Multiple promoters and induction by heat shock of the gene encoding the alternative sigma factor AlgU (sigma E) which controls mucoidy in cystic fibrosis isolates of Pseudomonas aeruginosa.

ABSTRACT Pseudomonas aeruginosa is a microorganism associated with the disease cystic fibrosis. While environmental P. aeruginosa strains are generally nonmucoid and motile, isolates recovered from the cystic fibrosis lung frequently display a mucoid, nonmotile phenotype. This phenotypic conversion is mediated by the alternative sigma factor AlgT. Previous work has shown that repression of fleQ by AlgT accounts for the loss of flagellum biosynthesis in these strains. Here, we elucidate the mechanism involved in the AlgT-mediated control of fleQ. Electrophoretic mobility shift assays using purified AlgT and extracts derived from isogenic AlgT+ and AlgT− strains revealed that AlgT inhibits fleQ indirectly. We observed that the AlgT-dependent transcriptional regulator AmrZ interacts directly with the fleQ promoter. To determine whether AmrZ functions as a repressor of fleQ, we mutated amrZ in the mucoid, nonmotile P. aeruginosa strain FRD1. Unlike the parental strain, the amrZ mutant was nonmucoid and motile. Complementation of the mutant with amrZ restored the mucoid, nonmotile phenotype. Thus, our data show that AlgT inhibits flagellum biosynthesis in mucoid, nonmotile P. aeruginosa cystic fibrosis isolates by promoting expression of AmrZ, which subsequently represses fleQ. Since fleQ directly or indirectly controls the expression of almost all flagellar genes, its repression ultimately leads to the loss of flagellum biosynthesis.

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Negative Control of Flagellum Synthesis in Pseudomonas aeruginosa Is Modulated by the Alternative Sigma Factor AlgT (AlgU)

Journal of Bacteriology ◽

10.1128/jb.181.23.7401-7404.1999 ◽

1999 ◽

Vol 181 (23) ◽

pp. 7401-7404 ◽

Cited By ~ 106

Author(s):

Edward S. Garrett ◽

Demetra Perlegas ◽

Daniel J. Wozniak

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Sigma Factor ◽

Negative Control ◽

Alternative Sigma Factor ◽

Flagellar Gene

ABSTRACT Many respiratory isolates of Pseudomonas aeruginosafrom cystic fibrosis patients are mucoid (alginate producing) yet lack flagella. It was hypothesized that an alginate regulator inhibits flagellar gene expression. Mutations in algB,algR, and algT resulted in nonmucoid derivatives, yet algT mutants expressed flagella. AlgT-dependent control of flagellum synthesis occurred through inhibition of fliC but not rpoN transcription.

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rpoE, the gene encoding the second heat-shock sigma factor, sigma E, in Escherichia coli.

The EMBO Journal ◽

10.1002/j.1460-2075.1995.tb07084.x ◽

1995 ◽

Vol 14 (5) ◽

pp. 1032-1042 ◽

Cited By ~ 184

Author(s):

P.E. Rouvière ◽

A. De Las Peñas ◽

J. Mecsas ◽

C.Z. Lu ◽

K.E. Rudd ◽

...

Keyword(s):

Escherichia Coli ◽

Heat Shock ◽

Sigma Factor ◽

Gene Encoding ◽

Sigma E

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Cloning the gene for the heat shock response positiwe regulator (sigma 32 homolog) from Pseudomonas aeruginosa

Canadian Journal of Microbiology ◽

10.1139/m95-010 ◽

1995 ◽

Vol 41 (1) ◽

pp. 75-87 ◽

Cited By ~ 13

Author(s):

Zerlina M. Naczynski ◽

Andrew M. Kropinski ◽

Chris Mueller

Keyword(s):

Pseudomonas Aeruginosa ◽

Heat Shock ◽

Sigma Factor ◽

Oligonucleotide Probe ◽

In Vitro Transcription ◽

Translation System ◽

Amino Acid Homology ◽

E Coli ◽

Transcriptional Start Sites

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040–1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (σ32) of E. coli. The plasmid containing the rpoH gene complemented the function of σ32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription–translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 °C) conditions. Two promoter sites were identified having sequence homology to the E. coli σ70 and σ24 consensus sequences.Key words: heat shock, Pseudomonas aeruginosa, sigma factor, transcription, oligonucleotide probe.

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