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Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep, has unique iron requirements in that it is mycobactin-dependent for cultivation in vitro. The iron-dependent regulator (IdeR) is a well-characterized global regulator responsible for maintaining iron homeostasis in Mycobacterium tuberculosis (MTB). We identified an orthologous segment in the MAP genome, MAP2827, with >93 % amino acid identity to MTB IdeR. Electrophoretic mobility shift assays and DNase protection assays confirmed that MAP2827 binds the 19 bp consensus motif (iron box) on the MAP genome. Sequencing of MAP2827 from multiple isolates revealed a non-synonymous change (R91 G) exclusive to sheep strains. Reporter gene assays and quantitative real-time RT-PCR assays in two diverse MAP strains and in an ideR deletion mutant of M. smegmatis ([mc.sup.2]155) suggested that both sheep MAP IdeR (sideR) and cattle MAP IdeR (cIdeR) repress mbtB transcription at high iron concentrations and relieve repression at low iron concentrations. On the other hand, bfrA (an iron storage gene) was upregulated by cIder when presented with MTB or the cattle MAP bfrA promoter, and was downregulated by sIdeR in the presence of MTB, or sheep or cattle MAP bfrA promoters, at high iron concentrations. The differential iron regulatory mechanisms between IdeR-regulated genes across strains may contribute to the differential growth or pathogenic characteristics of sheep and cattle MAP strains. Taken together, our study provides a possible reason for mycobactin dependency and suggests strong implications in the differential iron acquisition and storage mechanisms in MAP. DOI 10.1099/mic.0.031948-0

Microarray-based comparisons of three Mycobacterium avium subsp, paratuberculosis isolates, including one sheep strain and two cattle strains, identified three large genomic deletions in the sheep strain, totaling 29,208 bp and involving 24 open reading frames. These deletions may help explain some of the differences in pathogenicity and host specificity observed between the cattle and sheep strains of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp, paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp, paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp, silvaticum and Mycobacterium avium subsp, avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp, paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp, paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp, avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp, silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp, paratuberculosis by multiple clusters of divergent ORFs.

The sex pheromone plasmids in Enterococcus faecalis are one of the most efficient conjugative plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed [10.sup.-1] transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of plasmid pCF10 and the analysis of the transcription profile of plasmid genes, relative to conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis.

Research has been conducted on Pasteurella multocida grown in iron-free medium and supplemented with such iron sources as hemoglobin, ferric citrate, ferritin and transferrin. The addition of the defined iron source to the medium has been monitored via the use of whole-genome DNA microarrays, and the results demonstrate that P. multocida genes are expressed in response to different iron sources and have to be characterized.

Research has been conducted on Pasteurella multocida. The identification of the bacterium's genes has been carried out via utilization of the whole-genome microarrays in comparing gene expression levels during growth in rich and minimal media and the details are reported.

Results show that expression of the accessory gene regulator, agr locus, is repressed in serum and in vivo, in Staphylococcus aureus, even when virulence factors expression is high enough to cause toxic shock syndrome in the animals.

We present here the complete genome sequence of a common avian clone of Pasteurella multocida, Pm70. The genome of Pm70 is a single circular chromosome 2,257,487 base pairs in length and contains 2,014 predicted coding regions, 6 ribosomal RNA operons, and 57 tRNAs. Genome-scale evolutionary analyses based on pair-wise comparisons of 1,197 orthologous sequences between P. multocida, Haemophilus influenzae, and Escherichia coli suggest that P. multocida and H. influenzae diverged [approximately equals] 270 million years ago and the [Gamma] subdivision of the proteobacteria radiated about 680 million years ago. Two previously undescribed open reading frames, accounting for [approximately equals] 1% of the genome, encode large proteins with homology to the virulence-associated filamentous hemagglutinin of Bordetella pertussis. Consistent with the critical role of iron in the survival of many microbial pathogens, in silico and whole-genome microarray analyses identified more than 50 Pm70 genes with a potential role in iron acquisition and metabolism. Overall, the complete genomic sequence and preliminary functional analyses provide a foundation for future research into the mechanisms of pathogenesis and host specificity of this important multispecies pathogen.