Vibrio parahaemolyticus scrABC, a novel operon affecting swarming and capsular polysaccharide regulation

Abstract

Swarming is an adaptation of many bacteria to growth on surfaces. A search for genes controlling swarmer cell differentiation of Vibrio parahaemolyticus identified a novel three-gene operon that potentially encodes a pyridoxal-phosphate-dependent enzyme, an extracellular solute-binding protein, and a membrane-bound GGDEF- and EAL-motif sensory protein. The functions of these motifs, which are named after conserved amino acid sequences, are unknown, although the domains are found singly and in combination in a variety of bacterial signaling proteins. Studies with translational fusions supported the predicted localization of the gene products. When the operon was overexpressed, swarmer cell gene transcription was induced in liquid culture. Mutants with defects in any of the three genes exhibited decreased swarming and lateral flagellar (laf) gene expression. Complementation studies confirmed an operon organization and suggested that all three genes participated in laf regulation. The lesions that decreased swarming increased capsular polysaccharide (CPS) production, and overexpression of the operon inhibited transcription of the CPS gene cpsA. Thus, the scrABC locus appears to inversely regulate two gene systems that are pertinent to colonization of surface swarming and CPS.

FIG. 1.

Overexpression of scrABC induces lfgE::lux expression. Luminescence (expressed as SLU) was monitored during growth of cultures in HI liquid medium. (A) Cosmid-induced luminescence. Lux reporter strain LM1017 contained pLM2032 (▪), pLM2032 carrying scrB::TnphoA (♦), and vector control (▴). (B) Subclone-induced luminescence. Lux reporter strain LM1017 contained pLM2796 (▪), pLM2796 carrying scrA:: TnlacZ (▴), pLM2796 carrying scrC::TnlacZ (×), and vector control (♦).

FIG. 2.

Physical map of the scr locus and recombinant plasmids. Arrows point in the direction of transcription deduced from the open reading frames. The restriction sites relevant to constructs are indicated as follows: E, Eco47III; A, AvrII; X, XhoI; S, SmaI; D, DrdI; P, PshAI. Plasmids pLM2876 and pLM2878 were derived from pLM2796. PCR amplification was used to generate the scrC clone pLM2449.

FIG. 3.

Cellular localization of Scr proteins. Cellular fractions were isolated from strains producing the fusion proteins: ScrA-LacZ (LM5753), ScrB-PhoA (LM5153), and ScrC-LacZ (LM5140). Immunoblots were performed by probing with anti-β-galactosidase (A) or anti-alkaline phosphatase (B). Sample fractions are denoted as whole cells (WC), cytoplasm (C), membrane (M), or periplasm (P). Lanes: 1, LM1017 (WC); 2, LM5140 (WC); 3, LM5140 (C); 4, LM5140 (M); 5, LM5140 (P); 6, LM5753 (WC); 7, LM5753 (C); 8, LM5753 (M); 9, LM5753 (P); 10, LM5153 (WC); 11, LM5153 (C); 12, LM5153 (M); 13, LM5153 (P); 14, LM1017 (WC). The molecular size standards are given in kilodaltons on the right.

FIG. 4.

scr mutants are defective in lateral flagellar gene expression and swarming. (A) Luminescence (reported as SLU) was measured from HI plate-grown samples after 9 h at 30°C. The values used for light production were as follows: LM1017 (lfgE::lux), 1,540,000 ± 75,000 SLU; LM5545 (scrA1), 19,800 ± 800 SLU; LM5792 (scrA2), 195,000 ± 9,000 SLU; LM5547 (scrB1), 47,300 ± 3,100 SLU; and LM5549 (scrC1), 47,500 ± 2,200 SLU. (B) Swarming motility of scr mutants after 9 h at 30°C. The strains included BB22TR (wild type), LM5733 (scrA1), LM5793 (scrA2), LM4897 (scrB1), and LM5719 (scrC1).

FIG. 5.

Complementation of lateral flagellar gene expression defects in scr mutants. (A) Plasmids pLM2796 (scrA⁺B⁺C⁺) and pLM1877 (vector) in LM1017 and scr-defective backgrounds. (B) Plasmids pLM1877 (vector), pLM2876 (scrA⁺), pLM2878 (scrB⁺), and pLM2449 (scrC⁺) in LM1017 and scr-defective backgrounds. Luminescence (reported in SLU) was measured from HI plate-grown samples after 10 h of growth at 30°C.

FIG. 6.

scr mutants display a rugose colony morphology and produce an extracellular matrix. (A) Colony morphology of a smooth parent strain, LM1017, and a rough scrB mutant, LM5547. (B) Scanning electron microscopy of a smooth LM1017 colony and rugose LM5547 colony. Colonies were grown on HI plates at 30°C for 2 days. Bar, 2 μm.

FIG. 7.

scrABC and scrC overexpression inversely affects lateral flagellar and CPS gene expression. (A) The amounts of luminescence produced were as follows: 600,000 ± 11,200 SLU in LM4851 (carrying the vector), 1,400,000 ± 25,500 SLU in LM5435 (carrying scrA⁺B⁺C⁺), and 25 ± 4 SLU in LM5139 (scrC⁺). (B) The amounts of β-galactosidase produced (expressed as Miller units) were as follows: 35 ± 1.2 in LM5811 (vector), 25 ± 1 in LM5813 (scrA⁺B⁺C⁺), and 278 ± 6 in LM5812 (scrC⁺). Samples were harvested from HI-gentamicin plates with IPTG after 12 h of growth.

FIG. 8.

Model for the ScrABC sensory transduction system. ScrB has similarity to solute-binding proteins and is hypothesized to receive an input signal and interact with the periplasmic domain of ScrC. This interaction could modulate the activity of the cytoplasmic portion of ScrC, possibly controlling levels of a cyclic nucleotide signaling molecule by acting as a cyclic nucleotide phosphodiesterase or cyclase. ScrC aa 309 is the site of an active β-galactosidase fusion, suggesting that the GGDEF and EAL domains reside in the cytoplasm. ScrA contains a domain shared by pyridoxal-phosphate-dependent enzymes and is required for signal transduction, but its role is unclear. Levels of the small signaling molecule would ultimately determine output by affecting the expression of lateral flagella and CPS in an inverse manner.