Mycobacterium abscessus is an increasingly important cause of human disease; however, virulence determinants are largely uncharacterized. Previously, it was demonstrated that a rough, wild-type human clinical isolate (390R) causes persistent, invasive infection, while a smooth isogenic mutant (390S) has lost this capability. During serial passage of 390S, a spontaneous rough revertant was obtained, which was named 390V. This revertant regained the ability to cause persistent, invasive infection in human monocytes and the lungs of mice. Glycopeptidolipid (GPL), which plays a role in environmental colonization, was present in abundance in the cell wall of 390S, and was associated with sliding motility and biofilm formation. In contrast, a marked reducton in the amount of GPL in the cell wall of 390R and 390V was correlated with cord formation, a property associated with mycobacterial virulence. These results indicate that the ability to switch between smooth and rough morphologies may allow M. abscessus to transition between a colonizing phenotype and a more virulent, invasive form.

Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type Ⅱ polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding theβ-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production.

The chromosomal DNA of Streptomyces hygroscopicus 10-22, a derivative of strain 5102-6, was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestions with AseI gave 11 fragments with a total length of ca. 7.36 Mb. The AseI sites were mapped by analysis of overlapping chromosomal deletions in different mutants and confirmed by Southern hybridizations using partially digested genome fragments and linking cosmids as probes. PFGE analysis of DNA with and without proteinase K treatment, together with the hybridization results, suggested a linear organization with terminal proteins and large terminal inverted repeats. Some deletion mutants had circular chromosomes.

Streptomyces hygroscopicus 10-22 harbors a conjugative, autonomously replicating linear plasmid pHZ6 of ca. 70 kb, which shows no obvious homology with chromosomal DNA and is temperature-sensitive for replication, being stable in the host at 28³C but easily lost at 37³C. On a lawn of the wild-type S. hygroscopicus 10-22 cured of pHZ6, pHZ6 elicit pocks. Temperature sensitivity seemed to be a unique property for pHZ6 among six linear plasmids tested, including the well-known linear plasmids SLP2 in Streptomyces lividans 1326 and SCP1 in Streptomyces coelicolor A3(2). The distinct identity of pHZ6 from previously identified pHZ1-pHZ5 was demonstrated by the profile of relevant plasmids in six well-defined strains originated from S. hygroscopicus 10-22. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Two-component systems are important constituents of bacterial regulatory networks. Results of this investigation into the role of the MprAB two-component system of Mycobacterium tuberculosis indicate that it is associated with the regulation of several stress-responsive regulons. Using a deletion mutant lacking portions of the response regulator, MprA, and the histidine kinase, MprB, it was demonstrated by real-time PCR, primer extension analyses and DNA microarrays that MprAB activates sigma factor genes sigE and sigB, under SDS stress and during exponential growth. SDS-inducible, MprA-dependent transcriptional start points were identified for mprA, sigE and sigB, and variations in distance between these points and MprA-binding sites suggest that MprA is involved in different mechanisms of promoter activation. Although most of the SigE regulon was downregulated in the deletion mutant, the cluster of genes Rv1129c, Rv1130 and Rv1131, which is associated with growth in monoctyes, was upregulated in the deletion mutant under SDS stress, and this upregulation was dependent upon atmospheric growth conditions. Multiple stress-associated genes of the DosR, SigD and IdeR regulons were also upregulated in the deletion mutant, during exponential growth and/or in the presence of SDS. Surprisingly, the deletion mutant had increased resistance to SDS compared to the parental strain, and enhanced growth in human peripheral blood monocytes, characteristics which may result from a loss of repression of stress-associated genes.