Conversion to a mucoid, exopolysaccharide alginate-overproducing phenotype in Pseudomonas aeruginosa is associated with chronic respiratory infections in cystic fibrosis. Mucoidy is caused by muc mutations that derepress the alternative factor AlgU, which in turn activates alginate biosynthetic and ancillary regulatory genes. Here we report the molecular characterization of two newly identified genes, algW and mucD, that affect expression of mucoidy. The algW gene, mapping at 69 min, was isolated on the basis of its ability to suppress mucoidy and reduce transcription of the alginate biosynthetic gene algD. The predicted primary structure of AlgW displayed similarity to HtrA (DegP), a serine protease involved in proteolysis of abnormal proteins and required for resistance to oxidative and heat stress in enteric bacteria. Inactivation of algW on the chromosome of the wild-type nonmucoid strain PAO1 caused increased sensitivity to heat, H 2 O 2 , and paraquat, a redox cycling compound inducing intracellular levels of superoxide. This mutation also permitted significant induction of alginate production in the presence of subinhibitory concentrations of paraquat. Two new genes, mucC and mucD, were identified immediately downstream of the previously characterized portion (algU mucA mucB) of the gene cluster at 67.5 min encoding the alternative factor AlgU and its regulators. Interestingly, the predicted gene product of mucD also showed similarities to HtrA. Inactivation of mucD on the PAO1 chromosome resulted in conversion to the mucoid phenotype. The mutation in mucD also caused increased sensitivity to H 2 O 2 and heat killing. However, in contrast to algW mutants, no increase in susceptibility to paraquat was observed in mucD mutants. These findings indicate that algW and mucD play partially overlapping but distinct roles in P. aeruginosa resistance to reactive oxygen intermediates and heat. In addition, since mutations in mucD and algW cause conversion to mucoidy or lower the threshold for its induction by reactive oxygen intermediates, these factors may repress alginate synthesis either directly by acting on AlgU or its regulators or indirectly by removing physiological signals that may activate this stress response system.Pseudomonas aeruginosa is an opportunistic pathogen causing acute infections in individuals with compromised defense mechanisms, such as burned or neutropenic patients. Another notorious and in many aspects unique infection caused by P. aeruginosa is its characteristic association with the respiratory tract of patients with cystic fibrosis (CF) (8,41,53). This chronic infection differs from the acute disseminated disease by being localized almost exclusively to the lumen of the respiratory tract (1,26,53). Furthermore, the protracted colonization of the respiratory tract is accompanied by a distinct set of phenotypic changes of the bacterium (8, 53). For example, P. aeruginosa strains colonizing CF patients often convert to a mucoid, exopolysaccharide alginate-overproducing phenotype (17), los...

The alternative sigma factor AlgU (Pseudomonas aeruginosa E ) is required for full resistance of P. aeruginosa to oxidative stress and extreme temperatures. AlgU also controls conversion of P. aeruginosa to the mucoid, alginate-overproducing phenotype associated with lethal infections in cystic fibrosis patients. Mutations that cause conversion to mucoidy in cystic fibrosis isolates occur frequently in mucA, the second gene within the algU mucABCD gene cluster. Here we analyze the biochemical basis of conversion to mucoidy. MucA was shown to act as an anti-sigma factor by binding to AlgU and inhibiting its activity. MucB, another negative regulator of AlgU, was localized in the periplasm. MucB exerts its function from this compartment, since deletion of the leader peptide and the cytoplasmic location of MucB abrogated its ability to inhibit mucoidy. These data support a model in which a multicomponent system, encompassing an anti-factor and elements in the periplasmic compartment, modulates activity of AlgU. Since factors controlling AlgU are conserved in other gram-negative bacteria, the processes controlling conversion to mucoidy in P. aeruginosa may be applicable to the regulation of AlgU ( E ) equivalents in other organisms.The algU mucABCD cluster (Fig. 1) controls conversion of Pseudomonas aeruginosa to the mucoid, alginate-overproducing phenotype (4, 9, 29-31) associated with the establishment of lethal respiratory infections in cystic fibrosis (CF) patients (17). The uncovering of the genetic elements controlling conversion to mucoidy in P. aeruginosa has also facilitated the recognition of a new class of bacterial alternative factors (9, 27) distantly related to the 70 family (27). This new group of alternative factors, termed ECF (27) or the E -like family (9, 10), includes members with an average identity of approximately 25%. Within this diverse superfamily, P. aeruginosa AlgU (also known as AlgT [11] or Pa E [9]), the second or extreme heat shock sigma factor E (RpoE) in Escherichia coli (13) and Salmonella typhimurium (32), and several more recently described homologs (Fig. 1) found in diverse gramnegative organisms make up a more homogeneous subset of highly conserved (62 to 93% identity with AlgU) and most likely functionally related alternative factors.P. aeruginosa AlgU and E. coli E are functionally interchangeable (49). Biochemical studies in E. coli have indicated that E functions as an alternative sigma factor (13,39,40). Recent work has demonstrated that AlgU is an RNA polymerase subunit and that it directs transcription of cognate promoters in vitro (22,43). Interestingly, the conservation of the primary structure and function of AlgU homologs extends beyond the genes encoding these factors. The known and putative equivalents of algU are followed by elements closely resembling the mucABCD genes (Fig. 1). It has been proposed that these genes play a role in the regulation of cognate factors on the basis of the observation that spontaneous mutations in mucA (31) or experimental inactivatio...

Azotobacter vinelandii presents a differentiation process leading to the formation of desiccation-resistant cysts. Alginate, the exopolysaccharide produced by this bacterium, has been postulated to have a role in cyst formation. Here, we report the cloning and characterization of the A. vinelandii gene coding for the enzyme GDP-mannose dehydrogenase (algD), which is the key enzyme for alginate synthesis in Pseudomonas aeruginosa. This gene has a high degree of similarity with the algD gene from P. aeruginosa, and similar proteins seem to be involved in algD regulation in both bacteria. We show the existence of two mRNA start sites; one of these sites corresponds to a promoter transcribed by RNA polymerase containing a E subunit. An A. vinelandii algD mutant which is completely impaired in alginate production and which is unable to form desiccation-resistant cells was constructed. The effects of NH 4 , NO 3 , and NaCl concentrations on algD transcription for three A. vinelandii strains producing different alginate levels were evaluated. We found a strict correlation between alginate production and algD transcription for the three strains studied; however, the effects on algD transcription under the conditions studied were different for each strain. The nitrogen source regulates algD expression in the wild-type strain.

The study of the biosynthesis of alginate, the exopolysaccharide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, has biotechnological and medical significance. We report here the identification of the A. vinelandii genes coding for the putative sigma factor AlgU and its negative regulators MucA and MucB through the suppression of the highly mucoid phenotype of an A. vinelandii strain by a plasmid encoding MucA and MucB. The sequences of the A. vinelandii algU, mucA, and mucB genes are highly homologous to those of the corresponding P. aeruginosa genes, AlgU shows 93% identity, and MucA and MucB are 64.4 and 63.9% identical, respectively. Forming part of the same operon as algU, mucA, and mucB, two additional genes (mucC and mucD) were identified and sequenced; the product of the former gene is homologous to ORF4 of Photobacterium sp. strain SS9, and that of the latter gene belongs to the HtrA serine protease family. Interestingly, the nonmucoid A. vinelandii UW136 had a 0.9-kb insertion within the algU gene. A strong correlation between AlgU activity and alginate production by A. vinelandii was also found, as reflected in the level of algD transcription.

One remarkable characteristic of the genomes of some Rhizobium species is the frequent occurrence of rearrangements. In some instances these rearrangements alter the symbiotic properties of the strains. However, no detailed molecular mechanisms have been proposed for the generation of these rearrangements. To understand the mechanisms involved in the formation of rearrangements in the genome of Rhizobium phaseoli, we have designed a system which allows the positive selection for amplification and deletion events. We have applied this system to investigate the stability of the symbiotic plasmid of R. phaseoli. High-frequency amplification events were detected which increase the copy number of a 120-kb region carrying nodulation and nitrogen fixation genes two to eight times. Deletion events that affect the same region were also found, albeit at a lower frequency. Both kinds of rearrangements are generated by recombination between reiterated nitrogenase (nifHDK) operons flanking the 120-kb region.Rhizobium spp. are gram-negative soil bacteria studied primarily for their ability to establish nitrogen-fixing symbioses with leguminous plants. Intensive genetic analysis during the past decade has led to the identification of genes essential for the nodulation (nod genes) and nitrogen fixation (nif and fix genes) processes. In all the fast-growing Rhizobium species, these genes are carried on large plasmids, the so-called Sym plasmids or pSym (20,24).One interesting characteristic of the Rhizobium genome is the presence of a large number of reiterated DNA sequences. For Rhizobium phaseoli, the symbiont of the common bean (Phaseolus vulgaris L.), we have estimated the presence of about 700 reiterated elements, belonging to 200 different families (9). This high degree of reiteration is not restricted to R. phaseoli; other members of the Rhizobiaceae family, including the closely related bacteria Agrobacterium tumefaciens, possess a large amount of reiterated DNA (9, 24). For other organisms, it has been shown that recombination between pairs of repeated elements may lead to different kinds of genomic rearrangements, including additions, amplifications, deletions, and inversions (2, 31).Frequent genomic rearrangements (in the range of 10-2 to 10-3) have been commonly observed in different Rhizobium species, including Bradyrhizobium japonicum, Rhizobium trifolii, and R. phaseoli. These rearrangements, which are frequently deletions, may affect the symbiotic properties of the strain, either for nodulation or for nitrogen fixation (4,7,10,14,18,39,44). Unfortunately, the mechanisms involved in the generation of genetic instability in these species have been poorly analyzed.In order to understand the mechanisms involved in the genetic instability of R. phaseoli, we have developed an experimental approach for the general selection of genomic rearrangements. This system allows the positive selection of different kinds of rearrangements, including amplifications, deletions, insertions, and loss of plasmids. We have used this sys...