Streptococcus uberis is one of the principal causative agents of bovine mastitis. The organism is typically considered an environmental pathogen, however, recent studies also suggest possible host adaptation. In this study, two multilocus sequence typing (MLST) schemes and whole genome DNA microarrays were used to evaluate the degree and nature of genome flexibility between S. uberis strains. The 21 isolates examined in this study arise from a collection of 232 international isolates for which previous epidemiological and preliminary genotyping data existed. The microarray analysis resulted in an estimate of the core genome for S. uberis, consisting of 1530 ORFs, among 1855 tested, representing 82.5% of the S. uberis 0140J genome. The remaining ORFs were variable in gene content across the 21 tested strains. A total of 26 regions of difference (RDs), consisting of three or more contiguous ORFs, were identified among the variable genes. Core genes mainly encoded housekeeping functions, while the variable genes primarily fell within categories such as protection responses, degradation of small molecules, laterally acquired elements, and two component systems. Recombination detection procedures involving the MLST loci suggested S. uberis is a highly recombinant species, precluding accurate phylogenetic reconstructions involving these data. On the other hand, the microarray data did provide limited support for an association of gene content with strains found in multiple cows and/or multiple herds, suggesting the possibility of genes related to bovine transmissibility or host-adaptation.
Keywords: comparative genomic hybridization
Overall design
Combimatrix CustomArray™ 4X2K was used in this study (design1_#4231). This array is divided into 4 sectors, each of which contains 2,240 in situ synthesized oligonucleotide probes (spots) in 56 columns and 40 rows with the same probe design and layout. Based on the sequence of S. uberis strain 0140J, oligonucleotide probes were designed to have a similar annealing temperature of 56ºC and a length 35-40 bp. The first design included 100 negative controls probes as well as 1855 of the 1870 ORFs described in the un-annotated sequence of S. uberis strain 0140J. Replicate microarrays were hybridized for every 21 strains tested in this study.
Combimatrix CustomArray™ 4X2K was used in this study (design2_#4887). This array is divided into 4 arrays (sectors), each of which contains 2K in situ synthesized oligonucleotide probes (spots) in 56 columns and 40 rows with the same probe design and layout. This second design of S. uberis was based on genes that were not clearly present (loci with low intensity or no hybridization for at least one strain) for all 21 strains in the hybridization results involving the first design. Based on the sequence of S. uberis strain 0140J, oligonucleotide probes were designed to have a similar annealing temperature of 56ºC and a length 35-40 bp. The design included four or five probes, separated from one another in order to span the entire gene, for 434 of 451 ambiguous loci, synthesized in situ to occupy the 2,240 independent microarray spots. Replicate microarrays were hybridized for every 21 strains tested in this study.
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