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AlgR controls numerous virulence factors in Pseudomonas aeruginosa, including alginate, hydrogen cyanide production, and type 1V pilus-mediated twitching motility. In this study, the role of AlgR in biofiims was examined in continuous-flow and static biofilm assays. Strain PSL317 ([DELTA]algR) produced one-third the biofilm biomass of wild-type strain PAP1. Complementation with algR, but notfimTU-pilVWXY1Y2E, restored PSL317 to the wild-type biofilm phenotype. Comparisons of the transcriptional profiles of biofilm-grown PAP1 and PSL317 revealed that a number of quorum-sensing genes were upregulated in the algR deletion strain. Measurement of rhlA::lacZ and rhlI::lacZ promoter fusions confirmed the transcriptional profiling data when PSL317 was grown as a hiofiim, but not planktonically. Increased amounts of rhamnolipids and N-butyryl homoserine iactone were detected in the biofilm effluent but not the planktonic superuatants of the algR mutant. Additionally, AlgR specifically bound to the rhlA and rhlI promoters in mobility shift assays. Moreover, PAP1 containing a chromosomal mutated AlgR binding site in its rhlI promoter formed biofilms and produced increased amounts of rhamnolipids similarly to the algR deletion strain. These observations indicate that AlgR specifically represses the Rhl quorum-sensing system during biofilm growth and that such repression is necessary for normal biofilm development. These data also suggest that AlgR may control transcription in a contact-dependent or biofilm-specific manner.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis (CF) patients. One characteristic of P. aeruginosa CF isolates is the overproduction of the exopolysaccharide alginate, controlled by AlgR. Transcriptional profiling analyses comparing mucoid P. aeruginosa strains to their isogenic algR deletion strains showed that the transcription of cyanide-synthesizing genes (hcnAB) was ~3-fold lower in the algR mutants. S1 nuclease protection assays corroborated these findings, indicating that AlgR activates hcnA transcription in mucoid P. aeruginosa. Quantification of hydrogen cyanide (HCN) production from laboratory isolates revealed that mucoid laboratory strains made sevenfold more HCN than their nonmucoid parental strains. In addition, comparison of laboratory and clinically derived nonmucoid strains revealed that HCN was fivefold higher in the nonmucoid CF isolates. Moreover, the average amount of cyanide produced by mucoid clinical isolates was 4.7 [+ or -] 0.85 [micro]mol of HCN/mg of protein versus 2.4 [+ or -] 0.40 [micro]mol of HCN/mg of protein for nonmucoid strains from a survey conducted with 41 P. aeruginosa CF isolates from 24 patients. Our data indicate that (i) mucoid P. aeruginosa regardless of their origin (laboratory or clinically derived) produce more cyanide than their nonmucoid counterparts, (ii) AlgR regulates HCN production in P. aeruginosa, and (iii) P. aeruginosa CF isolates are more hypercyanogenic than nonmucoid laboratory strains. Taken together, cyanide production may be a relevant virulence factor in CF lung disease, the production of which is regulated, in part, by AlgR.

An estimated one-third of the world's population is latently infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. Here, we demonstrate that, unlike wild-type M. tuberculosis, a strain of M. tuberculosis disrupted in the mce 1 operon was unable to enter a stable persistent state of infection in mouse lungs. Instead, the mutant continued to replicate and killed the mice more rapidly than did the wild-type strain. Histological examination of mouse lungs infected with the mutant strain revealed diffusely organized granulomas with aberrant inflammatory cell migration. Murine macrophages infected ex vivo with the mutant strain were reduced in their ability to produce tumor necrosis factor [alpha], IL-6, monocyte chemoattractant protein 1, and nitric oxide (NO), but not IL-4. The mce 1 mutant strain complemented with the mce 1 genes stimulated tumor necrosis factor [alpha] and NO production by murine macrophages at levels stimulated by the wild-type strain. These observations indicate that the mce 1 operon mutant is unable to stimulate T helper 1-type immunity in mice. The hypervirulence of the mutant strain may have resulted from its inability to stimulate a proinflammatory response that would otherwise induce organized granuloma formation and control the infection without killing the organism. The mce 1 operon of M. tuberculosis may be involved in modulating the host inflammatory response in such a way that the bacterium can enter a persistent state without being eliminated or causing disease in the host.