Genome Sequence of Streptococcus agalactiae Strain 09mas018883, Isolated from a Swedish Cow.

We announce the complete genome sequence of Streptococcus agalactiae strain 09mas018883, isolated from the milk of a cow with clinical mastitis. The availability of this genome may allow identification of candidate genes, leading to discovery of antigens that might form the basis for development of a vaccine as an alternative means of mastitis control.

Genome size and genetic map of Cowdria ruminantium.

Cowdria ruminantium is the cause of a serious tick-borne disease of domestic ruminants, known as heartwater or cowdriosis. The organism belongs to the tribe Ehrlichieae:, which contains obligate intracellular pathogens, causing several important animal and human diseases. Although a few C. ruminantium genes have been cloned and sequenced, very little is known about the size, gross structure and organization of the genome. This paper presents a complete physical map and a preliminary genetic map for C. ruminantium. Chromosomal C. ruminantium DNA was examined by PFGE and Southern hybridization. PFGE analysis revealed that C. ruminantium has a circular chromosome approximately 1576 kb in size. A physical map was derived by combining the results of PFGE analysis of DNA fragments resulting from digestion of the whole genome with KSP:I, RSR:II and SMA:I and Southern hybridization analysis with a series of gene probes and isolated macrorestriction fragments. A genetic map for C. ruminantium with a mean resolution of 290 kb was established, the first for a member of the Ehrlichieae: A total of nine genes or cloned C. ruminantium DNA fragments were mapped to specific KSP:I, RSR:II and SMA:I fragments, including the major antigenic protein gene, map-1.

Macrorestriction fragment profiles reveal genetic variation of Cowdria ruminantium isolates.

Macrorestriction profile analysis by pulsed-field gel electrophoresis (PFGE) was used to distinguish between seven isolates of Cowdria ruminantium from geographically different areas. Characteristic profiles were generated for each isolate by using the restriction endonucleases KspI, SalI, and SmaI with chromosomal sizes ranging between 1,546 and 1,692 kb. Statistical analysis of the macrorestriction profiles indicated that all the isolates were distinct from each other; these data contribute to a better understanding of the epidemiology of this pathogen and may be exploited for the identification of genotype-specific DNA probes.

Cowdria ruminantium DNA is unstable in a SuperCos1 library.

A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected. Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli host, the C. ruminantium clones were unstable in the SuperCos1 vector and most clones did not grow reproducibly. The library was contaminated with E. coli clones and these clones were maintained with greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two of these clones overlap. These clones constitute the first large regions of C. ruminantium DNA to be cloned and represent almost 10% of the C. ruminantium genome.

Purification of Cowdria ruminantium organisms for use in genome analysis by pulsed-field gel electrophoresis.

Cowdria ruminantium is an obligate intracellular rickettsial pathogen which is responsible for a tick-borne disease of domestic and wild ruminants called heartwater or cowdriosis. Although several genes have been cloned and partially sequenced, the genome size, gross structure, and organization of the C. ruminantium genome is unknown. Genome analysis of the organism has been hindered because it is difficult to obtain C. ruminantium DNA free from contaminating host cell DNA, and this probably accounts for the lack of genome size data for this organism. In this study we investigated several methods for purifying C. ruminantium from bovine cellular contaminants and organisms of a relatively high purity were obtained. These were used to prepare Cowdria DNA which was analyzed by pulsed-field gel electrophoresis (PFGE) and which revealed a genome approximately 1900 kbp in length plus an additional extra-chromosomal fragment migrating with an apparent size of 815 kbp. This is the first time that the genome size of C. ruminantium has been determined and the first demonstration of an extrachromosomal element.

Construction and initial analysis of a representative lambda ZAPII expression library of the intracellular rickettsia Cowdria ruminantium: cloning of map1 and three other Cowdria genes.

The causative agent of heartwater, the rickettsia Cowdria ruminantium, is very poorly understood at the molecular level owing to a profound lack of suitable tools. We have developed an immunoaffinity chromatographic method to purify C. ruminantium from host cell components and the purified rickettsial cells have been used to prepare substantially pure Cowdria DNA. This DNA has been used to construct what we believe to be the first fully representative C. ruminantium expression library. A clone containing the complete Cowdria map1 gene has been isolated and sequenced. This gene has been expressed in E. coli cells from the native Cowdria promoter, suggesting that the mechanisms for gene transcription and translation are similar between these two organisms. Parts of three other Cowdria genes have also been isolated and sequenced.