PMC

Effect of nhaR mutation on accumulation of PGA. Cell lysates were prepared from a series of isogenic strains and analyzed by immunoblotting with an anti-PIA antiserum (see Materials and Methods). Sample identities are as follows: (a) parent (TRMG); (b) nhaR::cam; (c) nhaR::cam(pNhaR); (d) ΔpgaABCD. This experiment was repeated in entirety three times, with essentially identical results. A representative blot is shown.

Primer extension analysis of pgaA transcript in nhaR wild-type and mutant (nhaR::cam) strains. Cultures were grown at 26°C with shaking and harvested for RNA isolation in the late exponential phase of growth. Strain identities are as shown in Fig. . The dideoxy sequencing ladder (lanes G, A, T, and C) was generated with PEXT3, using pPGA372 as a template. The 5′ end of the pgaA transcript is marked with an asterisk.

Purified NhaR-His₆ binds specifically to pgaABCD promoter DNA. (A) A 132-bp PCR product containing the promoter region and putative NhaR binding site of pgaA was 3′ labeled with DIG-11-ddUTP and used for gel mobility shift assays. The apparent equilibrium binding constant (K_d) was determined as described in Materials and Methods. (B) Five hundred- and 1,000-fold molar excesses of unlabeled specific DNA (pgaA) and nonspecific DNA (pgaBC) were used as competitors in the binding reactions.

Effects of nhaR::cam, monovalent cations and pH on expression of a pgaA′-′lacZ translational fusion. (A) Activity of the pgaA′-′lacZ chromosomal fusion in XWZ4 (▪) and its isogenic nhaR mutant (CGCFR) (□) after 14 h of growth at 26°C in L broth (LB without NaCl) or in L broth supplemented with the indicated concentrations of NaCl. (B) Activity of the reporter fusion in L broth containing the indicated concentrations (mM) of NaCl, KCl, LiCl, or sucrose. wt, wild type. (C) Effect of pH on pgaA′-′lacZ expression. Strains were grown for 24 h at 26°C in LB buffered with 60 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane, and the pH was adjusted to the indicated values with HCl. Values represent the means for three separate reactions ± standard errors of the means. Error bars smaller than the symbols are not visible. These experiments were repeated at least three times with similar results.

NhaR-His₆ activates in vitro transcription from the pgaA promoter. NhaR-His₆ and σ⁷⁰-saturated RNA polymerase were used for in vitro transcription of plasmid DNA containing the pgaABCD operon (pPGA372). The resulting transcripts were analyzed by primer extension (see Materials and Methods). The full image (A) and a close-up (B) obtained from a single gel are shown. In the full gel image, two transcripts originating from the vector are indicated by arrowheads labeled V1 and V2; an asterisk denotes the 5′ end of the transcript originating from pga (P_pga). Identical results were obtained when 25 mM KCl was used instead of NaCl in the transcription reaction (data not shown). The first two lanes (in vivo) depict primer extension analysis of total cellular RNAs isolated from the indicated strains. The last four lanes (in vitro) correspond to transcription reactions conducted in the presence or absence of NhaR (455 nM) and NaCl (25 mM).

DNase I protection footprint of NhaR on the pgaA promoter. A 132-bp PCR product containing the promoter region of pgaA (−115 to +18) was 5′ end labeled with ³²P on the top (A and C) or bottom (B and D) strand, as diagrammed in panel E, and subjected to DNase I footprinting in the presence of increasing concentrations of NhaR-His₆ (0, 0.29, 0.57, and 1.14 μM). The binding reaction mixtures contained no NaCl (A and B) or 50 mM NaCl (C and D). Protected regions are marked by vertical lines adjacent to the sequences. Hypersensitive sites are indicated by asterisks. Numbers depict positions relative to the initiating nucleotide (+1). These footprints were repeated four times, with no significant difference in the protection pattern observed in the presence or absence of NaCl. (E) Summary of DNase I footprinting. Protected regions are shaded gray; boxed sequences contain a LysR-type transcriptional regulator binding motif (T-N₁₁-A). The −35, −10, and +1 promoter elements are in boldface. Hypersensitive sites are indicated by asterisks.

Disruption of nhaR of E. coli affects biofilm formation. (A) Cultures were grown in microtiter plates in LB (pH 7.4) for 24 h at 26°C, and biofilm formation was assessed by crystal violet staining (see Materials and Methods). Bars depict the results obtained with a series of isogenic strains: 1, MG1655 (wild type); 2, nhaR::cam; 3, nhaR::cam(pKK223-3); 4, nhaR::cam(pNhaR); 5, csrA::kan; 6, csrA::kan nhaR::cam; 7, csrA::kan nhaR::cam(pKK223-3); 8, csrA::kan nhaR::cam(pNhaR). Error bars indicate standard errors of the means. (B) Time course of adherence to coverslips by isogenic E. coli K-12 MG1655 strains (described in Materials and Methods). Representative fields are shown. wt, wild type. (C) Effect of sodium chloride on biofilm formation. Cultures were grown in L broth (LB without NaCl) supplemented with the indicated concentrations of NaCl. Symbols represent the results obtained with a series of isogenic strains: ▪, MG1655 (wild type); ▴, nhaR::cam; ▾, nhaR::cam(pKK223-3); ⧫, nhaR::cam(pNhaR); •, nhaR::cam(pNhaR-His₆). Bars represent the averages for two wells, and error bars correspond to the standard errors of the means. (D) Epistasis analyses of nhaR and pgaABCD on biofilm formation. Biofilm formation in the following isogenic strains was assessed as for panel A: 1, MG1655; 2, ΔpgaABCD; 3, nhaR::cam; 4, MG1655(pUC19); 5, MG1655(pPGA372) (contains pgaABCD); 6, nhaR::cam(pUC19); 7, nhaR::cam(pPGA372); 8, ΔpgaABCD(pUC19); 9, ΔpgaABCD(pPGA372); 10, ΔpgaABCD(pKK223-3); 11, ΔpgaABCD(pNhaR). Bars represent the averages for 16 wells normalized to the biofilm formed by MG1655, and error bars correspond to the standard errors of the means. The asterisks denote significant differences relative to the corresponding parent strain (P < 0.001 [Tukey's multiple-comparison test]).