We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.

Dimethyl sulfide (DMS) is a key compound in global sulfur and carbon cycles. DMS oxidation products cause cloud nucleation and may affect weather and climate. DMS is generated largely by bacterial catabolism of dimethylsulfoniopropionate (DMSP), a secondary metabolite made by marine algae. We demonstrate that the bacterial gene dddD is required for this process and that its transcription is induced by the DMSP substrate. Cloned dddD from the marine bacterium Marinomonas and from two bacterial strains that associate with higher plants, the N(2)-fixing symbiont Rhizobium NGR234 and the root-colonizing Burkholderia cepacia AMMD, conferred to Escherichia coli the ability to make DMS from DMSP. The inferred enzymatic mechanism for DMS liberation involves an initial step in which DMSP is modified by addition of acyl coenzyme A, rather than the immediate release of DMS by a DMSP lyase, the previously suggested mechanism.

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Rhizobium leguminosarum genome

The genome sequence of the α-proteobacterial N2-fixing symbiont of legumes, Rhizobium leguminosarum, is described, revealing a 'core' and an 'accessory' component.

Enhanced biological phosphorus removal (EBPR) selects for polyphosphate accumulating microorganisms to achieve phosphate removal from wastewater. We used high-resolution community proteomics to identify key metabolic pathways in 'Candidatus Accumulibacter phosphatis' (A. phosphatis)-mediated EBPR and to evaluate the contributions of co-existing strains within the dominant population. Overall, 702 proteins from the A. phosphatis population were identified. Results highlight the importance of denitrification, fatty acid cycling and the glyoxylate bypass in EBPR. Strong similarity in protein profiles under anaerobic and aerobic conditions was uncovered (only 3% of A. phosphatis-associated proteins exhibited statistically significant abundance differences). By comprehensive genome-wide alignment of 13 930 orthologous proteins, we uncovered substantial differences in protein abundance for enzyme variants involved in both core-metabolism and EBPR-specific pathways among the A. phosphatis population. These findings suggest an essential role for genetic diversity in maintaining the stable performance of EBPR systems and, hence, demonstrate the power of integrated cultivation-independent genomics and proteomics for the analysis of complex biotechnological systems.

The genetic diversity of symbiosis (Sym) plasmids was investigated in samples from two field populations of Rhizobiurn legurninosarurn biovar viceae that had previously been characterized for chromosomally-encoded enzyme electrophoretic polymorphism. Five overlapping cloned DNA fragments from the Sym plasmid pRLlJI were used as hybridization probes to identify restriction fragment variation in the homologous genes of the isolates. In addition, a clone of the P-galactosidase gene region was used as a probe, extending the data on chromosomal relatedness. The plasmid-encoded Sym region was very polymorphic (1 1.4% average DNA sequence divergence). Some isolates had the same Sym fragment pattern as pRLl JI, but most were very different. One was closely similar to pRL6J1, another widely-studied viceae plasmid. The distribution of plasmids across chromosomal backgrounds was far from random, as though the species were subdivided into compartments with largely separate plasmid pools. Nevertheless, indistinguishable plasmids were found in quite different genetic backgrounds, implying that plasmid transfer must occur in the field. This has implications for the population genetics and evolution of bacteria and for the release of genetically engineered strains.

SummaryThe Escherichia coli Tat apparatus is a membranebound protein translocase that serves to export folded proteins synthesized with N-terminal twinarginine signal peptides. The essential TatC component of the Tat translocase is an integral membrane protein probably containing six transmembrane helices. Sequence analysis identified conserved TatC amino acid residues, and the role of these side-chains was assessed by single alanine substitution. This approach identified three classes of TatC mutants. Class I mutants included F94A, E103A and D211A, which were completely devoid of Tat-dependent protein export activity and thus represented residues essential for TatC function. Cross-complementation experiments with class I mutants showed that coexpression of D211A with either F94A or E103A regenerated an active Tat apparatus. These data suggest that different class I mutants may be blocked at different steps in protein transport and point to the coexistence of at least two TatC molecules within each Tat translocon. Class II mutations identified residues important, but not essential, for Tat activity, the most severely affected being L99A and Y126A. Class III mutants showed no significant defects in protein export. All but three of the essential and important residues are predicted to cluster around the cytoplasmic N-tail and first cytoplasmic loop regions of the TatC protein. Escherichia coli TatA, TatB and TatC have been shown to be genuine integral inner membrane proteins and are present in the cell at a molar ratio estimated at 40:2:1 respectively (Jack et al., 2001;Sargent et al., 2001). Despite this large molar excess of TatA over the other Tat components, affinity-purified E. coli TatC protein can be isolated in an approximately equimolar complex with TatA and TatB (Bolhuis et al., 2001). Interestingly, the molar ratio of the thylakoidal Tat pathway components is slightly different from that reported for the E. coli system. In pea thylakoids, the Tha4:Hcf106:TatC molar ratio (essentially TatA:TatB:TatC) was reported as 8:5:1 . Some significant similarity is shared with the E. coli system, however, as Hcf106 and TatC (TatB and TatC) have also been shown to form a stable complex in the plant system .In this study, we have sought to define in more detail the structure and function of the essential TatC protein of the bacterial Tat system. TatC family proteins are the most highly conserved of all known Tat components, and homologues are encoded by the genomes of all bacteria that synthesize proteins with twin-arginine signal peptides, some plant nuclear and mitochondrial genomes and the chloroplast genomes of algae . Using a genetic approach consisting of site-directed mutagenesis followed by in vivo transcomplementation analysis, we identified conserved residues essential for TatC biological function. Results and discussion Site-directed mutagenesis of conserved TatC residuesThe E. coli tatC gene encodes a 28.9 kDa polypeptide of 258 amino acid residues Sargent et al., 1998 Amino acid determinants of Tat-dep...

Mutations in a Rhizobium leguminosarum gene, rirA (rhizobial iron regulator), caused high-level, constitutive expression of at least eight operons whose transcription is normally Fe-responsive and whose products are involved in the synthesis or uptake of siderophores, or in the uptake of haem or of other iron sources. Close homologues of RirA exist in other rhizobia and in the pathogen Brucella ; many other bacteria have deduced proteins with more limited sequence similarity. None of these homologues had been implicated in Femediated gene regulation. Transcription of rirA itself is about twofold higher in cells grown in Fe-replete than in Fe-deficient growth media. Mutations in rirA reduced growth rates in Fe-replete and -depleted medium, but did not appear to affect symbiotic N 2 fixation.

BackgroundThrough identification of highly expressed proteins from a mixed culture activated sludge system this study provides functional evidence of microbial transformations important for enhanced biological phosphorus removal (EBPR).Methodology/Principal FindingsA laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P. The microbial communities were dominated by the uncultured polyphosphate-accumulating organism “Candidatus Accumulibacter phosphatis”. When EBPR failed, the sludge was dominated by tetrad-forming α-Proteobacteria. Representative and reproducible 2D gel protein separations were obtained for all sludge samples. 638 protein spots were matched across gels generated from the phosphate removing sludges. 111 of these were excised and 46 proteins were identified using recently available sludge metagenomic sequences. Many of these closely match proteins from “Candidatus Accumulibacter phosphatis” and could be directly linked to the EBPR process. They included enzymes involved in energy generation, polyhydroxyalkanoate synthesis, glycolysis, gluconeogenesis, glycogen synthesis, glyoxylate/TCA cycle, fatty acid β oxidation, fatty acid synthesis and phosphate transport. Several proteins involved in cellular stress response were detected.Conclusions/SignificanceImportantly, this study provides direct evidence linking the metabolic activities of “Accumulibacter” to the chemical transformations observed in EBPR. Finally, the results are discussed in relation to current EBPR metabolic models.