- Volume 150, Issue 12, 2004

Myxococcus xanthus is a gliding bacterium that possesses two motility systems, the adventurous (A-motility) and social (S-motility) systems. A-motility is used for individual cell gliding, while S-motility is used for gliding in multicellular groups. Video microscopy studies showed that nla24 cells are non-motile on agar surfaces, suggesting that the nla24 gene product is absolutely required for both A-motility and S-motility under these assay conditions. S-motility requires functional type IV pili, wild-type LPS O-antigen, and an extracellular matrix of exopolysaccharide (EPS) and protein called fibrils. The results of expression studies and tethering assays indicate that the nla24 mutant has functional type IV pili. The nla24 mutant also produces wild-type LPS. However, several lines of evidence suggest that the nla24 mutant is defective for production of the EPS portion of the fibril matrix. The nla24 mutant is also defective for transcription of two genes (aglU and cglB) known to be required for A-motility, which is consistent with the idea that nla24 cells are defective for A-motility. Based on these findings, it is proposed that the putative transcriptional activator Nla24 regulates a subset of genes that are important for A-motility and S-motility in M. xanthus.

One of the most common pathways for the export of O-specific lipopolysaccharide (LPS) across the plasma membrane requires the participation of the Wzx protein. Wzx belongs to a family of integral membrane proteins that share little conservation in their primary amino acid sequence, making it difficult to delineate functional domains. This paper reports the cloning and expression in Escherichia coli K-12 of various Wzx homologues from different bacteria as FLAG epitope-tagged protein fusions. A reconstitution system for O16 LPS synthesis was used to assess the ability of each Wzx protein to complement an E. coli K-12 Δwzx mutant. The results demonstrate that Wzx proteins from O-antigen systems that use N-acetylglucosamine or N-acetylgalactosamine for the initiation of the biosynthesis of the O repeat can fully complement the formation of O16 LPS. Partial complementation was seen with Wzx from Pseudomonas aeruginosa, a system that uses N-acetylfucosamine in the initiation reaction. In contrast, there was negligible complementation with the Wzx protein from Salmonella enterica, a system in which galactose is the initiating sugar. These results support a model whereby the first sugar of the O repeat can be recognized by the O-antigen translocation machinery.

CcaR is a positive-acting transcriptional regulator involved in cephamycin C and clavulanic acid biosynthesis in Streptomyces clavuligerus. Previous sequence analyses of the ccaR gene revealed two possible start codons, an ATG, and a GTG located in-frame 18 bp downstream of the ATG. To determine the true start codon, ccaR was expressed, either from the GTG or ATG codon, in Escherichia coli. A protein product was only obtained from the ATG construct. Similarly, ccaR constructs originating from ATG or GTG and designed for expression from a glycerol-regulated promoter in Streptomyces species were prepared and used to complement a S. clavuligerus ccaR mutant. Bioassays showed that only the ATG construct could complement the ccaR mutant to restore cephamycin C production, and Western analysis confirmed the presence of CcaR in the mutant complemented with the ATG construct only. To ensure that expression of ccaR from its native promoter also initiated at the ATG rather than GTG, a conservative point mutation was introduced into ccaR, converting the GTG to GTC. The GTC construct still fully complemented a ccaR mutant, confirming that ATG is the true start codon. Inspection of the region upstream of ccaR by S1 nuclease protection and primer extension analyses indicated the presence of two transcript start points that mapped to residues located 74 and 173 bp upstream of the ATG codon.

The phycobilisome is a large pigment-protein assembly that harvests light energy for photosynthesis. This supramolecular complex is composed of two main structures: a core substructure and peripheral rods. Linker polypeptides assemble phycobiliproteins within these structures and optimize light absorption and energy transfer. Mutations have been constructed in three rod-linker-coding genes located in the cpc operon of Synechocystis sp. strain PCC6803. The cpcC1 gene encoding the 33 kDa linker is found to be epistatic to cpcC2 encoding the 30 kDa linker, indicating a specific role for each of these two linkers in rod growth. This corroborates studies on the sequential degradation of phycobilisomes upon nitrogen starvation. Three allelic mutants affecting cpcC2 revealed a polar effect of commonly used cassettes (aphI, aadA) on the operon steady-state transcripts and an effect of rod linker availability on the amount of phycocyanin incorporated in the phycobilisome. This led to the proposal that regulation of rod length could occur through processing of transcripts upstream of the cpcC2 gene.

Pst1p was previously identified as a protein secreted by yeast regenerating protoplasts, which suggests a role in cell wall construction. ECM33 encodes a protein homologous to Pst1p, and both of them display typical features of GPI-anchored proteins and a characteristic receptor L-domain. Pst1p and Ecm33p are both localized to the cell surface, Pst1p being at the cell membrane and possibly also in the periplasmic space. Here, the characterization of pst1Δ, ecm33Δ and pst1Δ ecm33Δ mutants is described. Deletion of ECM33 leads to a weakened cell wall, and this defect is further aggravated by simultaneous deletion of PST1. As a result, the ecm33Δ mutant displays increased levels of activated Slt2p, the MAP kinase of the cell integrity pathway, and relies on a functional Slt2-mediated cell integrity pathway to ensure viability. Analyses of model glycosylated proteins show glycosylation defects in the ecm33Δ mutant. Ecm33p is also important for proper cell wall ultrastructure organization and, furthermore, for the correct assembly of the mannoprotein outer layer of the cell wall. Pst1p seems to act in the compensatory mechanism activated upon cell wall damage and, in these conditions, may partially substitute for Ecm33p.

DNA ligases are essential enzymes in cells due to their ability to join DNA strand breaks formed during DNA replication. Several temperature-sensitive mutant strains of Escherichia coli, including strain GR501, have been described which can be complemented by functional DNA ligases. Here, it is shown that the ligA251 mutation in E. coli GR501 strain is a cytosine to thymine transition at base 43, which results in a substitution of leucine by phenylalanine at residue 15. The protein product of this gene (LigA251) is accumulated to a similar level at permissive and non-permissive temperatures. Compared to wild-type LigA, at 20 °C purified LigA251 has 20-fold lower ligation activity in vitro, and its activity is reduced further at 42 °C, resulting in 60-fold lower ligation activity than wild-type LigA. It is proposed that the mutation in LigA251 affects the structure of the N-terminal region of LigA. The resulting decrease in DNA ligase activity at the non-permissive temperature is likely to occur as the result of a conformational change that reduces the rate of adenylation of the ligase.

In a previous environmental survey of a polluted area, the authors identified two catechol 2,3-dioxygenase (C23O) sequences predominant in environmental bacterial isolates mineralizing benzene and/or toluene and also in soil DNA extracts. In the present study, using information of stable operon arrangement and conserved gene sequences, the complete C23O ORFs of these two variants were cloned, sequenced and overexpressed. The variants differ in six nucleotide positions, and the putative protein sequences differ only by a single amino acid, Tyr or His, at position 218. Even though the three-dimensional model does not suggest a significant influence of such an amino acid substitution on enzyme function, the Tyr218 variant differed significantly from the His218 variant in lower turnover number and in lower apparent K m for catecholic substrates. These results are evidence of the importance for enzyme function of amino acids not directly influencing active site structure and prove the utility of recovering polymorphisms evolved and selected for special functions in natural ecosystems.

Genetic diversity among 86 Pasteurella multocida isolates was investigated by comparative sequence analysis of a 1468 bp fragment of the 16S rRNA gene. The strains included 79 field isolates recovered from birds (poultry) (22), cattle (21), pigs (26) and sheep (10) within England and Wales, four Asian isolates associated with bovine haemorrhagic septicaemia, and the type strains of the three subspecies of P. multocida. Dulcitol and sorbitol fermentation patterns were also determined to establish correlations between subspecies status and phylogenetic relatedness. Nineteen 16S rRNA types were identified, but these were clustered into two distinct phylogenetic lineages, A and B. Sequences within lineages A and B had a mean number of nucleotide differences of 21·12±3·90. Isolates within lineage A were associated with birds, cattle, pigs and sheep, whereas those belonging to lineage B were recovered from birds and a cat. Eighty-seven per cent of the isolates were classified as P. multocida subsp. multocida by dulcitol and sorbitol fermentation patterns, but these have diverse 16S rRNA gene sequences that were represented in both lineages A and B. Avian P. multocida subsp. septica isolates were associated exclusively with lineage B, but bovine P. multocida subsp. septica isolates were present in lineage A. P. multocida subsp. gallicida isolates of avian, bovine and porcine origin represent a homogeneous group within lineage A, but they have the same 16S rRNA type as certain P. multocida subsp. multocida isolates. These findings provide strong support for the view that dulcitol and sorbitol fermentation patterns are inaccurate indicators of genetic relatedness among P. multocida strains. Avian capsular type B isolates and capsular type B and E isolates associated with haemorrhagic septicaemia of cattle and water buffaloes are closely related and form a distinct cluster within lineage A. The current subspecies nomenclature of P. multocida neither accurately reflects the 16S rRNA-based phylogenetic relationships among isolates nor does it adequately encompass the full range of diversity within the species. The study provides a 16S rRNA-based evolutionary framework that will form the basis of further studies into the genetic diversity of P. multocida and will also help in the reclassification of the species.

Using an established spotted cDNA microarray platform, the nature of changes in the transcriptional profiles of 2200 unique genes from the chestnut blight fungus Cryphonectria parasitica in response to the absence of either the Gα subunit CPG-1 or the Gβ subunit CPGB-1 has been explored. It is reported that 216 transcripts were altered in accumulation in the Δcpg-1 strain and 163 in the Δcpgb-1 strain, with a considerable overlap (100 genes) that were changed in both cases. Of note, these commonly altered transcripts were changed in the same direction in every instance, thus suggesting a considerable redundancy in pathway control or extensive crosstalk. To further knowledge of the potential impact on G-protein-signalling of infection by hypovirus CHV1-EP713, the accumulation of CPG-1 and CPGB-1 was also investigated by Western analysis. It was demonstrated that both signalling components were reduced in abundance to approximately 25 % of wild-type levels, while their transcripts were slightly elevated. Comparison of a list of genes with altered expression in the presence of CHV1-EP713 to the data obtained in the absence of either G-protein subunit showed that more than one-half of all the transcripts changed by hypovirus infection were also changed in at least one G-protein mutant strain, with one-third being changed in both. Significantly, 95 % of the co-changed genes were altered in the same direction. These data provide the first evidence for modulation of Gβ protein levels as well as the Gβγ-signalling pathways by hypovirus infection, and support the hypothesis that modification of G-protein-signalling via both Gα and Gβγ provides for a significant contribution to hypovirus-mediated phenotype.

Variable-number tandem repeats (VNTRs) have been shown to be a powerful tool in the determination of evolutionary relationships and population genetics of bacteria. The sequencing of a number of Staphylococcus aureus genomes has allowed the identification of novel VNTR sequences in S. aureus, which are similar to those used in the study of the evolution of Mycobacterium tuberculosis clades. Seven VNTRs, termed staphylococcal interspersed repeat units (SIRUs), distributed around the genome are described, occurring in both unique and multiple sites, and varying in length from 48 to 159 bp. Variations in copy numbers were observed in all loci, within both the sequenced genomes and the UK epidemic methicillin-resistant S. aureus (EMRSA) isolates. Clonally related UK EMRSA isolates were clustered using SIRUs, which provided a greater degree of discrimination than multi-locus sequence typing, indicating that VNTRs may be a more appropriate evolutionary marker for studying transmission events and the geographical spread of S. aureus clades.

Vibrio cholerae is the aetiological agent of the deadly diarrhoeal disease cholera. In this study the 7·5 kb Vibrio seventh pandemic island-II (VSP-II) that is unique to V. cholerae El Tor and O139 serogroups was analysed and it was found to be part of a novel 26·9 kb genomic island (GEI) encompassing VC0490–VC0516. The low-GC-content VSP-II encompassed 24 predicted ORFs, including DNA repair and methyl-accepting chemotaxis proteins, a group of hypothetical proteins and a bacteriophage-like integrase adjacent to a tRNA gene. Interestingly, V. cholerae ORFs VC0493–VC0498, VC0504–VC0510 and VC0516, which encodes an integrase, were homologous to Vibrio vulnificus strain YJ016 ORFs VV0510–VV0516, VV0518–VV0525 and VV0560, which also encodes an integrase, respectively. Some ORFs showed amino acid identities greater than 90 % between the two species in these regions. In V. vulnificus strain YJ016, a 43·4 kb low-GC-content (43 %) GEI encompassing ORFs VV0509–VV0560 was identified and named V. vulnificus island-I (VVI-I). The 52 ORFs of VVI-I included a phosphotransferase system gene cluster, genes required for sugar metabolism and transposase genes. There was synteny and homology between the 5′ region of V. cholerae VSP-II and the 5′ region of V. vulnificus VVI-I; however, VVI-I contained an additional 31·5 kb of DNA between VV0526 and VV0560 in strain YJ016. A second V. vulnificus strain, CMCP6, did not contain the 43·4 kb VVI-I; in this strain two ORFs were found between the 5′ and 3′ flanking genes VV10636 and VV10632, showing 100 % identity to VV0508 and VV0561, respectively, which flank VVI-I.

Mutations in rirA of Rhizobium have been shown to deregulate expression of several genes that are normally repressed by iron. A conserved sequence, the iron-responsive operator (IRO), was identified near promoters of vbsC (involved in the synthesis of the siderophore vicibactin), rpoI (specifies an ECF σ factor needed for vicibactin synthesis) and the two fhuA genes (encoding vicibactin receptor). Removal of these IRO sequences abolished Fe-responsive repression. Most of these genes were constitutively expressed in the heterologous host, Paracoccus denitrificans, but introduction of the cloned rirA gene repressed expression of these Rhizobium genes in this heterologous host if the corresponding IRO sequences were also intact. These observations are the first to examine the mechanisms of RirA, which has no sequence similarity to well-known iron-responsive regulators such as Fur or DtxR. They provide strong circumstantial evidence that RirA is a transcriptional regulator that binds to cis-acting regulatory sequences near the promoters of at least some of the genes whose expression it controls in response to Fe availability.