Alteration of the rugose phenotype in waaG and ddhC mutants of Salmonella enterica serovar Typhimurium DT104 is associated with inverse production of curli and cellulose

Abstract

The rugose (also known as wrinkled or rdar) phenotype in Salmonella enterica serovar Typhimurium DT104 Rv has been associated with cell aggregation and the ability, at low temperature under low-osmolarity conditions, to form pellicles and biofilms. Two Tn5 insertion mutations in genes that are involved in lipopolysaccharide (LPS) synthesis, ddhC (A1-8) and waaG (A1-9), of Rv resulted in diminished expression of colony rugosity. Scanning electron micrographs revealed that the ddhC mutant showed reduced amounts of extracellular matrix, while there was relatively more, profuse matrix production in the waaG mutant, compared to Rv. Both mutants appeared to produce decreased levels of curli, as judged by Western blot assays probed with anti-AgfA (curli) antibodies but, surprisingly, were observed to have increased amounts of cellulose relative to Rv. Comparison with a non-curli-producing mutant suggested that the alteration in curli production may have engendered the increased presence of cellulose. While both mutants had impaired biofilm formation when grown in rich medium with low osmolarity, they constitutively formed larger amounts of biofilms when the growth medium was supplemented with either glucose or a combination of glucose and NaCl. These observations indicated that LPS alterations may have opposing effects on biofilm formation in these mutants, depending upon either the presence or the absence of these osmolytes. The phenotypes of the waaG mutant were further confirmed in a constructed, nonpolar deletion mutant of S. enterica serovar Typhimurium LT2, where restoration to the wild-type phenotypes was accomplished by complementation. These results highlight the importance of an integral LPS, at both the O-antigen and core polysaccharide levels, in the modulation of curli protein and cellulose production, as well as in biofilm formation, thereby adding another potential component to the complex regulatory system which governs multicellular behaviors in S. enterica serovar Typhimurium.

FIG. 1.

Growth of S. enterica serovar Typhimurium DT104 Rv and LT2 and their derivatives on LB Congo red plates at 4 days (4D) and 6 days (6D) at 25°C. (A) Stv is a spontaneous mutant of Rv; A1-8 and A1-9 are ddhC::Tn5 and waaG::Tn5 mutants, respectively. (B) YA155 is a knockout mutant of waaG (ΔwaaG::kan), and YA156 is a complemented strain derived from YA155. Cells from overnight cultures grown at 37°C were serially diluted (10⁻⁶ to 10⁻⁸), spread on LB plates to obtain individual colonies, and grown at 25°C for 4 days (top) or 6 days (bottom).

FIG. 2.

Sites of transposon insertion in strains A1-8 (ddhC::Tn5) and A1-9 (waaG::Tn5) and deletion in strain YA155 (ΔwaaG::kan). Values indicate the sizes of the respective genes in base pairs. Tn5 was inserted at nt 553 in the ddhC gene and at nt 612 in waaG. In strain YA155, the entire waaG gene, except for the last 25 bp, was deleted and replaced with a kanR gene flanked by Flip recombinase sites (FRT) that were created in the gene knockout procedure. Dotted lines indicate the presence of additional genes in the operon.

FIG. 3.

General structure of LPS in S. enterica serovar Typhimurium and roles of both ddhC and waaG in LPS synthesis. (A) Structure of Salmonella LPS with the sites affected by ddhC and waaG mutations identified. (B) LPS electrophoretic profiles of the A1-8 (ddhC::Tn5) and A1-9 (waaG::Tn5) mutants of S. enterica serovar Typhimurium DT104 showing the production of low-molecular-weight LPS compared with the ladder pattern of WT strain Rv. (C) A profile of LPS alteration similar to that of A1-9 was shown in the waaG knockout strain of the WT LT2 strain, YA155, which was complemented to the WT phenotype in strain YA156 by plasmid-borne waaG. The production of low-molecular-weight LPS in mutants further confirmed the roles of both ddhC and waaG in determining the structure of LPS. Proteinase K-treated whole-cell lysates were prepared from cells growing in early log phase at 37°C in LB, run on an SDS-PAGE gel, and silver stained. Abe, abequose; Kdo, 2-keto-3-deoxyoctulosonic acid.

FIG. 4.

Scanning electron micrographs of cells from intact colonies of S. enterica serovar Typhimurium DT104 Rv and mutants A1-8 and A1-9 grown on LB agar for 4 days at 25°C. Cells were streaked on LB agar for isolated colonies, and those reaching relatively full rugosity for the corresponding strain after 4 days were prepared for scanning electron microscopic observation. A thick fibrous matrix covered most of the cells in Rv colonies, as shown in panel A, and was contrasted with some smooth cells occasionally seen lying on the cellular matrix. The ddhC::Tn5 mutant (A1-8) produced a thinner fibrous matrix (B). The waaG::Tn5 mutant (A1-9), however, produced a much thicker, adhesive-like matrix (C) than that of the WT. Bars, 1 μm.

FIG. 5.

Curli production as judged from the major subunit CsgA production of WT Rv and LT2 and their derivatives. Curli production is shown for WT Rv and its ddhC::Tn5 (A1-8) and waaG::Tn5 (A1-9) mutants after 2, 4, and 6 days of growth (A) and for WT LT2 with its ΔwaaG::kan (YA155) derivative and the waaG-complemented strain of YA155 (YA156) (B) after 2 and 4 days of growth on LB at 25°C. Strains S. enterica serovar Typhimurium LT2 YA151 (ΔcsgA) and S. enterica serovar Enteritidis 4b (SE) were included as negative and positive controls, respectively, in panel A. Curli protein was isolated by formic acid treatment of whole-cell lysates, separated by SDS-PAGE, and probed with anti-S. enterica serovar Enteritidis 4b AgfA monoclonal antibodies.

FIG. 6.

Cellulose production by WT Rv and LT2 and their derivatives. Greater amounts of cellulose were produced by both the ddhC::Tn5 (A1-8) and waaG::Tn5 (A1-9) mutants than by WT Rv. Greater cellulose production was also obtained from the waaG deletion mutant YA155 than from WT LT2 and waaG-complemented strain YA156. Cellulose was isolated from 200 mg of lyophilized cell mass obtained from colonies of each strain streaked with a high-density inoculum on LB plates, followed by a 4-day incubation at 28°C. Control strains YA151 (ΔcsgA::kan) and YA159 (ΔbcsA::kan) were included as positive and negative controls, respectively. Error bars represent the standard deviations.

FIG. 7.

Production of biofilms by ddhC::Tn5 (A1-8) and waaG::Tn5 (A1-9) mutants. (A) Biofilm production by waaG mutants A1-9 (waaG::Tn5) and YA155 (ΔwaaG::kan) was observed as rings on the tube walls at the liquid-air interface. Strain A1-8 produced fragile biofilms which detached easily and cannot be seen here. Note the less dense medium in both A1-8 and A1-9 caused by the formation of cell clumps, which caused the cells to settle to the bottom of the tube. Bacteria were grown in LGS at 37°C with shaking at 250 rpm for 20 h. Complementation of waaG in strain YA156 eliminated the ability of cells to form biofilms under this condition. (B) The ddhC::Tn5 (A1-8) and waaG::Tn5 (A1-9) mutants were shown to exhibit higher levels of adherence to PVC surfaces compared to WT Rv. The higher level of adherence of A1-8 than of WT LT2 was similar to that of ΔwaaG::kan mutant strain YA155. Cells were grown in M63 medium supplemented with 0.2% glucose and 0.5% CAA in microtiter wells for 8 h. Attached cells were quantified by staining the biofilms with crystal violet and determining the absorbance of the solubilized dye at 595 nm.