Genetic variability of the emm-related genes of the large vir regulon of group A streptococci: potential intra- and intergenomic recombination events

One of the most prevalent genetic lineages of group A streptococci (GAS) harbors a genomic locus termed the large vir regulon, which contains an emm gene encoding the antiphagocytic M protein, and structurally related fcrA and enn (emm-related) genes encoding immunoglobulin-binding proteins. In the present study more than 100 large vir regulons from 42 different GAS serotypes were analyzed by PCR and partial DNA sequencing. On comparing these data to published sequences, sites of mutational and putative recombinational events were identified and ordered with respect to their intra/intergenic or intra/intergenomic nature. The emm-related genes were found to display small intragenic deletions or insertions, were completely deleted from, or newly inserted into the genome, or were fused to adjacent genes. Intergenomic exchanges of complete emm-related genes, or segments thereof, between different vir regulons were detected. Most of these processes seem to involve short flanking direct repeats. Occasionally, the structural changes could be correlated with changes in the functions of the encoded proteins.     

Three different types of organization of the vir regulon in group A streptococci

Summary The DNA of group A streptococci (GAS) encodes several important virulence factors such as the antiphagocytic M protein, the Ig-Fc-binding M-related proteins (FcrA-like and EnnX-like) and the complement factor-inactivating C5a peptidase. The corresponding genes emm, fcrA, ennX, and scpA, respectively, were assumed to be located close together in the GAS genome. Additionally, emm and scpA have been found to be under the positive, coordinate control of the virR locus, which led to the designation vir regulon for the corresponding genomic segment. In order to map the vir regulons of many GAS serotypes and to analyse any correlation between the organization of vir regulons and circumscribed heterogeneities within the emm, virR, and scpA genes, an approach using several distinct sets of polymerase chain reaction (PCR) experiments was chosen. By examination of the genomic DNA of 42 GAS isolates from 36 different M serotypes three patterns of vir regulon topography were found. The first, designated large vir regulon (LVR), consists of virR -fcrA(-like) emm - ennX(-like) - scpA. The second, designated small vir regulon (SVR), contains virR - emm- scpA, and the last, designated unusual vir regulon (UVR), resembles SVR but contains additional heterogeneous sequences between emm and scpA. The patterns correlate with heterogeneities at the 3 ends of the virR and scpA genes, with the M classification system and the occurrence of specific non-coding intervening sequences within the vir regulons. The potential impact of these patterns on models to account for generation of vir regulons is discussed.     

Molecular population genetic analysis of the enn subdivision of group A streptococcal emm-like genes: horizontal gene transfer and restricted variation among enn genes

The group A streptococcal emm-like genes, which encode the cell-surface M and M-like proteins, are divided into distinct mrp, emm and enn subdivisions and are clustered together in a region of the chromosome called the vir regulon. In order to understand the mechanisms involved in the evolution of emm-like genes, a 180bp fragment of the 5 variable region of the enn gene was characterized in 31 strains for which emm sequences and multilocus enzyme electrophoretic profiles have been previously determined. The results demonstrate that nucleotide polymorphisms at the enn locus are generated predominantly by point mutations and short deletions or insertions, and that variation among enn and emm genes has arisen by similar mechanisms. However, diversity at the enn locus is restricted in comparison to the emm locus. Moreover, there is strong evidence for intragenic recombination at the enn locus and the pattern of distribution of emm and enn alleles among strains suggests that these genes may be independently acquired by horizontal transfer and recombination from distinct donor strains, thereby generating a mosaic structure for the vir regulon. The results add to a growing body of evidence that horizontal gene transfer has played a major role in the evolution of Streptococcus pyogenes vir regulons.     

高毒力A组链球菌致病机制研究进展

A组链球菌(Group A Streptococcus,GAS)常导致咽炎和皮肤感染,也能引起严重侵袭性感染.根据其表面M蛋白编码基因emm可将GAS分为200多型,严重侵袭性感染多由高毒力株引起,以emm1、emm3、emm12、emm28和emm89型常见.研究发现高毒力GAS株中CovRS基因突变可导致细菌逃逸固...     

Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation

For successful transformation of a plant by Agrobacterium tumefaciens it is essential that the explant used in cocultivation has the ability to induce Agrobacterium tumour-inducing (Ti) plasmid virulence (vir) genes. Here we report a significant variation in different tissues of Indica rice (Oryza sativa L. cv. Co43) in their ability to induce Agrobacterium tumefaciens vir genes and T-strand generation, using explants preincubated in liquid Murashige and Skoog (MS) medium. An analysis of rice leaf segments revealed that they neither induced vir genes nor inhibited vir gene induction. Of different parts of rice plants of different ages analysed only scutellum from four-day old rice seedlings induced vir genes and generation of T-strands. We observed that the physical presence of preincubated scutella is required for vir gene induction. Conditioned medium from which preincubated scutella were removed did not induce the vir genes. Scutellum-derived calli, cultured for 25 days on medium containing 2,4-D, also induced virE to an appreciable level. These results suggest that scutellum and scutellum-derived calli may be the most susceptible tissues of rice for Agrobacterium-mediated transformation.     

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.     

Environmental conditions differentially affect vir gene induction in different Agrobacterium strains. Role of the VirA sensor protein

The induction of vir gene expression in different types of Agrobacterium strains shows different pH sensitivity profiles. The pH sensitivity pattern demonstrated by octopine Ti strains was similar to that of a supervirulent leucinopine Ti strain, whereas this was different from that shown by nopaline Ti strains and agropine Ri strains. Data are given which indicate that these differences are due to different properties of the virA genes of these wild types. An exceptional case was formed by strains with the limited-host-range plasmid pTiAG57 which showed AS-dependent vir induction only if reduced inoculum sizes were used and the temperature was 28°C or below.     

The effect of additional virulence genes on transformation efficiency,transgene integration and expression in rice plants using the pGreen/pSoup dual binary vector system

We assessed the effect of four different virulence (vir) gene combinations on plant transformation efficiency and transgene behaviour in rice using the pGreen/pSoup dual binary vector system. Transformation experiments were conducted using a pGreen vector containing the bar and gusA expression units with, or without, the virG542, virGN54D, virGwt or the virG/B/C genes added to the backbone. Additonal vir gene(s) significantly altered plant transformation efficiency and the integration of vector backbone sequences. However, no differences in transgene copy number, percentage of expressing lines and expression levels could be detected. Addition of virGwt was the most beneficial, doubling the overall performance of the pGreen/pSoup vector system based on transformation frequency, absence of backbone sequence integration and expression of unselected transgenes. In 39 of the plant lines, the additional vir genes were integrated into the rice genome. The contribution of super dual binary pGreen/pSoup vectors to the development of efficient rice transformation systems and to the production of plants free of selectable marker genes are discussed.     

Ti plasmid containing Rhizobium meliloti are non-tumorigenic on plants,despite proper virulence gene induction and T-strand formation

We examined the expression of the vir genes of the Agrobacterium tumefaciens Ti plasmid in Rhizobium meliloti, which remains non-tumorigenic on plants after introduction of a Ti- or Ri-plasmid. Both the levels of virulence (vir) gene expression, induced by the plant phenolic compound acetosyringone, and of subsequent T-strand formation were comparable to what is observed in Agrobacterium. In contrast to the situation in Agrobacterium, though, vir induction in R. meliloti did not require a low pH (5.3) of the induction medium and the optimum temperature for induction in R. meliloti was significantly lower than in Agrobacterium. At 37°C no induction of the vir genes was found both in Agrobacterium and R. meliloti. We postulate that the lack of tumorigenicity of Ti carrying R. meliloti strains is due either to a lack of proper attachment of the bacteria to plant cells, or to an improper assembly of a virB-determined essential structure in the cell wall of R. meliloti.     

Efficient and rapid <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of durum wheat (<Emphasis Type="Italic">Triticum turgidum L. var. durum)</Emphasis> using additional virulence genes

Genetic transformation of wheat, using biolistics or Agrobacterium, underpins a range of specific research methods for identifying genes and studying their function in planta. Transgenic approaches to study and modify traits in durum wheat have lagged behind those for bread wheat. Here we report the use of Agrobacterium strain AGL1, with additional vir genes housed in a helper plasmid, to transform and regenerate the durum wheat variety Ofanto. The use of the basic pSoup helper plasmid with no additional vir genes failed to generate transformants, whereas the presence of either virG⁵⁴² or the 15 kb Komari fragment containing virB, virC and virG⁵⁴² produced transformation efficiencies of between 0.6 and 9.7%. Of the 42 transgenic plants made, all but one (which set very few seeds) appeared morphologically normal and produced between 100 and 300 viable seeds. The transgene copy number and the segregation ratios were found to be very similar to those previously reported for bread wheat. We believe that this is the first report describing successful genetic transformation of tetraploid durum wheat (Triticum turgidum L. var. durum) mediated by Agrobacterium tumefaciens using immature embryos as the explant.     

The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames

Summary Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon mutagenesis studies have shown that virB gene products are required for virulence but their functions remain largely unknown. To provide information relevant to understanding the function of VirB polypeptides, the nucleotide sequence of the virB operon from a nopaline plasmid, pTiC58, is presented here. Eleven open reading frames (ORFs) are predicted from this sequence. The predicted sizes of 10 of the 11 VirB polypeptides are verified by specific expression in Escherichia coli. Only the product of the smallest ORF potentially encoding a 5.8 kDa polypeptide has not been detected. The initiation of translation of five virB ORFs occurs at codons that overlap the termination codons of the ORF immediately upstream; thus, translational coupling may be an important mechanism for efficient translation of the large virB polycistronic mRNA. Based on hydropathy plot analysis nine of the virB ORFs encode proteins that may interact with membranes; these data support the earlier hypothesis (Engstromm et al. 1987) that virB gene products may form a membrane pore or channel to mediate exit of the T-DNA copy (T-strands) from Agrobacterium into the plant cell. A comparison of the two published octopine virB sequences with the nopaline sequence presented here is made.     

Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium

Summary T-DNA circularization is one of the molecular events specifically induced in agrobacterial cells upon their infection of dicotyledonous plant cells. We developed a seedling co-cultivation procedure to determine whether or not monocotyledonous plants have the ability to induce T-DNA circularization and vir gene expression. Co-cultivation of Agrobacterium tumefaciens with seedlings of dicotyledonous plants showed that the circularization event takes place efficiently. The exudates and extracts of the seedlings also effectively induced T-DNA circularization and vir gene expression, indicating that dicotyledonous seedlings contain diffusible factors capable of inducing these molecular events. In contrast, neither T-DNA circularization nor vir gene expression was detectable when Agrobacterium was incubated with seedlings of monocotyledonous plants. Supplementing with acetosyringone, a known inducer of vir gene expression and T-DNA circularization, resulted in the induction of circularization during co-cultivation with monocotyledonous seedlings. These results indicate that the seedlings of monocotyledonous plants have no detectable amounts of diffusible inducers, unlike dicotyledonous seedlings. Therefore, it is unlikely that the vir genes are expressed in Agrobacterium inoculated in monocotyledonous plants. This may be one of the blocks in tumorigenesis of monocotyledonous plants by Agrobacterium.     

Sequencing of genes within the vir regulon of Streptococcus pyogenes type M15 — an opacity factor-positive serotype with low opacity factor expression

Major virulence determinants of group A streptococci, such as M-protein, immunoglobulin Fc-receptors (FcRA, EmmL) and C5a peptidase, appear to be genetically co-regulated, their genes being located within a vir regulon. We studied the organization of these genes in a group A, type M15 strain of Streptococcus pyogenes, previously defined as OF^?, by hybridization analysis of chromosomal DNA and of an S. pyogenes gene library in Escherichia coli, and by gene sequencing. Within the vir regulon, in addition to the virR and scpA genes, three so-called emm-related genes were found: fcrA, emmL and enn. Whereas IgG Fc-binding proteins were encoded by fcrA and emmL, the product of enn was not identified. The presence of three emm-related genes in this region is reminescent of vir regulon organization in OF⁺ rather than OF^? strains as earlier defined by others. Furthermore, analysis of the deduced product of the emmL gene showed deletions and amino acid substitutions within the PGTS-rich domain and membrane anchor, which thus resembles corresponding products of OF⁺ rather than OF^? strains. In view of these findings, the opacity factor (OF) activity of the strain was tested using growth supernatant, with negative outcome. However, a concentrated SDS cell extract revealed definite OF activity. One of two other type M15 reference strains also showed definite OF activity in SDS extracts. We therefore propose that type M15 strains belong to the OF⁺ category but often show low levels of expression of OF.     

Nuclear genes required for post-translational steps in the biogenesis of the chloroplast cytochrome b6f complex in maize

Nuclear genes essential for the biogenesis of the chloroplast cytochrome b ₆ f complex were identified by mutations that cause the specific loss of the complex. We describe four transposon-induced maize mutants that lack cytochrome b ₆ f proteins but contain normal levels of other photosynthetic complexes. The four mutations define two nuclear genes. To identify the step at which each mutation blocks protein accumulation, mRNAs encoding each subunit were examined by Northern hybridization analysis and the rates of subunit synthesis were examined in pulse-labeling experiments. In each mutant the mRNAs encoding the known subunits of the complex were normal in size and abundance and the major subunits were synthesized at normal rates. Thus, these mutations block the biogenesis of the cytochrome b ₆ f complex at a post-translational step. The two nuclear genes identified by these mutations may encode previously unknown subunits, be involved in prosthetic group synthesis or attachment, or facilitate assembly of the complex. These mutations were also used to provide evidence for the authenticity of a proposed fifth subunit of the complex and to demonstrate a role for the cytochrome b ₆ f complex in protecting photosystem 11 from light-induced degradation.     

Localised mutagenesis of the fts YEX operon conditionally lethal missense substitutions in the FtsE cell division protein of Escherichia coli are similar to those found in the cystic fibrosis transmembrane conductance regulator protein (CFTR) of human patients

Summary After localised mutagenesis of the 76 min region of the Escherichia coli chromosome, we isolated a number of conditionally lethal mutants. Some of these mutants had a filamentation temperature sensitive (fts) phenotype and were assigned to the cell division genes ftsE of ftsX whereas others were defective in the heat shock regulator gene rpoH. Both missense and amber mutant alleles of these genes were produced. The missense mutant ftsE alleles were cloned and sequenced to determine whether or not the respective mutations mapped to the region of the gene encoding the putative nucleotide binding site. Surprisingly, most of these mutant FtsE proteins had missense substitutions in a different domain of the protein. This region of the FtsE protein is highly conserved in a large family of proteins involved in diverse transport processes in all living cells, from bacteria to man. One of the proteins in this large family of homologues is the human cystic fibrosis transmembrane conductance regulator (CFTR), and the FtsE substitutions were found to be in very closely linked, or identical, amino acid residues to those which are frequently altered in the CFTR of human patients. These results confirm the structural importance of this highly conserved region of FtsE and CFTR and add weight to the current structural model for the human protein.     

Expression of VirB2 protein-containing structures on Agrobacterium in mating cultures

Exocellular structures containing VirB2 proteins were, for the first time, localized on the surface of Agrobacterium by transmission electron microscopy. Using colloidal gold (CG)-labeled VirB2-specific antibodies, it was shown that VirB2 proteins enter into the composition of short surface pili, which emerge at the poles of acetosyringone (AS)-induced Agrobacterium cells. However, cells of the Ti plasmidless A. tumefaciens strain UBAPF-2 and cells not induced with AS were incapable of pilus synthesis. In suspension, mating Agrobacterium cells were connected together by short thick bridges. It was found that these bridges did not include as part of their structure CG-labeled VirB1 and VirB2 proteins. We did not find the tetracycline-resistant transconjugants after mating of A. tumefaciens donor cells harboring binary systems with plasmid-free A. tumefaciens GM-I 9023 in vir-induced and vir-uninduced conditions. However, the same strains can transfer pSUP106 plasmid via a vir-dependent way. We found that activated vir genes slightly stimulate pTd33 plasmid transfer via a tra-dependent pathway to plasmid-free strain UBAPF-2. It seems, that vir-induced T-DNA/plasmid DNA transfer machinery is not essential for the conjugation process between agrobacterial cells but may participate in this process.     

Conservation of the Type IV Secretion System throughout Wolbachia evolution

The Type IV Secretion System (T4SS) is an efficient pathway with which bacteria can mediate the transfer of DNA and/or proteins to eukaryotic cells. In Wolbachia pipientis, a maternally inherited obligate endosymbiont of arthropods and nematodes, two operons of vir genes, virB3-B6 and virB8-D4, encoding a T4SS were previously identified and characterized at two separate genomic loci. Using the largest data set of Wolbachia strains studied so far, we show that vir gene sequence and organization are strictly conserved among 37 Wolbachia strains inducing various phenotypes such as cytoplasmic incompatibility, feminization, or oogenesis in their arthropod hosts. In sharp contrast, extensive variation of genomic sequences flanking the virB8-D4 operon suggested its distinct location among Wolbachia genomes. Long term conservation of the T4SS may imply maintenance of a functional effector translocation system in Wolbachia, thereby suggesting the importance for the T4SS in Wolbachia biology and survival inside host cells.     

The Pho Regulons of Bacteria

Bacterial Pho regulons contain genes whose products are involved in the transport and assimilation of inorganic phosphate. The expression of these genes is regulated by a specific two-component signal transduction system. The paper summarizes data on the organization and function of Pho regulons in gram-negative and gram-positive bacteria, with particular emphasis on the Pho regulons of the best studied bacteria Escherichia coli and Bacillus subtilis.