer of deep-sea sediments across the Black Sea, the population of BS-1 has occupied the major part of the basin for the last decade. The continued presence of intact but non-growing BS-1 cells at the periphery of the Black Sea indicates that the cells can survive long-distant transport and exhibit unusually low maintenance energy requirements. According to laboratory measurements, Chlorobium BS-1 has a maintenance energy requirement of ,1.6,4.9·10,15 kJ cell,1 day,1 which is the lowest value determined for any bacterial culture so far. Chlorobium BS-1 thus is particularly well adapted to survival under the extreme low-light conditions of the Black Sea, and can be used as a laboratory model to elucidate general cellular mechanisms of long-term starvation survival. Because of its adaptation to extreme low-light marine environments, Chlorobium BS-1 also represents a suitable indicator for palaeoceanography studies of deep photic zone anoxia in ancient oceans. [source] Reduction of fumarate, mesaconate and crotonate by Mfr, a novel oxygen-regulated periplasmic reductase in Campylobacter jejuniENVIRONMENTAL MICROBIOLOGY, Issue 3 2010Edward Guccione Summary Methylmenaquinol : fumarate reductase (Mfr) is a newly recognized type of fumarate reductase present in some ,-proteobacteria, where the active site subunit (MfrA) is localized in the periplasm, but for which a physiological role has not been identified. We show that the Campylobacter jejuni mfrABE operon is transcribed from a single promoter, with the mfrA gene preceded by a small open reading-frame (mfrX) encoding a C. jejuni -specific polypeptide of unknown function. The growth characteristics and enzyme activities of mutants in the mfrA and menaquinol : fumarate reductase A (frdA) genes show that the cytoplasmic facing Frd enzyme is the major fumarate reductase under oxygen limitation. The Mfr enzyme is shown to be necessary for maximal rates of growth by fumarate respiration and rates of fumarate reduction in intact cells measured by both viologen assays and 1H-NMR were slower in an mfrA mutant. As periplasmic fumarate reduction does not require fumarate/succinate antiport, Mfr may allow more efficient adaptation to fumarate-dependent growth. However, a further rationale for the periplasmic location of Mfr is suggested by the observation that the enzyme also reduces the fumarate analogues mesaconate and crotonate; fermentation products of anaerobes with which C. jejuni shares its gut environment, that are unable to be transported into the cell. Both MfrA and MfrB subunits were localized in the periplasm by immunoblotting and 2D-gel electrophoresis, but an mfrE mutant accumulated unprocessed MfrA in the cytoplasm, suggesting a preassembled MfrABE holoenzyme has to be recognized by the TAT system for translocation to occur. Gene expression studies in chemostat cultures following an aerobic-anaerobic shift showed that mfrA is highly upregulated by oxygen limitation, as would be experienced in vivo. Our results indicate that in addition to a role in fumarate respiration, Mfr allows C. jejuni to reduce analogous substrates specifically present in the host gut environment. [source] Genetic, immunological and biochemical evidence for a Rnf complex in the acetogen Acetobacterium woodiiENVIRONMENTAL MICROBIOLOGY, Issue 6 2009Eva Biegel Summary Acetogenic bacteria grow by the oxidation of various substrates coupled to the reduction of carbon dioxide (acetogenesis) or other electron acceptors but the mechanisms of energy conservation are still enigmatic. Here, we report the presence of a rnf gene cluster rnfCDGEAB in Acetobacterium woodii that is speculated to encode a novel, energy-conserving ferredoxin:NAD+ -oxidoreductase complex composed of at least six different subunits. Transcriptional analysis revealed that the genes constitute an operon. RnfC and RnfG were heterologously produced and antibodies were generated. Western blot analyses demonstrated that these subunits were produced and are associated with the cytoplasmic membrane. The subunits were present in cells respiring with either carbon dioxide or caffeate. A preparation with NADH dehydrogenase activity was obtained from detergent solubilized membranes that contained RnfC and RnfG. [source] Enterococcus faecalis with the gelatinase phenotype regulated by the fsr operon and with biofilm-forming capacity are common in the agricultural environmentENVIRONMENTAL MICROBIOLOGY, Issue 6 2009Lilia Macovei Summary The prevalence of gelatinase activity and biofilm formation among environmental enterococci was assessed. In total, 396 enterococcal isolates from swine and cattle faeces and house flies from a cattle farm were screened for gelatinase activity. The most prevalent phenotype on Todd,Hewitt agar with 1.5% skim milk was the weak protease (WP) (72.2% of isolates), followed by the strong protease (SP) 18.7%, and no protease (NP) (9.1%). The majority of WP isolates was represented by Enterococcus hirae (56.9%), followed by Enterococcus faecium (25.9%), Enterococcus casseliflavus (10.4%), Enterococcus gallinarum (5.2%) and Enterococcus saccharolyticus (1.7%). All WP isolates were negative for gelE (gelatinase) and sprE (serine protease) as well as the fsrABDC operon that regulates the two proteases, and only four isolates (7.0%) formed biofilms in vitro. All SP isolates were Enterococcus faecalis positive for the fsrABDC, gelE, sprE genes and the majority (91.2%) formed a biofilm. Diversity of NP isolates was relatively evenly distributed among E. hirae, E. faecium, E. casseliflavus, E. gallinarum, Enterococcus durans, E. saccharolyticus and Enterococcus mundtii. All NP isolates were negative for the fsr operon and only four E. hirae (11.1%) formed a biofilm. Of further interest was the loss of the gelatinase phenotype (18.9% of isolates) from SP isolates after 4 month storage at 4,8°C and several passages of subculture. Results of reverse transcription PCR analysis indicated that mRNA was produced for all the genes in the frs operon and sequencing of the gelE gene did not reveal any significant mutations. However, gelatinase was not detectable by Western blot analysis. Our study shows that E. faecalis with the complete fsr operon and the potential to form a biofilm are relatively common in the agricultural environment and may represent a source/reservoir of clinically relevant strains. In addition, many environmental enterococci, especially E. hirae, produce an unknown WP that can hydrolyse casein but does not contribute to biofilm formation. The stability of the gelatinase phenotype in E. faecalis and its regulation will require additional studies. [source] Comparative analysis of genome fragments of Acidobacteria from deep Mediterranean planktonENVIRONMENTAL MICROBIOLOGY, Issue 10 2008Achim Quaiser Summary Acidobacteria constitute a still poorly studied phylum that is well represented in soils. Recent studies suggest that members of this phylum may be also abundant in deep-sea plankton, but their relative abundance and ecological role in this ecosystem are completely unknown. A recent screening of three metagenomic deep-sea libraries of bathypelagic plankton from the South Atlantic (1000 m depth), the Adriatic (1000 m depth) and the Ionian (3000 m depth) seas in the Mediterranean revealed an unexpected relative proportion of acidobacterial fosmids, which affiliated to the Solibacterales (Group 3), to the Group 11 and, most frequently, to the Group 6 of this diverse phylum. Here, we present the comparative analysis of 11 acidobacterial genome fragments containing the rrn operon from these Mediterranean libraries. A highly conserved syntenic region spanning up to 30 kb and containing up to 25 open reading frames was shared by Group 6 Acidobacteria. Synteny was also partially conserved in distantly related acidobacterial genome fragments derived from a metagenomic soil library, indicating a remarkable conservation of this genomic region within these Acidobacteria. A search for Acidobacteria -specific hits in directly comparable, available fosmid-end sequences from soil and marine metagenomic libraries showed a significant increase of their relative proportion in plankton libraries as a function of increasing depth reaching, at high depth, levels nearly comparable to those of soil. Thus, our results suggest that Acidobacteria are abundant and represent a significant proportion of the microbial community in the deep-sea ecosystem. [source] New ways to break an old bond: the bacterial carbon,phosphorus hydrolases and their role in biogeochemical phosphorus cyclingENVIRONMENTAL MICROBIOLOGY, Issue 10 2007John P. Quinn Summary Phosphonates are organophosphorus molecules that contain the highly stable C,P bond, rather than the more common, and more labile, C,O,P phosphate ester bond. They have ancient origins but their biosynthesis is widespread among more primitive organisms and their importance in the contemporary biosphere is increasingly recognized; for example phosphonate-P is believed to play a particularly significant role in the productivity of the oceans. The microbial degradation of phosphonates was originally thought to occur only under conditions of phosphate limitation, mediated exclusively by the poorly characterized C,P lyase multienzyme system, under Pho regulon control. However, more recent studies have demonstrated the Pho-independent mineralization by environmental bacteria of three of the most widely distributed biogenic phosphonates: 2-aminoethylphosphonic acid (ciliatine), phosphonoacetic acid, and 2-amino-3-phosphonopropionic acid (phosphonoalanine). The three phosphonohydrolases responsible have unique specificities and are members of separate enzyme superfamilies; their expression is regulated by distinct members of the LysR family of bacterial transcriptional regulators, for each of which the phosphonate substrate of the respective degradative operon serves as coinducer. Previously no organophosphorus compound was known to induce the enzymes required for its own degradation. Whole-genome and metagenome sequence analysis indicates that the genes encoding these newly described C,P hydrolases are distributed widely among prokaryotes. As they are able to function under conditions in which C,P lyases are inactive, the three enzymes may play a hitherto-unrecognized role in phosphonate breakdown in the environment and hence make a significant contribution to global biogeochemical P-cycling. [source] Detergent-induced cell aggregation in subpopulations of Pseudomonas aeruginosa as a preadaptive survival strategyENVIRONMENTAL MICROBIOLOGY, Issue 9 2007Janosch Klebensberger Summary During growth of Pseudomonas aeruginosa strain PAO1 with the toxic detergent SDS, a part of the population actively formed macroscopic cell aggregates while the other part grew as freely suspended cells. The physiological function of aggregation for growth with SDS was investigated. Three mutants growing with SDS without aggregation were isolated: the spontaneous mutant strain N and two mutants with transposon insertions in the psl operon for exopolysaccharide synthesis. SDS-induced aggregation in strain N but not in a pslJ mutant was restored by complementation with two genes encoding diguanylate cyclases responsible for synthesis of cyclic-di-guanosine monophosphate (c-di-GMP). By expressing a c-di-GMP-specific phosphodiesterase SDS-induced aggregation of strain PAO1 was reduced. Upon exposure to SDS in the presence of the uncoupler carbonyl cyanide chlorophenylhydrazone, the aggregating strains had ca. 500-fold higher survival rates than the non-aggregating strains. Co-incubation experiments revealed that strain N could integrate into aggregates of strain PAO1 and thereby increase its survival rate more than 1000-fold. These results showed that SDS-induced aggregation involved c-di-GMP signalling with the psl operon as a possible target. Cell aggregation could serve as a pre-adaptive strategy ensuring survival and growth of P. aeruginosa populations in environments with multiple toxic chemicals. [source] A multicopper oxidase is essential for manganese oxidation and laccase-like activity in Pedomicrobium sp.ENVIRONMENTAL MICROBIOLOGY, Issue 4 2007ACM 306 Summary Pedomicrobium sp. ACM 3067 is a budding-hyphal bacterium belonging to the ,- Proteobacteria which is able to oxidize soluble Mn2+ to insoluble manganese oxide. A cosmid, from a whole-genome library, containing the putative genes responsible for manganese oxidation was identified and a primer-walking approach yielded 4350 bp of novel sequence. Analysis of this sequence showed the presence of a predicted three-gene operon, moxCBA. The moxA gene product showed homology to multicopper oxidases (MCOs) and contained the characteristic four copper-binding motifs (A, B, C and D) common to MCOs. An insertion mutation of moxA showed that this gene was essential for both manganese oxidation and laccase-like activity. The moxB gene product showed homology to a family of outer membrane proteins which are essential for Type I secretion in Gram-negative bacteria. moxBA has not been observed in other manganese-oxidizing bacteria but homologues were identified in the genomes of several bacteria including Sinorhizobium meliloti 1021 and Agrobacterium tumefaciens C58. These results suggest that moxBA and its homologues constitute a family of genes encoding an MCO and a predicted component of the Type I secretion system. [source] Genetic and functional properties of uncultivated thermophilic crenarchaeotes from a subsurface gold mine as revealed by analysis of genome fragmentsENVIRONMENTAL MICROBIOLOGY, Issue 12 2005Takuro Nunoura Summary Within a phylum Crenarchaeota, only some members of the hyperthermophilic class Thermoprotei, have been cultivated and characterized. In this study, we have constructed a metagenomic library from a microbial mat formation in a subsurface hot water stream of the Hishikari gold mine, Japan, and sequenced genome fragments of two different phylogroups of uncultivated thermophilic Crenarchaeota: (i) hot water crenarchaeotic group (HWCG) I (41.2 kb), and (ii) HWCG III (49.3 kb). The genome fragment of HWCG I contained a 16S rRNA gene, two tRNA genes and 35 genes encoding proteins but no 23S rRNA gene. Among the genes encoding proteins, several genes for putative aerobic-type carbon monoxide dehydrogenase represented a potential clue with regard to the yet unknown metabolism of HWCG I Archaea. The genome fragment of HWCG III contained a 16S/23S rRNA operon and 44 genes encoding proteins. In the 23S rRNA gene, we detected a homing-endonuclease encoding a group I intron similar to those detected in hyperthermophilic Crenarchaeota and Bacteria, as well as eukaryotic organelles. The reconstructed phylogenetic tree based on the 23S rRNA gene sequence reinforced the intermediate phylogenetic affiliation of HWCG III bridging the hyperthermophilic and non-thermophilic uncultivated Crenarchaeota. [source] Contrasting microcystin production and cyanobacterial population dynamics in two Planktothrix -dominated freshwater lakesENVIRONMENTAL MICROBIOLOGY, Issue 10 2005Ingmar Janse Summary Microcystin concentrations in two Dutch lakes with an important Planktothrix component were related to the dynamics of cyanobacterial genotypes and biovolumes. Genotype composition was analysed by using denaturing gradient gel electrophoresis (DGGE) profiling of the intergenic transcribed spacer region of the rrn operon (rRNA-ITS), and biovolumes were measured by using microscopy. In Lake Tjeukemeer, microcystins were present throughout summer (maximum concentration 30 µg l,1) while cyanobacterial diversity was low and very constant. The dominant phototroph was Planktothrix agardhii. In contrast, Lake Klinckenberg showed a high microcystin peak (up to 140 µg l,1) of short duration. In this lake, cyanobacterial diversity was higher and very dynamic with apparent genotype successions. Several genotypes derived from DGGE field profiles matched with genotypes from cultures isolated from field samples. The microcystin peak measured in Lake Klinckenberg could be confidently linked to a bloom of Planktothrix rubescens, as microscopic and genotypic analysis showed identity of bloom samples and a toxin-producing P. rubescens culture. Toxin-producing genotypes were detected in the microbial community before they reached densities at which they were detected by using microscopy. Cyanobacterial biovolumes provided additional insights in bloom dynamics. In both lakes, the microcystin content per cell was highest at the onset of the blooms. Our results suggest that while genotypic characterization of a lake can be valuable for detection of toxic organisms, for some lakes a monitoring of algal biomass has sufficient predictive value for an assessment of toxin production. [source] Calibration and deployment of custom-designed bioreporters for protecting biological remediation consortia from toxic shockENVIRONMENTAL MICROBIOLOGY, Issue 2 2005Siouxsie Wiles Summary We have previously described the development of a panel of site-specific lux -based bioreporters from an industrial wastewater treatment system remediating coking effluents. The Pseudomonad strains carry a stable chromosomal copy of the luxCDABE operon from Photorhabdus luminescens and display proportional responses in bioluminescence decay with increasing phenol concentration up to 800 mg l,1. In this work we describe their deployment to provide a strategic sensing network for protecting bacterial communities involved in the biological breakdown of coking effluents. This evaluation demonstrated the utility of strategic placement of reporters around heavy industry treatment systems and the reliability of the reporter strains under normal operational conditions. Mono-phenol or total phenolic variation within the treatment system accounted for >,65,80% of the luminescence response. The reporters exhibited stable luminescence output during normal operations with maximum standard deviations of luminescence over time of c. 5,15% depending on the treatment compartment. Furthermore, deployment of the bioreporters over a 5-month period allowed the determination of an operational range (OR) for each reporter for effluent samples from each compartment. The OR allowed a convenient measure of toxicity effects between treatment compartments and accurately reflected a specific pollution event occurring within compartments of the treatment system. This work demonstrates the utility of genetic modification to provide ecologically relevant bioreporters, extends the sensing capabilities currently obtained through marine derived biosensors and significantly enhances the potential for in situ deployment of reporting agents. [source] Targeting clusters of transferred genes in Thermotoga maritimaENVIRONMENTAL MICROBIOLOGY, Issue 11 2003Camilla L. Nesbø Summary We screened a Thermotoga sp. strain RQ2 lambda library for genes present in that strain but absent from the closely related completely sequenced relative Thermotoga maritima strain MSB8, by using probes generated in an earlier genomic subtraction study. Five lambda insert fragments were sequenced, containing, respectively, an archaeal type ATPase operon, rhamnose biosynthetic genes, ORFs with similarity to an arabinosidase, a Thermotoga sp. strain RQ2-specific alcohol dehydrogenase and a novel archaeal Mut-S homologue. All but one of these fragments contained additional Thermotoga sp. strain RQ2-specific sequences not screened for, suggesting that many such strain-specific genes will be found clustered in the genome. Moreover, phylogenetic analyses, phylogenetic distribution and/or G + C content suggests that all the Thermotoga sp. strain RQ2 specific sequences in the sequenced lambda clones have been acquired by lateral gene transfer. We suggest that the use of strain-specific small insert clones obtained by subtractive hybridization to target larger inserts for sequencing is an efficient, economical way to identify environmentally (or clinically) relevant interstrain differences and novel gene clusters, and will be invaluable in comparative genomics. [source] Identification of cyanobacteria and their toxigenicity in environmental samples by rapid molecular analysisENVIRONMENTAL TOXICOLOGY, Issue 6 2001Judith A. Baker Abstract We report molecular analyses which identify cyanobacterial strains present in environmental samples. These analyses do not require the isolation and culture of strains. Identification of cyanobacteria used the polymerase chain reaction (PCR), based on the phycocyanin operon. Differentiation was either by restriction endonuclease digestion (restriction fragment length polymorphisms) or sequencing of the PCR products. Identification was based on sequence homology of the intergenic spacer region (IGS) between the ,- and ,-phycocyanin subunits (PC-IGS) with database records. We have found that the length and sequence of the PC-IGS is capable of predicting the genus accurately, but not the species. Toxigenicity was determined with oligonucleotide probes for key steps in the microcystin toxin synthesis pathway. We have shown that it is possible to easily and routinely obtain PCR amplification products and differentiate the strains in bloom samples. The methods can detect even minor components in bloom samples, which may not be apparent on microscopic examination. Genetic probes for microcystin toxigenicity are effective on environmental samples, eliminating the need for isolation and culture of the organisms. The use of a suite of tests described here will allow water managers to determine the presence and the type of cyanobacteria and their microcystin toxigenicity. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 472,482, 2001 [source] pyr RNA binding to the Bacillus caldolyticus PyrR attenuation protein , characterization and regulation by uridine and guanosine nucleotidesFEBS JOURNAL, Issue 4 2008Casper M. Jørgensen The PyrR protein regulates expression of pyrimidine biosynthetic (pyr) genes in many bacteria. PyrR binds to specific sites in the 5, leader RNA of target operons and favors attenuation of transcription. Filter binding and gel mobility assays were used to characterize the binding of PyrR from Bacillus caldolyticus to RNA sequences (binding loops) from the three attenuation regions of the B. caldolyticus pyr operon. Binding of PyrR to the three binding loops and modulation of RNA binding by nucleotides was similar for all three RNAs. The apparent dissociation constants at 0 °C were in the range 0.13,0.87 nm in the absence of effectors; dissociation constants were decreased by three- to 12-fold by uridine nucleotides and increased by 40- to 200-fold by guanosine nucleotides. The binding data suggest that pyr operon expression is regulated by the ratio of intracellular uridine nucleotides to guanosine nucleotides; the effects of nucleoside addition to the growth medium on aspartate transcarbamylase (pyrB) levels in B. subtilis cells in vivo supported this conclusion. Analytical ultracentrifugation established that RNA binds to dimeric PyrR, even though the tetrameric form of unbound PyrR predominates in solution at the concentrations studied. [source] Cooperation of two carotene desaturases in the production of lycopene in Myxococcus xanthusFEBS JOURNAL, Issue 16 2007Antonio A. Iniesta In Myxococcus xanthus, all known carotenogenic genes are grouped together in the gene cluster carB,carA, except for one, crtIb (previously named carC). We show here that the first three genes of the carB operon, crtE, crtIa, and crtB, encode a geranygeranyl synthase, a phytoene desaturase, and a phytoene synthase, respectively. We demonstrate also that CrtIa possesses cis -to- trans isomerase activity, and is able to dehydrogenate phytoene, producing phytofluene and ,-carotene. Unlike the majority of CrtI-type phytoene desaturases, CrtIa is unable to perform the four dehydrogenation events involved in converting phytoene to lycopene. CrtIb, on the other hand, is incapable of dehydrogenating phytoene and lacks cis -to- trans isomerase activity. However, the presence of both CrtIa and CrtIb allows the completion of the four desaturation steps that convert phytoene to lycopene. Therefore, we report a unique mechanism where two distinct CrtI-type desaturases cooperate to carry out the four desaturation steps required for lycopene formation. In addition, we show that there is a difference in substrate recognition between the two desaturases; CrtIa dehydrogenates carotenes in the cis conformation, whereas CrtIb dehydrogenates carotenes in the trans conformation. [source] 2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulansFEBS JOURNAL, Issue 12 2001Characterization, comparison of both enzymes In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo -2-methylisocitrate; the erythro -diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources (, 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mm pyruvate, 150 mm succinate and 10 µm PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved. [source] agr -Genotyping and transcriptional analysis of biofilm-producing Staphylococcus aureusFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2007Viviana Cafiso Abstract We investigated the correlation between biofilm production and the accessory-gene-regulator (agr) in 29 strains isolated from catheter-associated infections compared to a control group (30 isolates). All strains were tested for their ability to produce biofilm in a static system, and their agr genotype was determined. ScaI-restriction fragment length polymorphism for agr -typing showed that strong biofilm-producing strains belong to agr - type II. We found two new agr -variants, and sequence analysis of the three PCR products revealed the insertion of IS256 within the agr- locus. Biofilm production was assessed and correlated with agr functionality, with the expression of the ica -operon and of two transcriptional regulators, sarA and rsbU. Our data show that agr -II strains produce large amounts of biofilm, possess a defective agr -system show early transcription of icaA and are defective in haemolysin activity, icaR transcription, and in the expression of the ,B activator rsbU. Strains with agrIII are medium biofilm producers, have an inactive agr -system, but express icaAR and rsbU in the late- and postexponential growth phases. In agrI,IV- and -IA-variants, medium or weak biofilm production was found. In these strains, the agr -locus was fully functional, rsbU- icaR and icaA were found in the late- and/or postexponential phases. Biofilm production was not affected by sarA. [source] Developing live Shigella vaccines using , Red recombineeringFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2006Ryan T. Ranallo Abstract Live attenuated Shigella vaccines have shown promise in inducing protective immune responses in human clinical trials and as carriers of heterologous antigens from other mucosal pathogens. In the past, construction of Shigella vaccine strains relied on classical allelic exchange systems to genetically engineer the bacterial genome. These systems require extensive in vitro engineering of long homologous sequences to create recombinant replication-defective plasmids or phage. Alternatively, the ,red recombination system from bacteriophage facilitates recombination with as little as 40 bp of homologous DNA. The process, referred to as recombineering, typically uses an inducible ,red operon on a temperature-sensitive plasmid and optimal transformation conditions to integrate linear antibiotic resistance cassettes flanked by homologous sequences into a bacterial genome. Recent advances in recombineering have enabled modification of genomic DNA from bacterial pathogens including Salmonella, Yersinia, enteropathogenic Escherichia coli, or enterohemorrhagic E. coli and Shigella. These advances in recombineering have been used to systematically delete virulence-associated genes from Shigella, creating a number of isogenic strains from multiple Shigella serotypes. These strains have been characterized for attenuation using both in vivo and in vitro assays. Based on this data, prototypic Shigella vaccine strains containing multiple deletions in virulence-associated genes have been generated. [source] Uncultured Archaea in a hydrothermal microbial assemblage: phylogenetic diversity and characterization of a genome fragment from a euryarchaeoteFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Hélène Moussard Abstract The polychaete Alvinella pompejana lives in organic tubes on the walls of active hydrothermal chimneys along the East Pacific Rise. To examine the diversity of the archaeal community associated with the polychaete tubes, we constructed libraries by direct PCR amplification and cloning of 16S rRNA genes. Almost half of the sequences of the 16S rRNA gene libraries clustered with uncultured archaeal groups. In an effort to access genomic information from uncultured archaeal members we further constructed a fosmid library from the same DNA source. One of the clones, Alv-FOS5, was sequenced completely. Its sequence analysis revealed an incomplete rRNA operon and 32 predicted ORFs. Seventeen of these ORFs have been assigned putative functions, including transcription and translation, cellular processes and signalling, transport systems and metabolic pathways. Phylogenetic analyses of the 16S rRNA gene suggested that Alv-FOS5 formed a new lineage related to members of Deep-Sea Hydrothermal Vent Euryarchaeota group II. Phylogenetic analyses of predicted proteins revealed the existence of likely cases of horizontal gene transfer, both between Crenarchaeota and Euryarchaeota and between Archaea and Bacteria. This study is the first step in using genomics to reveal the physiology of an as yet uncultured group of archaea from deep-sea hydrothermal vents. [source] The stress response protein Gls24 is induced by copper and interacts with the CopZ copper chaperone of Enterococcus hiraeFEMS MICROBIOLOGY LETTERS, Issue 1 2010Jivko V. Stoyanov Abstract Intracellular copper routing in Enterococcus hirae is accomplished by the CopZ copper chaperone. Under copper stress, CopZ donates Cu+ to the CopY repressor, thereby releasing its bound zinc and abolishing repressor,DNA interaction. This in turn induces the expression of the cop operon, which encodes CopY and CopZ, in addition to two copper ATPases, CopA and CopB. To gain further insight into the function of CopZ, the yeast two-hybrid system was used to screen for proteins interacting with the copper chaperone. This led to the identification of Gls24, a member of a family of stress response proteins. Gls24 is part of an operon containing eight genes. The operon was induced by a range of stress conditions, but most notably by copper. Gls24 was overexpressed and purified, and was shown by surface plasmon resonance analysis to also interact with CopZ in vitro. Circular dichroism measurements revealed that Gls24 is partially unstructured. The current findings establish a novel link between Gls24 and copper homeostasis. [source] Identification of a new promoter for the response regulator rcsB expression in Salmonella enterica serovar TyphimuriumFEMS MICROBIOLOGY LETTERS, Issue 2 2009María de las Mercedes Pescaretti Abstract The RcsCDB (Rcs) phosphorelay system regulates capsule synthesis, flagella production and other cellular activities in several enteric bacteria. This system consists of three proteins: the sensor RcsC, the cognate response regulator RcsB and the histidine-containing phosphotransfer protein RcsD (YojN), which is hypothesized to act as an intermediary in the phosphotransfer from RcsC to RcsB. The rcsC gene is convergently transcribed toward rcsB, which follows rcsD in what appears to be a two-gene operon. Here, it is reported that the overproduction of the rcsB gene represses rcsD transcription, but has a weak effect on its own expression. We demonstrated that the differential rcsD and rcsB expression is due to the activity of two promoters to transcribe the rcsB gene: (1) PrcsDB located upstream of rcsD and (2) PrcsB located within the rcsD coding region. In addition, here it was demonstrated that in Salmonella typhimurium, PrcsB is important to activate the rcsB expression during the stationary growth phase. [source] Construction and characterization of Bacillus subtilis deletion mutants lacking the prophage 2 - trnS regionFEMS MICROBIOLOGY LETTERS, Issue 2 2006Genki Akanuma Abstract During development of a novel method for constructing a series of deletions in Bacillus subtilis using an isogenic set of gene-disrupted mutants created by integration of pMutin, deletion of the trnS operon, consisting of seven tRNA genes, was found to affect cell growth, development of competence and spore formation. A suppressor (sts1) of the ,trnS mutant was isolated, sequenced and found to have undergone a single base change, CAG to GAG, in the first anticodon of tRNALeu, in the trnB operon. [source] Analysis of the germination of spores of Bacillus subtilis with temperature sensitive spo mutations in the spoVA operonFEMS MICROBIOLOGY LETTERS, Issue 1 2004Venkata Ramana Vepachedu Abstract A Bacillus subtilis strain with a base substitution in the ribosome-binding site of spoVAC was temperature sensitive (ts) in sporulation and spores prepared at the permissive temperature were ts in l -alanine-triggered germination, but not in germination with Ca2+ -dipicolinic acid (DPA) or dodecylamine. Spores of a ts spo mutant with a missense mutation in the spoVAC coding region were not ts for germination with l -alanine, dodecylamine or Ca2+ -DPA. These findings are discussed in light of the proposal that SpoVA proteins are involved not only in DPA uptake during sporulation, but also in DPA release during nutrient-mediated spore germination. [source] Phylogenetic reconstruction of Gram-positive organisms based on comparative sequence analysis of molecular chaperones from the ruminal microorganism Ruminococcus flavefaciens FD-1FEMS MICROBIOLOGY LETTERS, Issue 1 2003Dionysios A. Antonopoulos Abstract Primers designed on the basis of nucleotide sequences conserved in DnaK and GroEL from Gram-positive organisms were used to PCR amplify internal regions of the cognate genes from the anaerobic ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1. Genome walking was then utilized to elucidate the remainder of the sequences in addition to upstream and downstream regions. The full sequence of the gene encoding the GroES protein (groES) was found directly upstream from groEL. The deduced amino acid sequence of the groEL gene showed the highest homology with the amino acid sequence of the Clostridium thermocellum GroEL protein (72% amino acid identity). Similarly, translation of the groES nucleotide sequence showed highest homology to the C. thermocellum GroES protein (61% amino acid identity). Analysis of the upstream region of this chaperonin operon revealed a CIRCE regulatory element 45 bp upstream from the putative start of the groES ORF. The deduced amino acid sequence of the putative dnaK gene showed the highest homology with the amino acid sequence of the Clostridium acetobutylicum DnaK protein (68% amino acid identity). Phylogenetic analyses based on the translated sequences reiterate this relationship between R. flavefaciens and the Clostridia. However, when the nucleotide sequences of Gram-positive organisms are analyzed, a different topology occurs of the relationship between high- and low-G+C Gram-positive organisms to the 16S rRNA interpretation. [source] Identification and characterization of KvgAS, a two-component system in Klebsiella pneumoniae CG43FEMS MICROBIOLOGY LETTERS, Issue 1 2003Yi-Chyi Lai Abstract A two-component system encoding gene cluster kvgAS that is present only in virulent Klebsiella pneumoniae CG43 was isolated and its sequence determined. RT-PCR and Southern analysis demonstrated that kvgAS is organized as an operon. No apparent effect of a kvgS deletion on bacterial virulence was observed in a mouse peritonitis model. In the presence of paraquat or 2,2-dipyridyl, the activity of kvgAS promoter in the kvgS mutant was found to be reduced to half of the level in the wild-type strain. The data suggest that the KvgAS system is autoregulated and plays a role in countering free radical stresses and sensing iron-limiting conditions. [source] Bacillus subtilis contains a cyclodextrin-binding protein which is part of a putative ABC-transporterFEMS MICROBIOLOGY LETTERS, Issue 1 2001Annette Kamionka Abstract Bacillus subtilis is able to grow on ,-, ,- and ,-cyclodextrins as a carbon source via a yet unknown metabolizing system. Sequence analysis of the B. subtilis genome reveals that the putative yvfK-yvfO operon seems to be involved in cyclodextrin utilization, containing the open reading frame yvfK, now termed cycB. The amino acid sequence derived from the DNA sequence bears high similarities to solute-binding proteins from B. subtilis, as well as to cymE from Klebsiella oxytoca and malE from Escherichia coli, both encoding solute-binding proteins able to interact with cyclodextrins. A [His]6 -tagged variant of CycB from B. subtilis was constructed, overproduced in E. coli and purified. The modified protein has been used to study its substrate specificity by surface plasmon resonance and fluorescence spectroscopy. From these data, CycB can be classified as a cyclodextrin-binding protein which interacts with all three natural cyclodextrins: ,, , and ,, thereby showing the highest affinity to ,-cyclodextrin. [source] Differential regulation of amoA and amoB gene copies in Nitrosomonas europaeaFEMS MICROBIOLOGY LETTERS, Issue 2 2000Lisa Y Stein Abstract Nitrosomonas europaea contains two nearly identical copies of the operon, amoCAB, which encodes the ammonia monooxygenase (AMO) enzyme. Cells of N. europaea containing single mutations in either amoA or amoB gene copies were incubated in ammonium both prior to and after exposure to acetylene or light. For each strain, the O2 consumption rates and amounts of AmoA polypeptide, the active site-containing subunit of AMO, produced in each strain were determined. Strains carrying a mutation in either the amoA2 or amoB2 genes responded similarly to wild-type cells, but the strains carrying mutations in the amoA1 or amoB1 genes responded differently from the wild-type, or from each other. These results suggest that the copies of amoA and amoB are differentially regulated upon exposure to different external stimuli. [source] Occurrence of the wattle wilt pathogen, Ceratocystis albifundus on native South African treesFOREST PATHOLOGY, Issue 5 2007J. Roux Summary Ceratocystis albifundus causes the disease known as wattle wilt of non-native Acacia mearnsii trees in South Africa, Uganda and Kenya. Infection results in rapid wilt and death of susceptible trees and stem cankers on more tolerant trees. It has been suggested that C. albifundus is indigenous to southern Africa, possibly having spread from native Protea spp. to non-native A. mearnsii and A. decurrens trees. Although C. albifundus has been collected from Protea spp., these reports are based on limited records for which only aged herbarium specimens exist. During surveys of wound-infecting fungi on native tree species in South Africa, a fungus resembling C. albifundus was collected from Protea gaguedi, Acacia caffra, Burkea africana, Combretum molle, C. zeyheri, Faurea saligna, Ochna pulchra, Ozoroa paniculosa and Terminalia sericea. The identity of the fungus was confirmed as C. albifundus, using comparisons of DNA sequence data for the ITS and 5.8S gene of the rRNA operon. In pathogenicity trials, lesions were produced on C. molle and A. caffra, with some trees beginning to die at the termination of the experiment. This study represents the first report of C. albifundus from native tree species in South Africa and provides unequivocal evidence that the fungus occurs naturally on native Protea spp. The wide host range of C. albifundus, as well as its abundance on these indigenous hosts lends further support to the view that it is a native African pathogen. [source] Sirococcus shoot blight on Picea spinulosa in BhutanFOREST PATHOLOGY, Issue 1 2007T. Kirisits Summary During a recent survey of forest tree diseases in Western and Central Bhutan, Sirococcus shoot blight and an associated Sirococcus sp. were found on saplings and mature trees of Eastern Himalayan spruce (Picea spinulosa). Based on morphological characteristics and DNA sequence comparisons of the ITS region of the rDNA operon, representative isolates from Bhutan were unequivocally identified as Sirococcus conigenus. The DNA sequence data also showed that these isolates belong to the P group of S. conigenus. To our best knowledge, this is the first report of Sirococcus shoot blight from the Himalayas or any other part of Asia. Sirococcus conigenus does not appear to cause dramatic damage at the moment, but this fungus has the potential to cause severe disease problems on P. spinulosa in Bhutan. [source] Mycosphaerella species associated with leaf disease of Eucalyptus globulus in EthiopiaFOREST PATHOLOGY, Issue 4 2006Alemu Gezahgne Summary Eucalyptus spp. are among the most widely planted exotic trees in Ethiopia. Several damaging leaf pathogens are known from Eucalyptus spp. worldwide. Of these, Mycosphaerella spp. are among the most important, causing the disease known as Mycosphaerella leaf disease (MLD). Characteristic symptoms of MLD include leaf spot, premature defoliation, shoot and twig dieback. Recent disease surveys conducted in Ethiopian Eucalyptus plantations have revealed disease symptoms similar to those caused by Mycosphaerella spp. These symptoms were restricted to E. globulus trees growing in several localities in south, south western and western Ethiopia. The aim of this study was to identify the fungi associated with this disease. This was achieved by examining ascospore germination patterns, anamorph associations and sequence data from the Internal Transcribed Spacer (ITS) region of the rRNA operon, for representative isolates. Several different ascospore germination patterns were observed, suggesting that more than one species of Mycosphaerella is responsible for MLD on E. globulus in Ethiopia. Analysis of sequence data showed that three Mycosphaerella spp., M. marksii, M. nubilosa and M. parva were present. This is the first report of these three species from Ethiopia and represents a valuable basis on which to build further studies in the region. Résumé Les Eucalyptus comptent parmi les essences d'arbres exotiques les plus plantées en Ethiopie. Plusieurs pathogènes foliaires sont connus dans le monde pour occasionner des dégâts sur Eucalyptus. Parmi ceux-ci, les espèces de Mycosphaerella sont parmi les plus importantes, causant la maladie connue comme Maladie Foliaire àMycosphaerella (MFM, MLD en anglais). Les symptômes caractéristiques de la MFM comprennent des taches foliaires, une défoliation précoce et des dépérissements de pousses et de rameaux. Des campagnes de surveillance menées récemment dans les plantations éthiopiennes d'Eucalyptus ont révélé la présence de tels symptômes. Ces symptômes sont uniquement observés sur E. globulus dans plusieurs localités du sud, sud-ouest et ouest de l'Ethiopie. L'objectif de cette étude était d'identifier les champignons associés à cette maladie. Pour cela, des isolats représentatifs ont étéétudiés pour les modalités de germination des ascospores, les anamorphes associés ainsi que les données de séquence de la région ITS de l'opéron ADNr. Différentes modalités de germination des ascospores ont été observées, suggérant que plusieurs espèces de Mycosphaerella seraient associées à la MFM sur E. globulus en Ethiopie. L'analyse des données de séquence a montré la présence de 3 espèces : M. marksii, M. nubilosa et M. parva. Ceci constitue la première mention de ces 3 espèces en Ethiopie et une première étape pour envisager d'autres études dans cette région. Zusammenfassung Eucalyptus -Arten sind die am häufigsten angepflanzten exotischen Bäume in Äthiopien. An Eucalyptus kommen verschiedene Blattkrankheiten vor, wobei die Mycosphaerella -Arten als Verursacher der Mycosphaerella -Blattkrankheit (MLD) am bedeutendsten sind. Charakteristische Symtpome der MLD sind Blattnekrosen und vorzeitiger Blattfall sowie Trieb- und Zweigsterben. Bei der Inventur von Krankheiten in äthiopischen Eucalyptusplantagen wurden Symptome entdeckt, die denen von Mycosphaerella spp. ähnlich waren. Diese traten nur an E. globulus lokal in S-, SW- und W-Äthiopien auf. Ziel dieser Untersuchung war es, die damit assoziierten Pilze zu identifizieren. Hierzu wurde an repräsentativen Isolaten das Keimverhalten der Ascosporen, das Vorkommen von Anamorphen und die ITS-Sequenz des rRNA-Operons untersucht. Es wurden verschiedene Keimungstypen der Ascosporen beobachtet, was darauf schliessen liess, dass mehr als eine Mycosphaerella -Art für die Krankheit an E. globulus in Äthiopien verantwortlich ist. Anhand der Sequenzen wurden M. marksii, M. nubilosa und M. parva identifiziert. Dies ist der Erstnachweis für diese drei Arten in Äthiopien und eine Grundlage für weitere Studien. [source] | ||||||||||||||||||||||||||||||||||||||||