Background The ability of Mycobacterium tuberculosis to survive and replicate in macrophages is crucial for the mycobacterium's ability to infect the host and cause tuberculosis. To identify Mycobacterium tuberculosis genes involved in survival in macrophages, a library of non-pathogenic Mycobacterium smegmatis bacteria, each carrying an individual integrated cosmid containing M. tuberculosis H37Rv genomic DNA, was passed through THP-1 human macrophages three times. Results Two of the clones recovered from this enrichment process, sur2 and sur3, exhibited significantly increased survival relative to wild-type bacteria. In coinfection experiments, the ratio of sur2 colonies to wild-type colonies was 1:1 at 0 hours but increased to 20:1 at 24 hours post phagocytosis. The ratio of sur3 colonies to wild-type colonies was 1:1 at 0 hours and 5:1 at 24 hours. The M. tuberculosis ORFs responsible for increased survival were shown to be Rv0365c for the sur2 clone and Rv2235 for the sur3 clone. These ORFs encode proteins with as-of-yet unknown functions. Conclusions We identified two M. tuberculosis ORFs which may be involved in the ability of tubercle bacilli to survive in macrophages.

Background The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide. Results Finished and unfinished genome sequencing data of 98 publicly available microbial genomes has been analyzed for the presence of orthologs of the ESAT-6 loci. The multiple duplicates of the ESAT-6 gene cluster found in the genome of M. tuberculosis H37Rv are also conserved in the genomes of other mycobacteria, for example M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses of the resulting sequences have established the duplication order of the gene clusters and demonstrated that the gene cluster known as region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an ortholog could be found in the genomes of Corynebacterium diphtheriae and Streptomyces coelicolor. Conclusions Comparative genomic analysis revealed that the presence of the ESAT-6 gene cluster is a feature of some high-G+C Gram-positive bacteria. Multiple duplications of this cluster have occurred and are maintained only within the genomes of members of the genus Mycobacterium.

Background Since Mycobacterium avium subspecies paratuberculosis (MAP) was isolated from intestinal tissue of a human patient suffering Crohn's disease, a controversial discussion exists whether MAP have a role in the etiology of Crohn's disease or not. Raw milk may be a potential vehicle for the transmission of MAP to human population. In a previous paper, we have demonstrated that MAP are found in raw milk samples obtained from a defined region in Switzerland. The aim of this work is to collect data about the prevalence of MAP specific IS900 insertion sequence in bulk-tank milk samples in different regions of Switzerland. Furthermore, we examined eventual correlation between the presence of MAP and the somatic cell counts, the total colony counts and the presence of Enterobacteriaceae. Results 273 (19.7%) of the 1384 examined bulk-tank milk samples tested IS900 PCR-positive. The prevalence, however, in the different regions of Switzerland shows significant differences and ranged from 1.7% to 49.2%. Furthermore, there were no statistically significant (p >> 0.05) differences between the somatic cell counts and the total colony counts of PCR-positive and PCR-negative milk samples. Enterobacteriaceae occur as often in IS900 PCR-positive as in PCR-negative milk samples. Conclusion This is the first study, which investigates the prevalence of MAP in bulk-tank milk samples all over Switzerland and infers the herd-level prevalence of MAP infection in dairy herds. The prevalence of 19.7% IS900 PCR-positive bulk-milk samples shows a wide distribution of subclinical MAP-infections in dairy stock in Switzerland. MAP can therefore often be transmitted to humans by raw milk consumption.

Background Cortical myosin-II filaments in Dictyostelium discoideum display enrichment in the posterior of the cell during cell migration and in the cleavage furrow during cytokinesis. Filament assembly in turn is regulated by phosphorylation in the tail region of the myosin heavy chain (MHC). Early studies have revealed one enzyme, MHCK-A, which participates in filament assembly control, and two other structurally related enzymes, MHCK-B and -C. In this report we evaluate the biochemical properties of MHCK-C, and using fluorescence microscopy in living cells we examine the localization of GFP-labeled MHCK-A, -B, and -C in relation to GFP-myosin-II localization. Results Biochemical analysis indicates that MHCK-C can phosphorylate MHC with concomitant disassembly of myosin II filaments. In living cells, GFP-MHCK-A displayed frequent enrichment in the anterior of polarized migrating cells, and in the polar region but not the furrow during cytokinesis. GFP-MHCK-B generally displayed a homogeneous distribution. In migrating cells GFP-MHCK-C displayed posterior enrichment similar to that of myosin II, but did not localize with myosin II to the furrow during the early stage of cytokinesis. At the late stage of cytokinesis, GFP-MHCK-C became strongly enriched in the cleavage furrow, remaining there through completion of division. Conclusion MHCK-A, -B, and -C display distinct cellular localization patterns suggesting different cellular functions and regulation for each MHCK isoform. The strong localization of MHCK-C to the cleavage furrow in the late stages of cell division may reflect a mechanism by which the cell regulates the progressive removal of myosin II as furrowing progresses.

,Symptoms of mycosis induced by two native fungal entomopathogens of the citrus orthezia scale, Praelongorthezia praelonga (Hemiptera: Ortheziidae), an important pest of citrus orchards, are described. The data presented in this article are related to the article entitled "Seasonal prevalence of the insect pathogenic fungus Colletotrichum nymphaeae in Brazilian citrus groves under different chemical pesticide regimes". The endemic fungal pathogen, C. nymphaeae, emerges through the thin cuticular intersegmental regions of the citrus orthezia scale body revealing orange salmon-pigmented conidiophores bearing conidial masses, as well as producing rhizoid-like hyphae that extend over the citrus leaf. By contrast, nymphs or adult females of this scale insect infected with Lecanicillium longisporum exhibit profuse outgrowth of bright white-pigmented conidiophores with clusters of conidia emerging from the insect intersegmental membranes, and mycosed cadavers are commonly observed attached to the leaf surface by hyphal extensions. These morphological differences are important features to discriminate these fungal entomopathogens in citrus orthezia scales.,

Background The acetamidase of Mycobacterium smegmatis is a highly inducible enzyme. Expression of this enzyme is increased 100-fold when the substrate acetamide is present. The acetamidase gene is found immediately downstream of three open reading frames. Two of these are proposed to be involved in regulation. Results We constructed a deletion mutant in one of the upstream ORFs (amiA). This mutant (Mad1) showed a constitutively high level of acetamidase expression. We identified four promoters in the upstream region using a β-galactosidase reporter gene. One of these (P2) was inducible in the wild-type, but was constitutively active in Mad1. Conclusions These results demonstrate that amiA encodes a negative regulatory protein which interacts with P2. Since amiA has homology to DNA-binding proteins, it is likely that it exerts the regulatory effect by binding to the promoter to prevent transcription.