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.

Coordinated regulation of molecular chaperones is an important feature of the bacterial stress response. The small molecular chaperone gene acr2 of Mycobacterium tuberculosis is activated by exposure to several stresses, including heat and the detergent sodium dodecyl sulfate (SDS). In this study, we show that acr2 is directly regulated by the MprAB two-component system, and that MprAB has both positive and negative effects on acr2 expression. mRNA analyses showed that acr2 expression levels were lower under SDS stress and control conditions but higher under heat shock in an mprAB deletion mutant than they were in the parental strain. Parental expression patterns were restored in an mprAB-complemented strain. Western blotting using an anti-Acr2 antibody showed that Acr2 protein synthesis correlated with mRNA levels. Primer extension identified one transcriptional start point (TSP) for acr2 in all three strains under control and stress conditions. Electrophoresis mobility shift assays revealed multiple MprA binding sites in the acr2 promoter, including one downstream and three upstream of the acr2 TSP, with one overlapping the binding sites predicted for SigE, SigH, and HspR. DNA footprinting confirmed that MprA protected large sections of the acr2 promoter region.Expression of several housekeeping genes under SDS stress also was evaluated, revealing the upregulation of large molecular chaperone genes and, unexpectedly, sigA, with slightly lower sigA mRNA levels detected in the mprAB deletion mutant than in the wild type. In contrast to Acr2, SigA protein synthesis did not correlate with mRNA expression. Overall, the data indicated that MprA has complex interactions with the acr2 promoter and indirect effects on major housekeeping genes.

Streptomyces ambofaciens produces an orange pigment and the antibiotic alpomycin, both of which are products of a type II polyketide synthase gene cluster identified in each of the terminal inverted repeats of the linear chromosome. Five regulatory genes encoding Streptomyces antibiotic regulatory proteins (alpV, previously shown to be an essential activator gene; alpT; and alpU) and TetR family receptors (alpZ and alpW) were detected in this cluster. Here, we demonstrate that AlpZ, which shows high similarity to γ-butyrolactone receptors, is at the top of a pathway-specific regulatory hierarchy that prevents synthesis of the alp polyketide products. Deletion of the two copies of alpZ resulted in the precocious production of both alpomycin and the orange pigment, suggesting a repressor role for AlpZ. Consistent with this, expression of the five alp-located regulatory genes and of two representative biosynthetic structural genes (alpA and alpR) was induced earlier in the alpZ deletion strain. Furthermore, recombinant AlpZ was shown to bind to specific DNA sequences within the promoter regions of alpZ, alpV, and alpXW, suggesting direct transcriptional control of these genes by AlpZ. Analysis of solvent extracts of S. ambofaciens cultures identified the existence of a factor which induces precocious production of alpomycin and pigment in the wild-type strain and which can disrupt the binding of AlpZ to its DNA targets. This activity is reminiscent of-γbutyrolactone-type molecules. However, the AlpZ-interacting molecule(s) was shown to be resistant to an alkali treatment capable of inactivatingγ-butyrolactones, suggesting that the AlpZ ligand(s) does not possess a lactone functional group.

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.

A type II polyketide synthase gene cluster located in the terminal inverted repeats of Streptomyces ambofaciens ATCC 23877 was shown to be responsible for the production of an orange pigment and alpomycin, a new antibiotic probably belonging to the angucycline/angucyclinone class. Remarkably, this alp cluster contains five potential regulatory genes, three of which (alpT, alpU, and alpV) encode proteins with high similarity to members of the Streptomyces antibiotic regulatory protein (SARP) family. Deletion of the two copies of alpV (one in each alp cluster located at the two termini) abolished pigment and antibiotic producti on, suggesting that AlpV acts as a transcriptional activator of the biosynthetic genes. Consistent with this idea, the transcription of alpA, which encodes a ketosynthase essential for orange pigment and antibiotic production, was impaired in the alpV mutant, while the expression of alpT, alpU, and alpZ, another regulatory gene encoding a -butyrolactone receptor, was not significantly affected. Real-time PCR experiments showed that transcription of alpV in the wild-type strain increases dramatically after entering the transition phase. This induction precedes that of alpA, suggesting that AlpV needs to reach a threshold level to activate the expression of the structural genes. When introduced into an S. coelicolor mutant with deletions of actII-ORF4 and redD, the SARP-encoding genes regulating the biosynthesis of actinorhodin and undecylprodigiosin, respectively, alpV was able to restore actinorhodin production only. However, actII-ORF4 did not complement the alpV mutant, suggesting that AlpV and ActII-ORF4 may act in a different way.