Showing papers by "Vojo Deretic published in 1993"

Characterization of a locus determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence similarities with a Bacillus sigma factor.

Abstract: Overproduction of the exopolysaccharide alginate by Pseudomonas aeruginosa results in mucoid colony morphology and is an important virulence determinant expressed by this organism in cystic fibrosis. Mucoidy is transcriptionally regulated by signal transduction systems and histone-like elements. One point of convergence of regulatory elements controlling mucoidy is the algD promoter. A newly described genetic locus required for algD transcription was characterized in this study. This DNA region, cloned from a nonmucoid PAO strain, was initially isolated on the basis of its ability to suppress mucoidy when present on a plasmid. The suppressing activity was observed in several mucoid PAO derivatives, including strain PAO568, in which the mapped muc-2 mutation is responsible for its mucoid phenotype, and in close to 40% of cystic fibrosis strains tested. Protein expression studies detected two polypeptides with apparent molecular masses of 27.5 and 20 kDa encoded by the region required for the suppression activity. The gene encoding the polypeptide with an apparent molecular mass of 27.5 kDa, termed algU, was further characterized. A functional chromosomal copy of algU was found to be necessary for the expression of mucoidy. Insertional inactivation of algU on the chromosome of the mucoid strain PAO568 abrogated alginate production and algD transcription. DNA sequence analysis revealed sequence similarity of the predicted algU gene product with sigma H (Spo0H), a sigma factor involved in the control of sporulation and competence in Bacillus spp. Physical mapping revealed that algU resided on the same SpeI fragment (F) as did the pruAB locus, known to be tightly linked with genetic determinants (muc) which can confer mucoidy in genetic crosses. When the chromosomal algU copy was tagged with a Tcr cassette (algU::Tcr), a tight genetic linkage of algU with pruAB was demonstrated by F116L-mediated generalized transduction. Moreover, algU::Tcr derivatives of PAO568 (originally carrying the muc-2 marker) lost the ability to transfer mucoidy in genetic crosses. These results suggest that algU, a regulator of algD transcription showing sequence similarity to an alternative sigma factor, and the genes immediately downstream of algU may be associated with a locus participating in the differentiation into the mucoid phenotype. Images

Differentiation of Pseudomonas aeruginosa into the alginate‐producing form: inactivation of mucB causes conversion to mucoidy

Abstract: Summary Mucoidy in Pseudomonas aeruginosa is a critical virulence factor associated with chronic respiratory infections in cystic fibrosis. A cluster of three tightly linked genes. algU, mucA and mucB located at 67.5 min, controls development of mucoid phenotype. This locus is allelic with a group of mutations (muc) associated with conversion into constitutively mucoid forms. One of the genes previously characterized in this region, algU, is absolutely required for the transcriptional activation of algD, a critical event in the establishment of mucoidy. AlgU is homologous to the alternative sigma factor σ;H (Spo0H) controlling sporulation and competence in Bacillus. Two genes downstream of algU, mucA and mucB were further characterized in this study. Previous complementation studies have demonstrated that mucA is required for suppression of mucoidy in the muc-2 strain PAO568. In this work, complementation analysis indicated that, in addition. mucB was required for suppression of mucoidy in the muc-25 strain PAO581, and for enhanced complementation of the muc-2 mutation in PAO568. The complete nucleotide sequence of mucA and mucH was determined. Insertional inactivation of mucB on the chromosome of the standard genetic strain PAO resulted in mucoid phenotype, and in a strong transcriptional activation of algD. Thus, a loss of mucB function is sufficient to cause conversion of P. aeruginosa into the mucoid phenotype. Since the algU-mucA-mucB region is a general site where muc mutations have been mapped, it is likely that mucB participates in the emergence of mucoid forms. Both mucA and mucB play a regulatory role in concert with the sigma-like factor AlgU; all three genes, along with signal transduction and histone-like elements, control differentiation of P. aeruginosa into the mucoid phenotype.

Conversion to mucoidy in Pseudomonas aeruginosa

Abstract: Chronic respiratory complications in cystic fibrosis, compounded by recurring infections with mucoid Pseudomonas aeruginosa and the associated inflammation, are the primary cause of high mortality in this inheritable disease. Since the conversion of P. aeruginosa into the exopolysaccharide alginate overproducing strains plays a critical role in the establishment of chronic infection, studies are directed towards understanding the processes underlying this phenomenon. The genes (algU, mucA, and mucB) and genetic alterations responsible for conversion to mucoidy have been recently characterized. The mutations leading to the emergence of mucoid strains are superimposed on a regulatory system with elements that resemble those controlling other aspects of bacterial developmental physiology.

Host dependenet inactivation by IS2 of induced E.coli penicillin amidase gene cloned on multicopy plasmids

Abstract: Structural instability of the cloned penicillin acylase gene (pac) from E.coli ATCC11105 was studied under various physiological conditions. Structural changes which adversely affect the expression of penicillin acylase gene were selected for only under conditions in which the gene was active and fully induced. In E.coli strain YMC the predominant mutations were the insertions of the IS2 element at various sites within the 700 bp proximal portion of the pac gene. The results indicated that the induction of the plasmid cloned gene was the main factor which rendered it a target for inactivation by insertions of the host encoded IS2 elements. The mutational events were host specific and they were not influenced by mutual positions and orientations of key marker genes on the plasmid.