Use of a gene replacement cosmid vector for cloning alginate conversion genes from mucoid and nonmucoid Pseudomonas aeruginosa strains: algS controls expression of algT

Abstract

Pseudomonas aeruginosa can convert to a mucoid colony morphology by a genetic mechanism called alginate conversion; this results in the production of copious amounts of the exopolysaccharide alginate. The mucoid phenotype of P. aeruginosa is commonly associated with its ability to cause chronic pulmonary tract infections in patients with cystic fibrosis. In this study we isolated the cis-acting locus involved in alginate conversion, called algS, from both mucoid and nonmucoid isogenic strains. We then examined the role of aigS in the control of algT, a trans-active gene required for alginate production in P. aeruginosa. We used a new cosmid cloning vector, called pEMR2, that permitted both the cloning of large DNA fragments and their subsequent gene replacement in P. aeruginosa. To verify the predicted properties of this vector, we isolated and tested a pEMR2 hisl+ clone. Using cloned algS-containing ONA and a method for gene replacement, we constructed isogenic strains of P. aeruginosa that had TnSO adjacent to aigS on the chromosome. Two pEMR2 clone banks containing genomic fragments from isogenic algS(On) (exhibiting the alginate production phenotype) and algS(Off) (exhibiting the non-alginate production phenotype) strains were constructed, and TnSO0 served as an adjacent marker to select for clones containing the respective aigS allele. The pEMR2 algS(On) and pEMR2 algS(Off) clones were shown to contain the indicated algS allele by gene replacement with the chromosome of strains that carried the opposite allele. To test whether aigS controls the expression of the adjacent aIgT gene, we constructed a pLAFRI algS(Off)T clone and showed it to be unable to complement an algT::TnSOl mutation in trans. In contrast, a pLAFR1 algS(On)T clone did complement algT::TnSO in trans. Thus, aigS appears to control the activation of algT expression, bringing about alginate conversion.Pseudomonas aeruginosa is capable of converting to a mOucoid colony morphology by a genetic mechanism called alginate conversion (11). This mucoid phenotype is indicative of the production of a viscous exopolysaccharide known as alginate (9). When P. aeruginosa causes chronic and lethal pulmonary infections in patients with cystic fibrosis, the bacteria usually undergo alginate conversion in vivo (8, 36), resulting in a poor prognosis for the patient. Alginate production appears to confer upon the organisms an increased resistance to opsonic phagocytosis (32), an adherence mechanism (38), and may also clog small airways in the lungs (37). Several reports have shown that typical nonmucoid laboratory strains of P. aeruginosa can also undergo alginate conversion. Such mucoid strains arise following enrichments with agents such as phages (25, 26), pyocins (21), and antibiotics (17) that apparently select against the less-resistant nonmucoid strains, indicating that most P. aeruginosa strains probably carry genes necessary for alginate production (although these genes are normally silent) and that some mechanism activates their expression. The algina...

The σE‐mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of σE

“…Operons encoding other ECF sigmas have previously been shown to also encode regulators of the sigma-factor activity. For example, the operon encoding AlgU/T, an alternative sigma factor required for alginate biosynthesis in Pseudomonas aeruginosa (Hershberger et al, 1995;Schurr et al, 1995), includes four downstream genes, mucA-mucB/ algN-mucC-mucD, involved in regulating AlgU/T activity (Flynn and Ohman, 1988;Goldberg et al, 1993;Martin et al, 1993a,b,c). MucA inhibits AlgU/T activity in vitro Xie et al, 1996) and previous work in E. coli had identified a partial ORF immediately downstream of rpoE, termed mclA (mucA-like), that showed significant homology to mucA .…”

Precise excision and instability of the transposon Tn5 in Pseudomonas aeruginosa

“…aeruginosa, called excision marker rescue, which takes advantage of Tn5 instability to promote the exchange of plasmid-borne alleles for homologous chromosomal sequences (Goldberg & Ohman, 1987;Flynn & Ohman, 19886). Others have also used this technique (Woodruff & Hancock, 1988).…”

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“…This region of a/pP displays clonal variation; in some cases deletions involving the direct repeats of algP appear to be associated with loss of mucoidy ). However, direct correlation between changes in algP and transitions to nonmucoidy remains to be established since other genetic evidence points to an importance of the mucand algST loci to the metastable nature of mucoidy (Fyfe and Govan, 1980;Flynn and Ohman, 1988). This remains a controversial issue, both in terms of mechanism and significance.…”

“…1, Fyfe and Govan, 1980;MacGeorge ef ai, 1986). In a series of more recent gene replacement experiments, Ohman and coworkers using the a/gST locus (which is, like muc loci, linked to the hisi marker) have demonstrated its role in the regulation of mucoidy (Flynn and Ohman, 1988). Since only a limited number of mucoid clinical strains could be manipulated by these genetic methods it is not clear whether the known muc (and aigST) genes are the only loci ubiquitously associated with conversion to mucoidy.…”