The srhSR gene pair from Staphylococcus aureus: genomic and proteomic approaches to the identification and characterization of gene function

Systematic analysis of the entire two-component signal transduction system (TCSTS) gene complement of Staphylococcus aureus revealed the presence of a putative TCSTS (designated SrhSR) which shares considerable homology with the ResDE His-Asp phospho-relay pair of Bacillus subtilis. Disruption of the srhSR gene pair resulted in a dramatic reduction in growth of the srhSR mutant, when cultured under anaerobic conditions, and a 3-log attenuation in growth when analyzed in the murine pyelonephritis model. To further understand the role of SrhSR, differential display two-dimensional gel electrophoresis was used to analyze the cell-free extracts derived from the srhSR mutant and the corresponding wild type. Proteins shown to be differentially regulated were identified by mass spectrometry in combination with protein database searching. An srhSR deletion led to changes in the expression of proteins involved in energy metabolism and other metabolic processes including arginine catabolism, xanthine catabolism, and cell morphology. The impaired growth of the mutant under anaerobic conditions and the dramatic changes in proteins involved in energy metabolism shed light on the mechanisms used by S. aureus to grow anaerobically and indicate that the staphylococcal SrhSR system plays an important role in the regulation of energy transduction in response to changes in oxygen availability. The combination of proteomics, bio-informatics, and microbial genetics employed here represents a powerful set of techniques which can be applied to the study of bacterial gene function.     

Origins and complexes: the initiation of DNA replication

Eukaryotic DNA is organized for replication as multiple replicons. DNA synthesis in each replicon is initiated at an origin of replication. In both budding yeast, Saccharomyces cerevisiae and fission yeast, Schizosaccharomyces pombe, origins contain specific sequences that are essential for initiation, although these differ significantly between the two yeasts with those of S. pombe being more complex then those of S. cerevisiae. However, it is not yet clear whether the replication origins of plants contain specific essential sequences or whether origin sites are determined by features of chromatin structure. In all eukaryotes there are several biochemical events that must take place before initiation can occur. These are the marking of the origins by the origin recognition complex (ORC), the loading onto the origins, in a series of steps, of origin activation factors including the MCM proteins, and the initial denaturation of the double helix to form a replication "bubble". Only then can the enzymes that actually initiate replication, primase and DNA polymerase-alpha, gain access to the template. In many cells this complex series of events occurs only once per cell cycle, ensuring that DNA is not re-replicated within one cycle. However, regulated re-replication of DNA within one cell cycle (DNA endoreduplication) is relatively common in plants, indicating that the "once-per-cycle" controls can be overridden.     

The environmental and genetic regulation of obake expressivity: morphogenetic fields as evolvable systems

The morphogenetic field, a fundamental concept of classical embryology, is once again being invoked to describe developmental processes. Because the evolution of adult structures requires the modification of development, the ways in which morphogenetic fields can change over time may yield insights into evolutionary possibilities. We considered how the duplication/multiplication of a morphogenetic field in fruit flies, caused by the previously described obake (obk) mutation, is regulated by genetic and environmental factors. Mutations of genes in the canonical antenna-producing imaginal disc pathway suppressed duplication as expected, although the results suggested that other pathways might also be involved. Overgrowth mutations, expected to increase duplication, actually suppressed it. Mutations in the heat-shock protein gene Hsp83 did not uniformly enhance obk expressivity as hypothesized. Using third chromosomes extracted from wild-derived lines, natural genetic variation for modifiers of obk function was found to be extensive. Larval crowding suppressed the obk phenotype, but there was no evidence of trade-offs between body or head size and arista number. Our results suggest that a complex interplay of genetic and environmental factors in the regulation of fields may be responsible for ample natural variation in the expressivity of adult phenotypes, affording multiple opportunities for selection and evolutionary modification.     

Structure activity studies of ring E analogues of methyllycaconitine. Part 2: Synthesis of antagonists to the alpha3beta4* nicotinic acetylcholine receptors through modifications to the ester

The development of novel agents for the differentiation of neuronal nicotinic acetylcholine receptors (nAChRs) is important for the treatment of a variety of pathological conditions. We have prepared and evaluated a number of simpler analogues of the norditerpeniod alkaloid methyllycaconitine (MLA) in an effort to understand molecular determinants of nAChR*small molecule interactions. We have previously reported the synthesis and evaluation of a series of ring E analogues of MLA. We report here the optimization of the alpha3beta4* functional activity of this series of compounds through modification of the ester.     

Prediction of functional sites by analysis of sequence and structure conservation

We present a method for prediction of functional sites in a set of aligned protein sequences. The method selects sites which are both well conserved and clustered together in space, as inferred from the 3D structures of proteins included in the alignment. We tested the method using 86 alignments from the NCBI CDD database, where the sites of experimentally determined ligand and/or macromolecular interactions are annotated. In agreement with earlier investigations, we found that functional site predictions are most successful when overall background sequence conservation is low, such that sites under evolutionary constraint become apparent. In addition, we found that averaging of conservation values across spatially clustered sites improves predictions under certain conditions: that is, when overall conservation is relatively high and when the site in question involves a large macromolecular binding interface. Under these conditions it is better to look for clusters of conserved sites than to look for particular conserved sites.     

A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion

Initiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis, which encompasses RD1 (Rv3871-Rv3879c), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT-6, like M. tuberculosis RD1 mutants. M. marinum mutants in genes homologous to Rv3866-Rv3868 also failed to accumulate intracellular ESAT-6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT-6/CFP-10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell-to-cell spread by wild-type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c, not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo, suggesting that this gene plays additional roles in M. marinum survival in the host.     

Strain characterization and 16S-23S probe development for differentiating geographically dispersed isolates of the phytopathogen Ralstonia solanacearum

The causative agent of potato brown rot and bacterial wilt, Ralstonia solanacearum , results in serious world-wide economic losses, particularly in the tropics. In the last decade, however, the incidence of bacterial wilt in potatoes grown in Northern Europe has increased, presenting an interesting epidemiological puzzle. Its occurrence may be as a result of changes in agricultural practice or the emergence of a novel bacterial variety, better adapted to cooler conditions. To understand the distribution and genetic diversity of this phytopathogen, we have analysed a collection of 82 isolates from Europe and tropical regions. Both phenotypic [SDS–PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) profiling, FAME (fatty acid methyl esters) analysis, growth profiles and EPS (exopolysaccharide) production] and genotypic [16S rRNA RFLP (restriction fragment length polymorphism), ARDRA (amplified ribosomal DNA restriction analysis) and sequence analysis of 16S−23S rRNA ITS and flanking regions] methods were compared. Principal component analysis of FAME profiles clustered isolates into three groups and ARDRA of a 0.85 kb amplified fragment from the 16S-23S ITS region differentiated isolates into four groups. Using sequence analysis, specific primers were designed within the variable region 147–170 of the 23S rRNA. These primers, RsolT2 and RsolT3, respectively, differentiated isolates into two distinct clusters as described previously by Wullings and colleagues ( Wullings et al ., 1998 ). The European strains (Biovar 2, race 3) analysed in this study specifically hybridized with RsolT3, and showed considerable genetic homogeneity when compared with strains of other races from 'the rest of the world'. These data indicate the possible selection and proliferation of a 'European'-adapted variant.     

Species names in phylogenetic nomenclature

Linnaean binomial nomenclature is logically incompatible with the phylogenetic nomenclature of de Queiroz and Gauthier (1992, Annu. Rev. Ecol. Syst. 23:449-480): The former is based on the concept of genus, thus making this rank mandatory, while the latter is based on phylogenetic definitions and requires the abandonment of mandatory ranks. Thus, if species are to receive names under phylogenetic nomenclature, a different method must be devised to name them. Here, 13 methods for naming species in the context of phylogenetic nomenclature are contrasted with each other and with Linnaean binomials. A fundamental dichotomy among the proposed methods distinguishes those that retain the entire binomial of a preexisting species name from those that retain only the specific epithet. Other relevant issues include the stability, uniqueness, and ease of pronunciation of species names; their capacity to convey phylogenetic information; and the distinguishability of species names that are governed by a code of phylogenetic nomenclature both from clade names and from species names governed by the current codes. No method is ideal. Each has advantages and drawbacks, and preference for one option over another will be influenced by one's evaluation of the relative importance of the pros and cons for each. Moreover, sometimes the same feature is viewed as an advantage by some and a drawback by others. Nevertheless, all of the proposed methods for naming species in the context of phylogenetic nomenclature provide names that are more stable than Linnaean binomials.