MUCOID-TO-NONMUCOID CONVERSION IN ALGINATE-PRODUCING PSEUDOMONAS-AERUGINOSA OFTEN RESULTS FROM SPONTANEOUS MUTATIONS IN ALGT, ENCODING A PUTATIVE ALTERNATE SIGMA-FACTOR, AND SHOWS EVIDENCE FOR AUTOREGULATION

The mucoid phenotype is common among strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibro sis and is due to overproduction of an exopolysaccharide called algina te. However, the mucoid phenotype is unstable in vitro, especially whe n the cells are incubated under low oxygen tension. Spontaneous conver sion to the nonmucoid form is typically due to mutations (previously c alled algS) that are closely linked to the alginate regulatory gene al gT, located at 68 min on the chromosome. Our sequence analysis of algT showed that its 22-kDa gene product shares homology with several alte rnate sigma factors in bacteria, suggesting that AlgT (also known as A lgU) interacts directly with RNA polymerase core to activate the promo ters of alginate genes. AlgT showed striking sequence similarity (79%) to sigma(E) of Escherichia coli, an alternate sigma factor involved i n high-temperature gene expression. Our analysis of the molecular basi s for spontaneous conversion from mucoid to nonmucoid, in the cystic f ibrosis isolate FRD, revealed that nonmucoid conversion was often due to one of two distinct missense mutations in algT that occurred at cod ons 18 and 29. RNase protection assays showed that spontaneous nonmuco id strains with the algT18 and algT29 alleles have a four- to fivefold reduction in the accumulation of algT transcripts compared with the w ild-type mucoid strain. Likewise, a plasmid-borne algT-cat transcripti onal fusion was about 3-fold less active in the algT18 and algT29 back grounds compared with the mucoid wild-type strain, and it was 20-fold less active in an algT::Tn501 background. These data indicate that alg T is autoregulated. The spontaneous algT missense alleles also caused about fivefold-reduced expression of the adjacent negative regulator, algN (also known as mucB). Transcripts of algN were essentially absent in the algT::Tn501 strain. Thus, algT regulates the algTN cluster, an d the two genes may be cotranscribed. A primer extension analysis show ed that algT transcription starts 54 bp upstream of the start of trans lation. Although the algT promoter showed little similarity to promote rs recognized by the vegetative sigma factor, it was similar to the al gR promoter. This finding suggests that AlgT may function as a sigma f actor to activate its own promoter and those of other alginate genes. The primer extension analysis also showed that algT transcripts were r eadily detectable in the typical nonmucoid strain PAO1, which was in c ontrast to a weak signal seen in the algT18 mutant of FRD. A plasmid-b orne algT gene in PAO1 resulted in both the mucoid phenotype and high levels of algT transcripts, further supporting the hypothesis that Alg T controls its own gene expression and expression of genes of the algi nate regulon.