Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli.

Mucoid strains of Pseudomonas aeruginosa produce a viscous exopolysaccharide called alginate and also express alginate lyase activity which can degrade this polymer. By transposon mutagenesis and gene replacement techniques, the algL gene encoding a P. aeruginosa alginate lyase enzyme was found to reside between algG and algA within the alginate biosynthetic gene cluster at 35 min on the P. aeruginosa chromosome. DNA sequencing data for algL predicted a protein product of ca. 41 kDa, including a 27-amino-acid signal sequence, which would be consistent with its possible localization in the periplasmic space. Expression of the algL gene in Escherichia coli cells resulted in the expression of alginate lyase activity and the appearance of a new protein of ca. 39 kDa detected on sodium dodecyl sulfate-polyacrylamide gels. In mucoid P. aeruginosa strains, expression of algL was regulated by AlgB, which also controls expression of other genes within the alginate gene cluster. Since alginate lyase activity is associated with the ability to produce and secrete alginate polymers, alginate lyase may play a role in alginate production.     

Alginate lyase (AlgL) activity is required for alginate biosynthesis in Pseudomonas aeruginosa

To determine whether AlgL's lyase activity is required for alginate production in Pseudomonas aeruginosa, an algLdelta::Gm(r) mutant (FRD-MA7) was created. algL complementation of FRD-MA7 restored alginate production, but algL constructs containing mutations inactivating lyase activity did not, demonstrating that the enzymatic activity of AlgL is required for alginate production.     

AlgX is a periplasmic protein required for alginate biosynthesis in Pseudomonas aeruginosa

Alginate, an exopolysaccharide produced by Pseudomonas aeruginosa, provides the bacterium with a selective advantage that makes it difficult to eradicate from the lungs of cystic fibrosis (CF) patients. Previous studies identified a gene, algX, within the alginate biosynthetic gene cluster on the P. aeruginosa chromosome. By probing cell fractions with anti-AlgX antibodies in a Western blot, AlgX was localized within the periplasm. Consistent with these results is the presence of a 26-amino-acid signal sequence. To examine the requirement for AlgX in alginate biosynthesis, part of algX in P. aeruginosa strain FRD1::pJLS3 was replaced with a nonpolar gentamicin resistance cassette. The resulting algXDelta::Gm mutant was verified by PCR and Western blot analysis and was phenotypically nonmucoid (non-alginate producing). The algXDelta::Gm mutant was restored to the mucoid phenotype with wild-type P. aeruginosa algX provided on a plasmid. The algXDelta::Gm mutant was found to secrete dialyzable oligouronic acids of various lengths. Mass spectroscopy and Dionex chromatography indicated that the dialyzable uronic acids are mainly mannuronic acid dimers resulting from alginate lyase (AlgL) degradation of polymannuronic acid. These studies suggest that AlgX is part of a protein scaffold that surrounds and protects newly formed polymers from AlgL degradation as they are transported within the periplasm for further modification and eventual transport out of the cell.     

Identification of algF in the alginate biosynthetic gene cluster of Pseudomonas aeruginosa which is required for alginate acetylation.

Mucoid strains of Pseudomonas aeruginosa produce a high-molecular-weight exopolysaccharide called alginate that is modified by the addition of O-acetyl groups. To better understand the acetylation process, a gene involved in alginate acetylation called algF was identified in this study. We hypothesized that a gene involved in alginate acetylation would be located within the alginate biosynthetic gene cluster at 34 min on the P. aeruginosa chromosome. To isolate algF mutants, a procedure for localized mutagenesis was developed to introduce random chemical mutations into the P. aeruginosa alginate biosynthetic operon on the chromosome. For this, a DNA fragment containing the alginate biosynthetic operon and adjacent argF gene in a gene replacement cosmid vector was utilized. The plasmid was packaged in vivo into lambda phage particles, mutagenized in vitro with hydroxylamine, transduced into Escherichia coli, and mobilized to an argF auxotroph of P. aeruginosa FRD. Arg+ recombinants coinherited the mutagenized alginate gene cluster and were screened for defects in alginate acetylation by testing for increased sensitivity to an alginate lyase produced by Klebsiella aerogenes. Alginates from recombinants which showed increased sensitivity to alginate lyase were tested for acetylation by a colorimetric assay and infrared spectroscopy. Two algF mutants that produced alginates reduced more than sixfold in acetyl groups were obtained. The acetylation defect was complemented in trans by a 3.8-kb XbaI-BamHI fragment from the alginate gene cluster when placed in the correct orientation under a trc promoter. By a merodiploid analysis, the algF gene was further mapped to a region directly upstream of algA by examining the polar effect of Tn501 insertions. By gene replacement, DNA with a Tn501 insertion directly upstream of algA was recombined with the chromosome of mucoid strain FRD1. The resulting strain, FRD1003, was nonmucoid because of the polar effect of the transposon on the downstream algA gene. By providing algA in trans under the tac promoter, FRD1003 produced nonacetylated alginate, indicating that the transposon was within or just upstream of algF. These results demonstrated that algF, a gene involved in alginate acetylation, is located directly upstream of algA.     

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

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 algS 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 hisI+ clone. Using cloned algS-containing DNA and a method for gene replacement, we constructed isogenic strains of P. aeruginosa that had Tn501 adjacent to algS 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 Tn501 served as an adjacent marker to select for clones containing the respective algS 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 algS controls the expression of the adjacent algT gene, we constructed a pLAFR1 algS(Off)T clone and showed it to be unable to complement an algT::Tn501 mutation in trans. In contrast, a pLAFR1 algS(On)T clone did complement algT::Tn501 in trans. Thus, algS appears to control the activation of algT expression, bringing about alginate conversion.     

Cloning of genes from mucoid Pseudomonas aeruginosa which control spontaneous conversion to the alginate production phenotype.

Strains of Pseudomonas aeruginosa causing chronic pulmonary infections in patients with cystic fibrosis are known to convert to a mucoid form in vivo characterized by the production of the exopolysaccharide alginate. The alginate production trait is not stable, and mucoid strains frequently convert back to the nonmucoid form in vitro. The DNA involved in these spontaneous alginate conversions, referred to as algS, was shown here to map near hisI and pru markers on the chromosome of strain FRD, an isolate from a cystic fibrosis patient. Although cloning algS+ by trans-complementation was not possible, a clone (pJF5) was isolated that caused algS mutants to convert to the Alg+ phenotype at detectable frequencies (approximately 0.1%) in vitro. Gene replacement with transposon-marked pJF5 followed by mapping studies showed that pJF5 contained DNA transducibly close to algS in the chromosome. Another clone was identified called pJF15 which did contain algS+ from mucoid P. aeruginosa. The plasmid-borne algS+ locus could not complement spontaneous algS mutations in trans, but its cis-acting activity was readily observed after gene replacement with the algS mutant chromosome by using an adjacent transposon as the selectable marker. pJF15 also contained a trans-active gene called algT+ in addition to the cis-active gene algS+. The algT gene was localized on pJF15 by using deletion mapping and transposon mutagenesis. By using gene replacement, algT::Tn501 mutants of P. aeruginosa were constructed which were shown to be complemented in trans by pJF15. Both algS and algT were located on a DNA fragment approximately 3 kilobases in size. The algS gene may be a genetic switch which regulates the process of alginate conversion.     

Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate.

The algB gene, which is involved in the production of alginate in Pseudomonas aeruginosa, was localized to approximately 2.2 kilobases of DNA from strain FRD by using transposon Tn501 insertion mutagenesis, subcloning, and complementation techniques. The previously reported alg-50(Ts) mutation, which confers the phenotype of temperature-sensitive alginate production, was here designated as an algB allele. A transduction-mediated gene replacement technique was used for site-directed mutagenesis to isolate and characterize algB::Tn501 mutants of P. aeruginosa FRD. Although algB::Tn501 mutants had a nonmucoid phenotype (indicating an alginate deficiency), they still produced about 1 to 5% of wild-type levels of alginate in most growth media and up to 16% in very rich media. The algB::Tn501 mutations had no apparent effect on growth rate or growth requirements. Using another gene replacement technique called excision marker rescue, we constructed a chromosomal algB deletion (delta algB) mutant of P. aeruginosa FRD. The delta algB mutant also produced low levels of alginate as did the algB::Tn501 mutants. The alginate produced by algB::Tn501 mutants resembled wild-type alginate by all criteria studied: molecular weight, acetylation, and proportion of mannuronic and guluronic acids. Thus, the algB gene product is apparently involved in the high-level production of alginate by P. aeruginosa and is not directly involved in the pathway leading to its biosynthesis. Chromosomal mapping of an algB::Tn501 insertion showed linkage to the trp-2 marker on the FRD chromosome as does the algB50(Ts) mutation. The excision marker rescue technique was also used to place the algB::Tn501 marker on the chromosome of characterized strains of P. aeruginosa PAO. The algB::Tn501 mutation mapped near 21 min on the PAO chromosome.     

A mutation in algN permits trans activation of alginate production by algT in Pseudomonas species.

Conversion of the mucoid phenotype, which results from the production of the exopolysaccharide alginate, is a feature typical of Pseudomonas aeruginosa strains causing chronic pulmonary infections in patients with cystic fibrosis. In this study, we further characterized a recombinant plasmid, called pJF15, that contains DNA from the 65- to 70-min region of the chromosome of mucoid P. aeruginosa FRD1 and has loci involved in alginate conversion. Plasmid pJF15 complements algT mutations in trans and confers the mucoid phenotype in cis following gene replacement. However, the phenotype of nonmucoid P. aeruginosa carrying pJF15 is unchanged. Here we report the identification of a locus immediately downstream of algT, called algN, that may be a negative regulator that blocks algT from activating alginate production. Inactivation of algN by transposon Tn501 insertion allowed algT to stimulate alginate production in trans. The DNA sequence of this region identified an open reading frame that predicts an algN gene product of 33 kDa, but no homology was found to other proteins in a sequence data base. Clones of algT in which algN was deleted caused the activation of alginate biosynthesis in transconjugants of several P. aeruginosa strains. DNA containing algT was shown to hybridize to the genomes of several Pseudomonas species, including P. putida, P. stutzeri, and P. fluorescens. Transconjugants of these species carrying algT DNA (with a deletion of algN) from pJF15 showed a mucoid phenotype and increased production of uronic acid-containing polymers that resembled alginate.     

Cloning of Pseudomonas aeruginosa algG, which controls alginate structure.

The biochemical mechanism by which alpha-L-guluronate (G) residues are incorporated into alginate by Pseudomonas aeruginosa is not understood. P. aeruginosa first synthesizes GDP-mannuronate, which is used to incorporate beta-D-mannuronate residues into the polymer. It is likely that the conversion of some beta-D-mannuronate residues to G occurs by the action of a C-5 epimerase at either the monomer (e.g., sugar-nucleotide) or the polymer level. This study describes the results of a molecular genetic approach to identify a gene involved in the formation or incorporation of G residues into alginate by P. aeruginosa. Mucoid P. aeruginosa FRD1 was chemically mutagenized, and mutants FRD462 and FRD465, which were incapable of incorporating G residues into alginate, were independently isolated. Assays using a G-specific alginate lyase from Klebsiella aerogenes and 1H-nuclear magnetic resonance analyses showed that G residues were absent in the alginates secreted by these mutants. 1H-nuclear magnetic resonance analyses also showed that alginate from wild-type P. aeruginosa contained no detectable blocks of G. The mutations responsible for defective incorporation of G residues into alginate in the mutants FRD462 and FRD465 were designated algG4 and algG7, respectively. Genetic mapping experiments revealed that algG was closely linked (greater than 90%) to argF, which lies at 34 min on the P. aeruginosa chromosome and is adjacent to a cluster of genes required for alginate biosynthesis. The clone pALG2, which contained 35 kilobases of P. aeruginosa DNA that included the algG and argF wild-type alleles, was identified from a P. aeruginosa gene bank by a screening method that involved gene replacement. A DNA fragment carrying algG was shown to complement algG4 and algG7 in trans. The algG gene was physically mapped on the alginate gene cluster by subcloning and Tn501 mutagenesis.     

Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the sputum of cystic fibrosis patients produce copious quantities of an exopolysaccharide known as alginic acid. Since clinical isolates of the mucoid variants are unstable with respect to alginate synthesis and revert spontaneously to the more typical nonmucoid phenotype, it has been difficult to isolate individual structural gene mutants defective in alginate synthesis. The cloning of the genes controlling alginate synthesis has been facilitated by the isolation of a stable alginate-producing strain, 8830. The stable mucoid strain was mutagenized with ethyl methanesulfonate to obtain various mutants defective in alginate biosynthesis. Several nonmucoid (Alg-) mutants were isolated. A mucoid P. aeruginosa gene library was then constructed, using a cosmid cloning vector. DNA isolated from the stable mucoid strain 8830 was partially digested with the restriction endonuclease HindIII and ligated to the HindIII site of the broad host range cosmid vector, pCP13. After packaging in lambda particles, the recombinant DNA was introduced via transfection into Escherichia coli AC80. The clone bank was mated (en masse) from E. coli into various P. aeruginosa 8830 nonmucoid mutants with the help of pRK2013, which provided donor functions in trans, and tetracycline-resistant exconjugants were screened for the ability to form mucoid colonies. Three recombinant plasmids, pAD1, pAD2, and pAD3, containing DNA inserts of 20, 9.5, and 6.2 kilobases, respectively, were isolated based on their ability to restore alginate synthesis in various strain 8830 nonmucoid (Alg-) mutants. Mutants have been assigned to at least four complementation groups, based on complementation by pAD1, pAD2, or pAD3 or by none of them. Introduction of pAD1 into the spontaneous nonmucoid strain 8822, as well as into other nonmucoid laboratory strains of P. aeruginosa such as PAO and SB1, was found to slowly induce alginate synthesis. This alginate-inducing ability was found to reside on a 7.5-kilobase EcoRI fragment that complemented the alg-22 mutation of strain 8852. The pAD1 chromosomal insert which complements the alg-22 mutation was subsequently mapped at ca. 19 min of the P. aeruginosa PAO chromosome.     

Genetic analysis of the transcriptional arrangement of Azotobacter vinelandii alginate biosynthetic genes: identification of two independent promoters

The study of alginate biosynthesis, the exopolysac charide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, might lead to different bio-technological applications. Here we report the cloning of A. vinelandii algA, the gene coding for the bifunctional enzyme phosphomannose isomerase-guano-sine diphospho-D-mannose pyrophosphorylase (PMI-GMP). This gene was selected by the complementation for xanthan gum production of Xanthomonas campestris pv. campestris xanB mutants, which lack this enzymatic activity. The complementing cosmid clones selected, besides containing algA, presented a gene coding for an alginate lyase activity (algL), and some of them also contained algD which codes for GDP-mannose dehydrogenase. We present here the characterization of the A. vinelandii chromosomal region comprising algD and its promoter region, algA and algL, showing that, as previously reported for P. aeruginosa, A. vinelandii has a cluster of the biosynthetic alginate genes. We provide evidence for the presence of an algD-independent promoter in this region which transcribes at least algL and algA, and which is regulated in a manner that differs from that of the algD promoter.     

Alginate production by an <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> mutant unable to produce alginate lyase

Alginate is an industrially relevant linear copolymer composed of beta-1,4-linked D-mannuronic acid and its C-5 epimer L-guluronic acid. The rheological and gel-forming properties of alginates depend on the molecular weight and the relative content of the two monomers. Alginate produced by Azotobacter vinelandii was shown to be degraded towards the end of the culture, an undesirable situation in terms of potential alginate applications. A gene ( algL) encoding the alginate lyase activity AlgL is present within the alginate biosynthetic gene cluster of A. vinelandii. We constructed strain SML2, an A. vinelandii strain carrying a non-polar mutation within algL. No alginate lyase activity was detected in SML2. Under 3% dissolved oxygen tension, higher values of maximum mean molecular weight alginate were obtained (1240 kDa) with strain SML2, compared to those from the parental strain ATCC 9046 (680 kDa). These data indicate that AlgL activity causes the drop in the molecular weight of alginate produced by A. vinelandii.     

Molecular cloning into Tn5 and integration in the Pseudomonas aeruginosa chromosome: a tool for heterologous gene expression

The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.     

Involvement of the exopolysaccharide alginate in the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae

Alginate, a co-polymer of O-acetylated beta-1,4-linked D-mannuronic acid and L-guluronic acid, has been reported to function in the virulence of Pseudomonas syringae, although genetic studies to test this hypothesis have not been undertaken previously. In the present study, we used a genetic approach to evaluate the role of alginate in the pathogenicity of P. syringae pv. syringae 3525, which causes bacterial brown spot on beans. Alginate biosynthesis in strain 3525 was disrupted by recombining Tn5 into algL, which encodes alginate lyase, resulting in 3525.L. Alginate production in 3525.L was restored by the introduction of pSK2 or pAD4033, which contain the alginate biosynthetic gene cluster from P. syringae pv. syringae FF5 or the algA gene from P. aeruginosa respectively. The role of alginate in the epiphytic fitness of strain 3525 was assessed by monitoring the populations of 3525 and 3525.L on tomato, which is not a host for this pathogen. The mutant 3525.L was significantly impaired in its ability to colonize tomato leaves compared with 3525, indicating that alginate functions in the survival of strain 3525 on leaf surfaces. The contribution of alginate to the virulence of strain 3525 was evaluated by comparing the population dynamics and symptom development of 3525 and 3525.L in bean leaves. Although 3525. L retained the ability to form lesions on bean leaves, symptoms were less severe, and the population was significantly reduced in comparison with 3525. These results indicate that alginate contributes to the virulence of P. syringae pv. syringae 3525, perhaps by facilitating colonization or dissemination of the bacterium in planta.     

Role of alginate lyase in cell detachment of Pseudomonas aeruginosa.

The exopolysaccharide alginate of Pseudomonas aeruginosa was shown to be important in determining the degree of cell detachment from an agar surface. Nonmucoid strain 8822 gave rise to 50-fold more sloughed cells than mucoid strains 8821 and 8830. Alginate anchors the bacteria to the agar surface, thereby influencing the extent of detachment. The role of the P. aeruginosa alginate lyase in the process of cell sloughing was investigated. Increased expression of the alginate lyase in mucoid strain 8830 led to alginate degradation and increased cell detachment. Similar effects were seen both when the alginate lyase was induced at the initial stage of cell inoculation and when it was induced at a later stage of growth. It appears that high-molecular-weight alginate polymers are required to efficiently retain the bacteria within the growth film. When expressed from a regulated promoter, the alginate lyase can induce enhanced sloughing of cells because of degradation of the alginate. This suggests a possible role for the lyase in the development of bacterial growth films.     

Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase.

We have cloned a lipopolysaccharide (LPS) biosynthetic gene from Pseudomonas aeruginosa PAO1 that complements the defect in the production and incorporation of LPS O side chains in the LPS-rough strain AK1012. This gene was characterized by pulsed-field gel electrophoresis, deletion and restriction mapping of the cloned DNA, and biochemical analysis of the protein product. The cloned DNA was found to map to the 7-to-11-min region of the P. aeruginosa chromosome, and the gene needed for complementation of the LPS-rough phenotype was contained on a 2.6-kb HindIII-SacI fragment. This same size restriction fragment contains the alginate gene algC, which encodes the enzyme phosphomannomutase (PMM) and also maps to this region of the P. aeruginosa chromosome. The LPS-rough strain AK1012 was deficient in PMM activity, and this activity was restored to parental levels when the cloned gene was transferred to strain AK1012. In addition, the cloned gene could complement the PMM deficiency in the algC mutant strain 8858, and the cloned algC gene could restore the LPS-smooth phenotype to strain AK1012. These results indicate that the gene we have cloned is equivalent to the alginate gene algC. We designate this gene pmm to emphasize that it encodes the enzyme PMM, which has been shown to be essential for alginate production, and we demonstrate that PMM activity is required for the LPS-smooth phenotype in P. aeruginosa PAO1.     

Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme

The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.     

Method for gene replacement in Pseudomonas aeruginosa used in construction of recA mutants: recA-independent instability of alginate production.

The availability of a technique for site-directed mutagenesis by gene replacement provides a powerful tool for genetic analysis in any bacterial species. We report here a general technique for gene replacement in Pseudomonas aeruginosa. Genes on fragments of cloned P. aeruginosa DNA, altered by transposon mutagenesis, can be transduced into a recipient strain and can replace homologous genes in the P. aeruginosa genome. In this study we applied this technique to the construction of recA mutants of P. aeruginosa. A cloned segment of P. aeruginosa FRD1 DNA was isolated which encoded a protein analogous to the recA gene product of Escherichia coli. The P. aeruginosa recA gene was able to complement several defects associated with recA mutation in E. coli. Transposon Tn1 and Tn501 insertions in the cloned recA gene of P. aeruginosa were used to generate chromosomal recA mutants by gene replacement. These recA strains of P. aeruginosa were more sensitive to UV irradiation and methyl methane sulfonate and showed reduced recombination proficiency compared with the wild type. Also examined was the effect of recA mutations on the expression of alginate, a virulence trait. Alginate is a capsulelike polysaccharide associated with certain pulmonary infections, and its expression is typically unstable. The genetic mechanism responsible for the instability of alginate biosynthesis was shown to be recA independent.