Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa

Abstract

The opportunistic pathogen Pseudomonas aeruginosa secretes a capsule-like polysaccharide called alginate that is important for evasion of host defenses, especially during chronic pulmonary disease of patients with cystic fibrosis (CF). Most proteins for alginate biosynthesis are encoded by the 12-gene algD operon. Interestingly, this operon also encodes AlgL, a lyase that degrades alginate. Mutants lacking AlgG, AlgK, or AlgX, also encoded by the operon, synthesize alginate polymers that are digested by the coregulated protein AlgL. We examined the phenotype of an DeltaalgL mutation in the highly mucoid CF isolate FRD1. Generating a true DeltaalgL mutant was possible only when the algD operon was under the control of a LacI(q)-repressed trc promoter. Upon induction of alginate production with isopropyl-beta-D-thiogalactopyranoside, the DeltaalgL mutant cells were lysed within a few hours. Electron micrographs of the DeltaalgL mutant showed that alginate polymers accumulated in the periplasm, which ultimately burst the bacterial cell wall. The requirement of AlgL in an alginate-overproducing strain led to a new model for alginate secretion in which a multiprotein secretion complex (or scaffold, that includes AlgG, AlgK, AlgX, and AlgL) guides new polymers through the periplasm for secretion across the outer membrane. In this model, AlgL is bifunctional with a structural role in the scaffold and a role in degrading free alginate polymers in the periplasm.

FIG. 1.

Genetic manipulations of the algD operon encoding proteins for alginate biosynthesis in P. aeruginosa FRD1. (A). Suicide plasmid pJLS3 contained a Ptrc-algD′ fragment and, when integrated into the chromosome of FRD1 at algD, formed strain FRD1050, which put the operon under LacI^q repression. (B) Suicide plasmid pSJ243, containing a fragment of the algD operon with a nonpolar ΔalgL::Gm^r allele, was used to generate a ΔalgL mutant of FRD1050 by allelic exchange. (C) Among the Gm^r transconjugants, FRD1300 was a ΔalgL mutant that had lost the vector-encoded (Suc^s) phenotype. Induction of the algD operon in FRD1300 with IPTG prevented cell growth.

FIG. 2.

Alginate phenotypes of FRD1 derivatives. (A) The mucoid (Alg⁺) phenotype on L agar was observed with parent strain FRD1 but not with FRD1050 (Ptrc-algD-A), which has the algD operon repressed by LacI^q. (B) The mucoid phenotype on L agar plus IPTG (1 mM) was observed with both FRD1 and FRD1050 (Ptrc-algD-A), which has the algD operon induced.

FIG. 3.

Effect of the induction of alginate production on the growth of FRD1300 (Ptrc-algD-A ΔalgL::Gm^r). A 0.3-ml sample of an FRD1300 overnight culture was inoculated into 25 ml L broth, which was incubated with aeration at 37°C. After 1 h, the culture was split and 5 mM IPTG was added to one flask. A plot of culture density (OD₆₀₀) versus minutes postinduction is shown.

FIG. 4.

Electron micrographs of thin-sectioned FRD1300 (Ptrc-algD-A ΔalgL::Gm^r) following incubation in L broth and comparing cells at 0, 2, 4, and 6 h post IPTG induction of the algD operon. (A) Uninduced, normal-appearing cells (×40,000). P. aeruginosa rods are approximately 1 by 2 μm in size. (B) Induction for 2 h shows zones of separation between membranes. (C) Induction for 4 h shows larger zones of separation. (D) Induction for 4 h; this enlargement of panel C shows periplasmic polymer accumulation. (E) Induction for 6 h showed general lysis of the cells. (F) Enlarged image of FRD1300 cells after 6 h of induction with IPTG.

FIG. 5.

Effect of IPTG concentrations on growth and alginate production in FRD1050 and FRD1300. Overnight cultures were used to inoculate MAP medium and incubated with aeration at 37°C to an OD₆₀₀ of 0.20. Twenty-five-milliliter volumes of log-phase cultures were incubated with 4.0 mM IPTG (A) or 0.4 mM IPTG (B) to induce alginate production. Samples were taken periodically for measurements of growth as OD₆₀₀ (closed symbols) and alginate accumulation in culture supernatants (open symbols). FRD1050 algL⁺, squares; FRD1300 ΔalgL, circles. A repeat of this experiment produced similar results.

FIG. 6.

Model for alginate secretion. (Left) Polymerization of mannuronates (•) from GDP-mannuronate probably occurs via Alg8, which appears to be an inner membrane (IM) glycosyltransferase. A multicomponent protein scaffold is proposed to transport polymer across the periplasm to AlgE in the outer membrane (OM). AlgG, a periplasmic C-5 mannuronan epimerase, converts some d-mannuronate residues to l-guluronate (grey circles). Likely candidates as components of the scaffold complex include the periplasmic proteins Alg44, AlgK, AlgG, AlgX, and AlgL. (Middle) The scaffold fails to assemble correctly when AlgK or AlgG protein is absent. AlgL's alginate lyase activity is then free to digest any newly formed polymer in the periplasm, thus releasing dialyzable uronates into the extracellular environment. Occasionally, the scaffold may disassemble spontaneously in the wild type, allowing AlgL to digest periplasmic polymer. (Right) When AlgL is absent, the scaffold again does not assemble correctly but the absence of periplasmic lyase activity causes polymer to accumulate in the periplasmic space, ultimately bursting the cells.