Cloning, sequence and mutagenesis of the structural gene of Pseudomonas aeruginosa CysB, which can activate algD transcription

Abstract

SummaryPseudomonas aeruginosa strains infecting patients with cystic fibrosis (CF) acquire a mucoid phenotype due to overproduction of alginate. The key enzyme in alginate synthesis is AlgD, whose promoter is transcriptionally active in mucoid strains and under the control of several trans-acting factors, including the integration host factor (IHF). The algD promoter (palgD ) contains two IHF-binding sites (ihf1 and ihf2 ). Study of IHF binding to ihf2 of palgD, by electrophoretic mobility-shift assays, led to the discovery of a protein of 36 kDa (p36) able to bind downstream from ihf2, to the 3Ј region of palgD. The gene encoding p36 was isolated from the mucoid strain CHA of P. aeruginosa and sequenced. It can encode a 324-amino-acid protein, which shares a high degree of sequence identity (63%) with CysB from Escherichia coli and from Salmonella typhimurium, a transcriptional factor of the LysR superfamily. Furthermore, both p36 and S. typhimurium CysB bind the same site of palgD ; p36 was therefore termed CysB and its structural gene was called cysB. Next to cysB, on the opposite DNA strand, cysH was capable of encoding a protein sharing 26% identity with CysH (PAPS reductase) of E. coli and an even greater identity (54%) with the nucleotide-deduced protein from Arabidopsis. A CysB-deficient mutant of CHA, constructed by insertional inactivation of cysB, was a cysteine auxotroph and was unable to form a specific complex with palgD in vitro. Activity of palgD in the cysB mutant, in CHA and in the non-mucoid strain PAO was assessed by the use of a transcriptional algDxylE fusion. Cells of PAO and of the cysB mutant grown in minimal media in the presence of 0.3 M NaCl exhibited a palgD activity, which was 10% or less that of the mucoid strain CHA. Thus, P. aeruginosa CysB can act as an activator of algD expression.

Identification of a New Class of 5′-Adenylylsulfate (APS) Reductases from Sulfate-Assimilating Bacteria

“…The P. aeruginosa cysH gene exists as a lone ORF and does not appear to exist in an operon. A cysB homolog is located downstream, but it is located on the opposite strand and is transcribed convergently with cysH (6). Therefore, it is unlikely that polar effects resulting from the insertional mutation can account for cysteine auxotrophy.…”

“…Nine nucleotide substitutions were found, resulting from either PCR-derived changes or due to sequence polymorphisms. All of the nucleotide differences were silent with respect to the reported amino acid sequence of the gene product (6). pB-PaAPR was tested for the ability to complement a range of E. coli cys mutants.…”

“…The P. aeruginosa cysH gene was amplified from genomic DNA by using the following PCR primers designed from the published sequence (6) and with the inclusion of HindIII and SacI recognition sites 5Ј-GCAAGCTTACGCCGGCTTATTCCTGG-3Ј and 5Ј-CCGAGCTCTATCGACGGTTTCAGGCC-3Ј, respectively. The amplified 0.9-kb DNA fragment was cloned into pUC19 and also digested with HindIII and b Gerben Zylstra laboratory strain.…”

“…The cysH homologs from Bacillus subtilus (20), Burkholderia cepacia (this study), Mycobacterium tuberculosis (5), Pseudomonas aeruginosa (6), and Rhizobium tropici (18) were identified as cysH homologs, based on homology analysis, and none had yet been functionally characterized. Thus, the presence of APS or PAPS reductase activity was examined in a range of bacterial species, and the cysH homolog from P. aeruginosa was chosen for detailed analysis.…”

Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach

“…This method is general and applicable to any simple organism for which a mutant can be made in the early stages of the reductive branch of the sulfate assimilation pathway. Such mutant strains of many commonly studied microorganisms already exist, as they are easily isolated as sulfur auxotrophs (28)(29)(30)(31). (Table 1).…”