Variable expression of extracellular polysaccharide in the marine bacterium Pseudomonas atlantica is controlled by genome rearrangement

Abstract

Production ofextracellular polysaccharide by the marine bacterium Pseudomonas atantica is a variable trait. Strains that produce extracellular polysaccharide (EPS +) have a mucoid colony phenotype, but during cultivation in the laboratory nonmucoid, EPS -variants arise that have a crenated colony morphology. This change is reversible since crenated variants rapidly switch to the original mucoid phenotype. We have cloned the locus (eps) controlling variable expression of EPS production by screening a recombinant cosmid library for clones that restore EPS production in the crenated variant. By using eps as a probe of genomic structure in variant strains, expression of EPS production was found to be controlled by a specific DNA rearrangement. Insertion of a 1.2-kilobase-pair DNA sequence in the eps locus results in EPS -, whereas excision of the sequence restores the EPS + phenotype. Properties of the rearrangement suggest the involvement of a mobile genetic element. The possible significance of this DNA rearrangement to the survival ofP. atlantica in the ocean is discussed.Pseudomonas atlantica attaches to and colonizes a variety of surfaces in the marine environment (1). The ability of a bacterium to elaborate adhesins for binding to a given substratum may be critical to its survival in the ocean because nutrients, which are scarce in seawater, are more abundant on animate surfaces, biofilms, or surfaces with adsorbed organic matter (2). Components associated with the cell envelope such as flagellar and fimbrial appendages, outer membrane proteins, lipopolysaccharides, and extracellular polysaccharides (EPS) function in a large variety of interactions between bacteria and surfaces. With P. atlantica, production of an acidic EPS is thought to be a primary determinant of cell adhesiveness (3).As part of an investigation of the genetic regulation of polysaccharide synthesis in P. atlantica we sought to isolate mutants defective in EPS production. However, EPSstrains were observed to arise spontaneously at frequencies greater than those expected for mutational events. The EPS "variants" are distinguished by their colony morphology. The parental EPS + phenotype is mucoid (M), and the EPSvariants display either a crenated (C) or translucent (T) colony morphology. M and nonmucoid (non-M) variants of Pseudomonas aeruginosa and Pseudomonas fluorescens have also been described (4,5). The genetic mechanisms regulating variable expression of some properties of pathogenic bacteria-i.e., phase variation of flagella in Salmonella and pili variation in Escherichia coli and Neisseria-have been determined (6-8). These variable systems are controlled by complex genomic rearrangements that apparently function to generate antigenic diversity in surface components to allow the pathogens to evade the host immune system. Mechanisms that generate diversity-for example, of adhesive forms-could be important to the survival of marine bacteria as well. Furthermore, insight into novel strategies regulating gene expression might be dis...

Computer-based analyses of the protein constituents of transport systems catalysing export of complex carbohydrates in bacteria

“…The various carbohydrate-containing constituents localized to the cell surface are frequently essential virulence factors of pathogenic bacteria as they provide protective functions that allow the organism to combat host defence mechanisms (Hornick et al, 1970;Robbins & Robbins, 1984; Pearce & Roberts, 1995). These macromolecules often exhibit the characteristic of phase variation, allowing the bacteria to successfully evade the immune system of the host (Saier & Jacobson, 1984;Bartlett et al, 1988;Seifert & So, 1988;Dybvig, 1993).…”

Site-specific insertion of IS1301 and distribution in Neisseria meningitidis strains

“…Transposition of insertion elements (IS) is known to cause a number of effects, including insertions, deletions, and cointegrate formation, resulting either in silent mutations, in knockout of gene expression (14), or in regulation of downstream-located genes (5,14,34). Transposition activity may thus create diversity in a bacterial population, with the result that a small fraction of the bacteria is preadapted to environmental changes (2,21).…”

“…Although a huge number of IS in bacteria have been identified, besides IS1301, only a few examples of reversible regulatory functions of gene expression have been described: IS492 causes reversible inactivation of extracellular polysaccharide production in Pseudomonas atlantica (1,2), IS-PA-4/5 is involved in the reversible mucoid conversion of Pseudomonas aeruginosa (36), IS1 insertion in virF of Shigella flexneri results in loss of virulence (26), and insertion of an IS1-like element causes inactivation of the Citrobacter freundii capsular antigen Vi in Escherichia coli (29). However, except for IS1301 in siaA and IS492 in the eps locus, the exact mechanisms of regulation remain unclear, either with the link between insertion site and gene expression missing (IS-PA-4/5) or with only indirect evidence for reversibility (IS1 in virF) or no evidence in the natural host (Vi of C. freundii).…”

Analysis of the Piv Recombinase-Related Gene Family of Neisseria gonorrhoeae

“…7A). Preference for insertion next to repeated sequences or site-specific insertion has been reported for other members of the IS110 family (2,21). The alignment of the flanking sequences for the irg genes reveals 14 bp of identity immediately upstream of the predicted start codons for irg2, irg3, irg4, irg5, irg6 (irg2-6), and irg8 and 133 bp of near-identity (1-bp difference in the irg8 flanking sequence) in the sequence downstream of the stop codons for irg1-6 and irg8 (Fig.…”

“…All donor probes were labeled with fluorescein isothiocyanate at the 3Ј end, while all acceptor probes were labeled at the 5Ј end with LightCycler-Red 640-N-hydroxysuccinimide ester. Acceptor probes also had phosphate groups attached to the 3Ј end to prevent probe extension (IT Biochem 2 (for a final concentration of 5 mM), 2 l of each primer (0.5 mM), 2 l of each hybridization probe (0.2 mM), 2 l of template, and PCR-grade sterile water for a final volume of 20 l. Fluorescence was acquired at the end of every extension step during the PCR. Primers used for PCR were LCirg (5Ј-CGTAGTGAACCCGCTGAAAA TAAGCAAGTATGC-3Ј) and LCirgrev (5Ј-GACCGGCAATACTGCGC-3Ј).…”