A 16-kbp DNA region that contains genes involved in the biosynthesis

A 16-kbp DNA region that contains genes involved in the biosynthesis of the capsule of (A1 has been characterized. an mutant with the A1 genes resulted in the restoration of ECA biosynthesis. Region 3 contains two genes (and (A1 is the principal microorganism responsible for bovine pneumonic pasteurellosis a major cause of sickness and economic loss to the feed lot industry (15 46 Some of its characterized virulence factors include a leukotoxin a sialoglycoprotease neuraminidase and transferrin-binding proteins (9). In addition the bacterium produces an extracellular capsular polysaccharide (CPS) which has been implicated to play a role in pathogenesis. The role of CPS in the virulence of a number of LY317615 gram-negative pathogens has been well documented. Some of these activities include adherence (11) prevention of LY317615 desiccation (30) and resistance to host immune defense (29). For A1 the activities of CPS in virulence and protection have not been LY317615 well defined. It has been reported that CPS is important in the adherence of the bacterium to alveolar surfaces (6 45 and inhibition of complement-mediated serum killing (7) as well as inhibition of the phagocytic and bactericidal activities of neutrophils (12 43 Preliminary studies by Yates et al. (47) using crude CPS preparations of A1 suggested that the capsule conferred some protection against experimental pasteurellosis; however it was unclear which molecule(s) in the preparation was responsible for this protection. On the contrary Conlon and Shewen (10) showed that purified A1 CPS did not elicit protection against experimental challenge. It has been suggested by Gatewood et al. (19) that the antigenic nature of the CPS could be influenced by the culture conditions and that only CPS produced during growth in the host could stimulate a protective immune response. The CPS of A1 is composed of a disaccharide repeat of A1. Proposal of nomenclature scheme. There are numerous reports in the books that determined and called the many genes and protein involved with CPS biosynthesis. Including the genes that code for the ATP-binding transporter which have been called are in (38) in (22) in (17) in (44) and in (8) to mention a few. These cognate proteins and genes have already been shown generally to become functionally compatible by complementation studies. These different gene designations generate misunderstandings in the books especially when analysts are analyzing homologous features or the building of cross genes and proteins. As even more hereditary loci involved LY317615 with CPS biosynthesis are characterized extra nomenclature will become released. During a consultation P. Reeves suggested a uniform nomenclature for the genes in the CPS cluster that follows the scheme that has been established for the genes in bacterial polysaccharide biosynthesis (34). Using the A1 CPS biosynthetic cluster as an example it is proposed that the four genes in region 1 that code for the ATP-binding transporter be designated in the order of their genetic organization that the two genes in region 2 that code for PIK3C2G homologues of the ManNAcA pathway be designated and and until their functions are determined. When the same gene from different organisms is being referred to a suitable subscript will be added e.g. strain XL1-Blue (Strategene La Jolla Calif.) was used for the cloning of all recombinant plasmids. strain CSR603 (41) was used for the maxi-cell labeling experiments. RS2436 (EV36 Δ21566 (mutant and cluster of A1 λ library was obtained from George Weinstock (University of Texas Houston Tex.). The λ library was constructed by the use of strains were cultured in Luria-Bertani broth supplemented with thymine (50 μg/ml) and with ampicillin (100 μg/ml) when required. A1 was cultured in brain heart infusion broth. All cultures were grown at 37°C unless stated otherwise. Enzymes chemicals and antibodies. Restriction endonucleases T4 DNA ligase and protein and DNA molecular weight standards were purchased from Pharmacia Chemicals (Baie d’Urfe Quebec Canada) GIBCO/Bethesda Research Laboratories (Burlington Ontario Canada) or Bio-Rad Laboratories (Mississauga Ontario Canada) and used according to the manufacturer’s instructions. Screening of λ library and characterization of cloned DNA. The λ library was plated out on XL1-Blue cells to produce approximately 300 plaques per plate. Plaques were lifted from the agar plates and the phage DNA was prepared for hybridization as described by the supplier. A 1.5-kbp cells using Qiagen columns (Chatsworth Calif.). Standard techniques were used for.