Spatio-temporal diversity of free-living and particle-attached prokaryotes in the tropical lagoon of Ahe atoll (Tuamotu Archipelago) and its surrounding oceanic waters.

Spatio-temporal variability of prokaryotic water column communities inside and outside a Polynesian tropical lagoon subjected to pearl oysters farming was assessed in terms of abundance by quantitative PCR and diversity by DGGE. Communities and operational taxonomic units (OTUs) were analysed according to dry/rainy seasons and free-living/particle-attached state. Bacterial density was higher in the lagoon compared to ocean and a seasonal trend was observed. No influence of the localisation within lagoon or of the planktonic/attached states was noticed on bacterial abundance and diversity. The OTUs belonged to Cyanobacteria, to heterotrophic groups in Proteobacteria and Flavobacteria. Archaeal abundance showed seasonal tendency and particle-prevalence, but no effect of lagoon or oceanic location was observed. Lagoon and oceanic archaeal diversity were different and Euryarchaeota (MG-II, MBG, and Halobacteria) were detected. During the dry season, planktonic and particle-associated community differed, whereas at rainy season, both communities were similar and included members usually associated with coral.

Molecular, biochemical, and physiological approaches for understanding the ecology of denitrification.

One of the major challenges in microbial ecology for the future is to establish links between structural and functional biodiversity. This is particularly difficult when one is interested in a phylogenetically diversified function such as denitrification. The data banks are very rich in functional gene sequences (nirS in this study), but most of them were obtained from not yet cultivated bacteria, and thus must be supplemented by sequences of organisms from the environment for which we could associate a taxonomic position and physiological characteristics. Combined analysis including molecular (16S-rRNA or nirS genes), physiological, and biochemical approaches was carried out on a bacterial set of 89 strains isolated from marine sediment. The denaturing gradient gel electrophoresis (DGGE) technique was successfully applied on unclamped polymerase chain reaction (PCR) products of nirS genes to compare the picture of the biodiversity obtained with 16S rRNA and nirS genes. The diversity of nirS genes and denitrifier characteristics were found within several of the 16S rDNA phylotypes. In contrast, the nirS phylotypes were no diverse both with respect to 16S rDNA and to physiology and biochemistry of denitrification. Sequences of the nirS PCR products were very close to marine environmental clones and were analyzed within the same phylogenetic tree.

Comparison of methods for quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples.

Two PCR primer sets were developed for the detection and quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques. Quantification of cytochrome cd(1) denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method. Enumerations using both molecular techniques yielded similar results in seawater and sediment samples. However, both molecular techniques showed 1,000 or 10 times more cytochrome cd(1) denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting.

Aerobic and anaerobic metabolism of 6,10,14-trimethylpentadecan-2-one by a denitrifying bacterium isolated from marine sediments.

This report describes the metabolism of 6,10,14-trimethylpentadecan-2-one by a denitrifying bacterium (Marinobacter sp. strain CAB) isolated from marine sediments. Under aerobic and denitrifying conditions, this strain efficiently degraded this ubiquitous isoprenoid ketone. Several bacterial metabolites, 4,8,12-trimethyl-tridecan-1-ol, 4,8,12-trimethyltridecanal, 4,8,12-trimethyltridecanoic acid, Z-3,7-dimethylocten-2-oic acid, Z-3,7,11-trimethyldodecen-2-oic acid, and 6,10,14-trimethylpentadecan-2-ol, were formally identified, and different pathways were proposed to explain the formation of such isoprenoid compounds.

Isolation and characterization of a marine bacterium capable of utilizing 2-methylphenanthrene.

A marine bacterium isolated from a coastal hydrocarbon-polluted sediment has been described and attributed on the basis of its phenotypic and genotypic characteristics to the genus Sphingomonas sp. This strain was capable of using an alkylated phenanthrene 2-methylphenanthrene, as sole source of carbon and energy. In experiments, 2-methylphenanthrene (0.2 g/l) was added as crystals to the culture medium. After 5 days of aerobic growth at 30 degrees C, 70% was degraded and the complete dissipation occurred after 20 days. Furthermore, the strain could degrade various kinds of polyaromatic compounds, but failed to grow on aliphatic hydrocarbons.

Characterisation of the mcpA and mcpB genes capable of encoding methyl-accepting type chemoreceptors in Rhodobacter capsulatus.

Two contiguous mcp genes, mcpA and mcpB, transcribed from the same DNA strand and capable of encoding methyl-accepting chemotaxis proteins (Mcp) have been isolated from Rhodobacter capsulatus (Rc), sequences and overexpressed in Escherichia coli (Ec). The deduced proteins (McpA, 69 171 Da; McpB, 81 629 Da) show a structure similar to that of Ec Mcp. The products of mcpA and mcpB, overproduced in Ec, were recognized by anti-Ec Mcp (Trg) antibodies.

The global regulatory protein FruR modulates the direction of carbon flow in Escherichia coli.

The Escherichia coli fructose repressor, FruR, is known to regulate expression of several genes concerned with carbon utilization. Using a previously derived consensus sequence for FruR binding, additional potential operators were identified and tested for FruR binding in DNA band migration retardation assays. Operators in the control regions of operons concerned with carbon metabolism bound FruR, while those in operons not concerned with carbon metabolism did not. In vivo assays with transcriptional lacZ fusions showed that FruR controls the expression of FruR operator-containing genes encoding key enzymes of virtually every major pathway of carbon metabolism. Moreover, a fruR null mutation altered the rates of utilization of at least 36 carbon sources. In general, oxidation rates for glycolytic substances were enhanced while those for gluconeogenic substances were depressed. Alignment of FruR operators revealed that the consensus sequence for FruR binding is the same for operons that are activated and repressed by FruR and permitted formulation of a revised FruR-binding consensus sequence. The reported observations indicate that FruR modulates the direction of carbon flow by transcriptional activation of genes encoding enzymes concerned with oxidative and gluconeogenic carbon flow and by repression of those concerned with fermentative carbon flow.

Novel proteins of the phosphotransferase system encoded within the rpoN operon of Escherichia coli. Enzyme IIANtr affects growth on organic nitrogen and the conditional lethality of an erats mutant.

Two rpoN-linked delta Tn10-kan insertions suppress the conditionally lethal erats allele. One truncates rpoN while the second disrupts another gene (ptsN) in the rpoN operon and does not affect classical nitrogen regulation. Neither alter expression of era indicating that suppression is post-translational. Plasmid clones of ptsN prevent suppression by either disruption mutation indicating that this gene is important for lethality caused by erats. rpoN and six neighboring genes were sequenced and compared with sequences in the database. Two of these genes encode proteins homologous to Enzyme IIAFru and HPr of the phosphoenolpyruvate:sugar phosphotransferase system. We designate these proteins IIANtr (ptsN) and NPr (npr). Purified IIANtr and NPr exchange phosphate appropriately with Enzyme I, HPr, and Enzyme IIA proteins of the phosphoenolpyruvate: sugar phosphotransferase system. Several sugars and tricarboxylic acid cycle intermediates inhibited growth of the ptsN disruption mutant on medium containing an amino acid or nucleoside base as a combined source of nitrogen, carbon, and energy. This growth inhibition was relieved by supplying the ptsN gene or ammonium salts but was not aleviated by altering levels of exogenously supplied cAMP. These results support our previous proposal of a novel mechanism linking carbon and nitrogen assimilation and relates IIANtr to the unknown process regulated by the essential GTPase Era.

Effect of the FruR regulator on transcription of the pts operon in Escherichia coli.

The promoters of the pts operon of Escherichia coli are controlled by the cyclic AMP receptor protein (CRP) complexed with cAMP (CRP.cAMP). In addition, glucose stimulates pts operon expression in vivo. The pts promoter region has a fructose repressor (FruR)-binding site (the FruR box) that partially overlaps with one of the CRP.cAMP-binding sites. The effects of the pleiotropic transcriptional regulator FruR on pts operon expression were studied to determine whether the in vivo glucose effect on pts operon expression is mediated by FruR. In vitro, FruR can repress P1b transcription, which is activated by CRP.cAMP, and restore P1a transcription, which is repressed by CRP.cAMP. FruR can displace CRP.cAMP from its binding site in the presence of RNA polymerase even though FruR and CRP.cAMP can bind simultaneously to their partially overlapping binding sites in the absence of RNA polymerase. FruR had very little effect on the transcription of the P0 promoter, which is most important for regulation by glucose. Consistent with the in vitro results, pts P0 transcription did not increase as much in cells grown in the presence of fructose or in fruR- mutant cells as in cells grown in the presence of glucose. These results suggest that FruR alone does not mediate the in vivo glucose effect on pts operon expression.

Secretion of a temporally controlled cell-associated protein in Arthrobacter aureus: production of the protein and inactivation of its structural gene.

Arthrobacter aureus secretes 2 proteins of 22 and 100 kDa (P22 and P100, respectively). P22 is a protease, while P100, of unknown function, is associated with the cells before being released into the medium. An immunologically related P100 protein was also found in the culture supernatant of A. ureafasciens. Despite the fact that production of P22 and P100 proteins is induced by growth in the presence of bovine serum albumin, their synthesis is not always induced coordinately. Inactivation of the P100 chromosomal structural gene had no effect on growth characteristics of the mutant under inducing conditions.

Novel phosphotransferase system genes revealed by bacterial genome analysis: unique, putative fructose- and glucoside-specific systems.

Analyses of sequences made available through the Escherichia coli genome project in the 87.2-89.2-min and 81.5-84.5-min regions have revealed 2 putative operons encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The first putative operon, designated frv, includes 4 open reading frames (ORFs), ORFf147, ORFf485, ORFf356, and ORFf582, ORFf147 and ORFf485 comprise an Enzyme IIA-Enzyme IIBC pair of the PTS. The sequence similarity of ORFf485 to previously characterized fructose-specific Enzymes IIBC suggests that ORFf485 may be specific for fructose. ORFf147 encodes a protein with comparable degrees of sequence similarity to fructose and mannitol-specific Enzymes IIA as well as homologous proteins implicated in sigma 54-dependent transcriptional regulation. Unique features of this system include a detached IIA protein and the absence of a IIB domain duplication. ORFf356 and ORFf582 are functionally unidentified and nonhomologous to other ORFs in the current protein databases, but ORFf582 contains 2 N-terminal helix-turn-helix motifs, suggestive of a role in frv operon transcriptional regulation. The second putative operon, designated glv, includes 3 ORFs, ORFf455, ORFf161, and ORFf212. We suggest that ORFf455 was incorrectly assigned and should be designated ORFf368. ORFf368 and ORFf161 encode an Enzyme IIC and IIB pair of the PTS showing greatest sequence similarity to Enzymes II specific for sugars of the gluco configuration. ORFf212 encodes a protein with sequence similarity to a phospho-beta-glucosidase and an alpha-galactosidase. No putative transcriptional regulator of the glv operon was found. This operon is the first one encoding a putative PTS permease with detached Enzymes IIB and IIC and lacking an Enzyme IIA. It is suggested that both the frv and glv operons are cryptic in E. coli and that additional genes encoding novel PTS-related proteins will be revealed by bacterial genome sequence analyses.

Characterization of an endoserine protease secreted by Arthrobacter aureus.

A 22-kDa endoserine protease secreted by an Arthrobacter aureus strain was identified. The optimal temperature for this protease was 70 degrees C. Protease production was maximal at the end of the exponential growth phase, and the protease was induced by amino acids and peptides and repressed by carbon sources.