The surface (S)-layer gene region of the Gram-positive bacterium Corynebacterium glutamicum ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of C. glutamicum ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5·97 kb DNA region that is absent from the C. glutamicum ATCC 13032 chromosome was identified. This region includes cspB, the structural gene encoding the S-layer protomer PS2, and six additional coding sequences. PCR experiments demonstrated that the respective DNA region is conserved in different C. glutamicum wild-type strains capable of S-layer formation. The DNA region is flanked by a 7 bp direct repeat, suggesting that illegitimate recombination might be responsible for gene loss in C. glutamicum ATCC 13032. Transfer of the cloned cspB gene restored the PS2− phenotype of C. glutamicum ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living C. glutamicum cells by atomic force microscopy. Furthermore, the promoter of the cspB gene was mapped by 5′ rapid amplification of cDNA ends PCR and the corresponding DNA fragment was used in DNA affinity purification assays. A 30 kDa protein specifically binding to the promoter region of the cspB gene was purified. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and peptide mass fingerprinting of the purified protein led to the identification of the putative transcriptional regulator Cg2831, belonging to the LuxR regulatory protein family. Disruption of the cg2831 gene in C. glutamicum resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of cspB gene expression in C. glutamicum.
Pseudomonas aeruginosa utilizes several xenosiderophores under conditions of iron limitation, including the citrate hydroxamate siderophore aerobactin. Analysis of the P. aeruginosa genome sequence revealed the presence of two genes, chtA (PA4675) and PA1365, encoding proteins displaying significant similarity to the aerobactin outer-membrane receptor, IutA, of Escherichia coli. The chtA and PA1365 genes were mutated by insertional inactivation and it was demonstrated that ChtA is the outer-membrane receptor for aerobactin. ChtA also mediated the utilization of rhizobactin 1021 and schizokinen, which are structurally similar to aerobactin. In contrast to the utilization of other xenosiderophores by P. aeruginosa, there was no apparent redundancy in the utilization of aerobactin, rhizobactin 1021 and schizokinen. The utilization of citrate hydroxamate siderophores by P. aeruginosa was demonstrated to be TonB1 dependent. A Fur box was identified in the region directly upstream of chtA and it was demonstrated by the in vivo Fur titration assay that this region is capable of binding Fur and accordingly that expression of chtA is iron regulated. The PA1365 mutant was unaffected in the utilization of citrate hydroxamate siderophores.
Inspection of the genomic DNA sequence of the oral anaerobe Porphyromonas gingivalis reveals that the micro-organism possesses the peroxide-sensing transcription activator OxyR, but not the superoxide-sensing transcription factor SoxR. Investigatation of oxidative-stress-responsive proteins in P. gingivalis by two-dimensional gel electrophoresis showed that two proteins were predominantly upregulated in oxidative conditions. In a P. gingivalis oxyR mutant these two proteins were not induced by treatment with hydrogen peroxide under aerobic conditions. By N-terminal amino acid sequencing, the two proteins were found to be superoxide dismutase and alkyl hydroperoxide reductase, encoded by sod and ahpC, respectively. Northern blot and lacZ fusion analyses revealed that P. gingivalis sod and ahpC were positively regulated by OxyR. Primer extension analysis located the promoter regions of sod and ahpC, and putative −35 boxes of these promoters were found immediately adjacent to their putative OxyR-binding sequences. Moreover, the promoter regions of sod and ahpC had the ability to bind P. gingivalis OxyR protein. These results demonstrate that P. gingivalis sod is one of the OxyR regulons, suggesting that OxyR functions as an intracellular redox sensor rather than a peroxide sensor in this organism. A sod gene of Bacteroides fragilis, which is taxonomically related to P. gingivalis, is inducible by redox stresses but not controlled by its OxyR. A DNA fragment including the B. fragilis sod promoter region could bind the P. gingivalis OxyR protein; however, a putative OxyR binding sequence within the DNA fragment was 14 bases distant from a putative −35 box of its promoter.
It has been shown previously that expression of the Streptomyces lividans clpP1P2 operon, encoding proteolytic subunits of the Clp complex, the clpC1 gene, encoding the ATPase subunit, and the lon gene, encoding another ATP-dependent protease, are all activated by ClgR. The ClgR regulon also includes the clgR gene itself. It is shown here that the degradation of ClgR and Lon is ClpP1/P2-dependent and that the two C-terminal alanines of these new substrates are involved in their stability. The ClpC1 protein, which does not end with two alanines, is also accumulated in a clpP1P2 mutant. The results presented here support the idea that ClpP1/P2 ensure post-translational control of ClgR regulon members, including ClgR itself.
Enterotoxigenic Escherichia coli (ETEC) is a primary cause of diarrhoea in infants in developing countries and in travellers to endemic regions. While several virulence genes have been identified on ETEC plasmids, little is known about the ETEC chromosome, although it is expected to share significant homology in backbone sequences with E. coli K-12. In the absence of genomic sequence information, the subtractive hybridization method and the more recently described optical mapping technique were carried out to determine the degree of genomic variation between virulent ETEC strain H10407 and the non-pathogenic E. coli K-12 strain MG1655. In one round of PCR-based suppression subtractive hybridization, 153 fragments representing sequences unique to strain H10407 were identified. blast searches indicated that few unique sequences showed homology to known pathogenicity island genes identified in related E. coli pathogens. A total of 65 fragments contained sequences that were either linked to hypothetical proteins or showed no homology to any known sequence in the database. The remaining sequences were either phage or prophage related or displayed homology to classifiable genes that function in various aspects of bacterial metabolism. The 153 unique sequences showed variable distribution across different ETEC strains including ETEC strain B7A, which is attenuated in virulence and lacked several H10407-specific sequences. Restriction-enzyme-based optical maps of strain H10407 were compared to in silico restriction maps of strain MG1655 and related E. coli pathogens. The 5·1 Mb ETEC chromosome was ∼500 kb greater in length than the chromosome of E. coli K-12, collinear with it and indicated several discrete regions where insertions and/or deletions had occurred relative to the chromosome of strain MG1655. No major inversions, transpositions or gross rearrangements were observed on the ETEC chromosome. Based on comparisons with known genomic sequences and related optical-map-based restriction site similarity, the sequence of the H10407 chromosome is expected to demonstrate ∼96 % identity with that of E. coli K-12.
A novel shuttle entrapment vector, pMMB2, was used to identify a large transposable element, TnPpa1 (44·3 kb), of Paracoccus pantotrophus DSM 11072. TnPpa1 has a composite structure with divergently oriented copies of a cryptic transposon, Tn3434 (Tn3 family), located at both termini. The core region of the element contains a large set of putative genes, whose products show similarity to enzymes involved in central intermediary metabolism (e.g. tricarboxylic acid cycle or 2-methylcitrate cycle), transporters, transcriptional regulators and conserved proteins of unknown function. A 4·2 kb DNA segment of TnPpa1 is homologous to a region of chromosome cII of Rhodobacter sphaeroides 2.4.1, which exemplifies the mosaic structure of this element. TnPpa1 is bordered by 5 bp long directly repeated sequences and is located within a mega-sized replicon, pWKS5, in the DSM 11072 genome. Spontaneous inversion of the core region of TnPpa1 was detected in the host genome. Analysis of the distribution of TnPpa1 in three other strains of P. pantotrophus revealed that this element was present exclusively within DSM 11072, which suggests its relatively recent acquisition by lateral transfer. The identification of TnPpa1 (which may be considered a transposable genomic island) provides evidence for the transposition and lateral transfer of large DNA segments of chromosomal origin (carrying various housekeeping genes), which may have a great impact on the evolution of bacterial genomes.
Acidobacterium capsulatum is the most thoroughly studied species of a new bacterial phylogenetic group designated the phylum Acidobacteria. Through a tblastn search, the A. capsulatum lexA gene has been identified, and its product purified. Electrophoretic mobility shift assays have shown that A. capsulatum LexA protein binds specifically to the direct repeat GTTCN7GTTC motif. Strikingly, this is also the LexA box of the Alphaproteobacteria, but had not previously been described outside this subclass of the Proteobacteria. In addition, a phylogenetic analysis of the LexA protein clusters together Acidobacterium and the Alphaproteobacteria, moving the latter away from their established phylogenetic position as a subclass of the Proteobacteria, and pointing to a lateral gene transfer of the lexA gene from the phylum Acidobacteria, or an immediate ancestor, to the Alphaproteobacteria. Lastly, in vivo experiments demonstrate that the A. capsulatum recA gene is DNA-damage inducible, despite the fact that a LexA-binding sequence is not present in its promoter region.
Previous work has shown that the Bacillus subtilis EzrA protein directly inhibits FtsZ ring assembly, which is required for normal cell division, and that loss of EzrA results in hyperstabilization of the FtsZ polymer in vivo. Here, it was found that in ezrA-disrupted cells, artificial expression of YneA, which suppresses cell division during the SOS response, and disruption of noc (yyaA), which acts as an effector of nucleoid occlusion, resulted in accumulation of multiple non-constricting FtsZ rings, inhibition of cell division, and synthetic lethality. Overexpression of the essential cell division protein FtsL suppressed the effect of ezrA disruption. FtsL overexpression recovered the delayed FtsZ ring constriction seen in ezrA-disrupted wild-type cells. Conversely, the absence of EzrA caused lethality in cells producing a lower amount of FtsL than wild-type cells. It has previously been reported that FtsL is recruited to the division site during the later stages of cell division, although its exact role is currently unknown. The results of this study suggest that FtsL and EzrA synergistically regulate the FtsZ ring constriction in B. subtilis. Interestingly, FtsL overexpression also suppressed the cell division inhibition due to YneA expression or Noc inactivation in ezrA-disrupted cells.
Two kinds of nucleoside hydrolases (NHs) encoded by rih1 and rih2 were cloned from Corynebacterium ammoniagenes using deoD- and gsk-defective Escherichia coli. Sequence analysis revealed that NH 1 was a protein of 337 aa with a deduced molecular mass of 35 892 Da, whereas NH 2 consisted of 308 aa with a calculated molecular mass of 32 310 Da. Experiments with crude extracts of IPTG-induced E. coli CGSC 6885(pTNU23) and 6885(pTNI12) indicated that the Rih1 enzyme could catalyse the hydrolysis of uridine and cytidine and showed pyrimidine-specific ribonucleoside hydrolase activity. Rih2 was able to hydrolyse both purine and pyrimidine ribonucleosides with the following order of activity – inosine>adenosine>uridine>guanosine>xanthosine>cytidine – and was classified in the non-specific NHs family. rih1 and rih2 deletion mutants displayed a decrease in cell growth on minimal medium supplemented with pyrimidine and purine/pyrimidine nucleosides, respectively, compared with the wild-type strain. Growth of each mutant was substantially complemented by introducing rih1 and rih2, respectively. Furthermore, disruption of both rih1 and rih2 led to the inability of the mutant to utilize purine and pyrimidine nucleosides as sole carbon source on minimal medium. These results indicated that rih1 and rih2 play major roles in the salvage pathways of nucleosides in this micro-organism.