Search

Many pathogenic strains of Clostridium perfringens carry several highly similar toxin or antibiotic resistance plasmids that have 35 to 40 kb of very closely related syntenous sequences, including regions that carry the genes encoding conjugative transfer, plasmid replication and plasmid maintenance functions. Key questions are how are these closely related plasmids stably maintained in the same cell and what is the basis for plasmid incompatibility in C. perfringens. Comparative analysis of the Rep proteins encoded by these plasmids suggested that this protein was not the basis for plasmid incompatibility since plasmids carried in a single strain often encoded an almost identical Rep protein. These plasmids all carried a similar, but not identical, parMRC plasmid partitioning locus. Phylogenetic analysis of the deduced ParM proteins revealed that these proteins could be divided into ten separate groups. Importantly, in every strain that carried more than one of these plasmids, the respective ParM proteins were from different phylogenetic groups. Similar observations were made from the analysis of phylogenetic trees of the ParR proteins and the parC loci. These findings provide evidence that the basis for plasmid incompatibility in the conjugative toxin and resistance plasmid family from C. perfringens resides in subtle differences in the parMRC plasmid partitioning loci carried by these plasmids., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Kimberley virus (KIMV) is an arthropod-borne rhabdovirus that was isolated in 1973 and on several subsequent occasions from healthy cattle, mosquitoes (Culex annulirostris) and biting midges (Culicoides brevitarsis) in Australia. Malakal virus (MALV) is an antigenically related rhabdovirus isolated in 1963 from mosquitoes (Mansonia uniformis) in Sudan. We report here the complete genome sequences of KIMV (15442 nt) and MALV (15444 nt). The genomes have a similar organisation (3'-l-N-P-M-G-G(NS)-α1-α2-β-γ-L-t-5') to that of bovine ephemeral fever virus (BEFV). High levels of amino acid identity in each gene, similar gene expression profiles, clustering in phylogenetic analyses of the N, P, G and L proteins, and strong cross-neutralisation indicate that KIMV and MALV are geographic variants of the same ephemerovirus that, like BEFV, occurs in Africa, Asia and Australia., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Outer membrane lipoproteins are emerging as key targets for protective immunity to many bacterial pathogens. Heterologous expression of lipoproteins in Escherichia coli does not always result in high level expression of acylated recombinant protein. Thus, these proteins do not take up their correct membrane topology and are lacking the immunostimulatory properties endowed by the lipid. To this end, we have designed a lipoprotein expression vector (pDUMP) that results in the production of fusion proteins containing the E. coli major outer membrane lipoprotein (Lpp) signal sequence, lipoprotein signal peptidase recognition site, and the +2 outer membrane sorting signal at their N termini. To test the ability of pDUMP to express lipoproteins from heterologous hosts, the surface lipoprotein PsaA from the Gram-positive organism Streptococcus pneumoniae and the outer membrane lipoproteins MlpA from the Gram-negative Pasteurella multocida and BlpA from the spirochete Brachyspira hyodysenteriae were cloned into both hexahistidine fusion vectors and pDUMP. High level expression of antigenically active protein from both the hexahistidine fusion vectors and pDUMP resulted in abundant bands of the predicted molecular masses when analyzed by SDS-PAGE. When grown in the presence of 3[H]palmitic acid, proteins encoded by pDUMP were observed to incorporate palmitic acid whilst the hexahistidine fusion proteins did not. Using mass spectrometry and image analysis we determined the efficiency of lipidation between the three clones to vary from 31.7 to 100%. In addition, lipidated, but not hexahistidine, forms of the proteins were presented on the E. coli surface., (Copyright 2002 Published by Elsevier Science (USA))

Leptospiral LPS plays a critical role in immunity to leptospirosis and forms the basis for serological classification of Leptospira. However, neither the structure of leptospiral LPS nor the genetics of its biosynthesis have been elucidated. A probe derived from the rhamnose biosynthetic genes of L. interrogans serovar Copenhageni was used to identify the rfb locus of L. borgpetersenii serovar Hardjobovis. Chromosome walking and sequence analysis revealed an rfb locus spanning 36.7 kb, which consists of 31 ORFs transcribed in the same direction. Clusters of genes were identified which encode proteins related to enzymes involved in the biosynthesis of activated sugars including rhamnose. Additional ORFs in the locus encode glycosyltransferases for the assembly of the O-antigen subunit and integral membrane proteins for the transport of O-antigen subunits through the membrane and assembly into LPS.

Two major clades, designated Groups I and II, of nucleopolyhedroviruses (NPVs) from lepidopteran hosts have been previously identified. To reveal more detailed relationships, a series of DNA polymerase nucleotide sequences from the taxa MbMNPV, SeMNPV, HzSNPV, HearNPV, SpltNPV, BusuNPV, and OranNPV have been determined using a polymerase chain reaction (PCR)-based approach. This technique enabled gene sequence determination using microliter samples of NPV-infected insect cadavers. Polyhedrin genes from HearNPV, OranNPV, SeMNPV, and SpltNPV were also isolated and sequenced using a similar approach. These sequences, together with other database entries, were aligned for positional homology of peptide sequences. Phylogenetic analysis of DNA polymerase molecular sequence alignments supports LdMNPV as a taxon of Group II and three Group II subclades, designated A, B, and C. Comparison of DNA polymerase trees with those estimated from occlusion protein molecular sequences enabled identification of three subclades of Group II. These are Subgroup II-A [MbMNPV, LeseNPV, MacoNPV, PaflNPV, SeMNPV, SpltNPV (India isolate), SfMNPV]; Subgroup II-B [SpliNPV, SpltNPV (Japan isolate), SpltNPV (Queensland isolate), and possibly HzSNPV, HearNPV, and ManeNPV], and Subgroup II-C [OpSNPV, OranNPV (S-type), BusuNPV (S-type), and possibly EcobNPV (S-type)]. Notably, all Subgroup II-A taxa are from noctuid hosts. Correlations of virus and host evolution within Group II taxa are discussed. The methods and data developed in this study will allow rapid sequencing of NPV DNA polymerase genes., (Copyright 1999 Academic Press.)