A linear plasmid truncation induces unidirectional flagellar phase change in H:z66 positive Salmonella Typhi

Abstract

The process by which bacteria regulate flagellar expression is known as phase variation and in Salmonella enterica this process permits the expression of one of two flagellin genes, fliC or fljB, at any one time. Salmonella Typhi (S. Typhi) is normally not capable of phase variation of flagellar antigen expression as isolates only harbour the fliC gene (H:d) and lacks an equivalent fljB locus. However, some S. Typhi isolates, exclusively from Indonesia, harbour an fljB equivalent encoded on linear plasmid, pBSSB1 that drives the expression of a novel flagellin named H:z66. H:z66+S. Typhi isolates were stimulated to change flagellar phase and genetically analysed for the mechanism of variation. The phase change was demonstrated to be unidirectional, reverting to expression from the resident chromosomal fliC gene. DNA sequencing demonstrated that pBSSB1 linear DNA was still detectable but that these derivatives had undergone deletion and were lacking fljA(z66) (encoding a flagellar repressor) and fljB(z66). The deletion end-point was found to involve one of the plasmid termini and a palindromic repeat sequence within fljB(z66), distinct to that found at the terminus of pBSSB1. These data demonstrate that, like some Streptomyces linear elements, at least one of the terminal inverted repeats of pBSSB1 is non-essential, but that a palindromic repeat sequence may be necessary for replication.

Fig. 2

Indentification of S. Typhi flagellar antigens. A. Western blotting of various S. Typhi mid-log phase whole-cell lysates with anti-z66 flagellar antibody. Lane 1, S. Typhi Ty2; Lane 2, S. Typhi Ty2(ΔfliC); Lane 3, S. Typhi 403ty-fliC(j); Lane 4, S. Typhi 404ty-fliC(d); Lane 5, S. Typhi 404ty ΔfljB^z66; Lane 6, S. Typhi 404ty ΔfljA^z66; Lane 7, S. Typhi 404ty ΔfljB^z66, ΔfljA^z66; Lane 8, S. Typhi 403tya-fliC(j) (post-phase switch); Lane 9, S. Typhi 404tya-fliC(d) (post-phase switch). Protein sizes were estimated on SDS-PAGE gels prior to transfer with seeblue2 protein ladder (Invitrogen). B. Western blotting of various S. Typhi whole-cell lysates with anti-d flagellar antibody. Lanes as A. C. Western blotting of various S. Typhi whole-cell lysates with non-specific Salmonella flagellar antibody. Lanes as A. D. RT-PCR detecting mRNA transcription of aroC (lanes a, primers aroC_RT_F/R, 100 bp), fliC (lanes b, primers fliC_RT_F/R 150, bp), fljB^z66 (lanes c, primers fljB_RT_F/R, 200 bp) and fljA^z66 (lanes d, primers fljA_RT_F/R, 250 bp). Panel 1, genomic DNA from S. Typhi 404ty-fliC(d); panel 2, cDNA from S. Typhi Ty2; panel 3, cDNA from S. Typhi 404ty-fliC(d); panel 4, cDNA from S. Typhi 404ty-fliC(d)ΔfljB^z66; panel 5, cDNA from S. Typhi 404ty-fliC(d) ΔfljA^z66 and panel 6, cDNA from S. Typhi 404ty-fliC(d) ΔfljB^z66, ΔfljA^z66. Sizes are compared with the migration of Hyperladder IV (Bioline).

Fig. 1

Immunoflorescence staining to visualize expression of the z66 and d S. Typhi flagellar antigens. A. Confocal microscopy image of 404ty-fliC(d) (pre-phase change) expressing the z66 flagellar antigen. The bacterial cell post incubation with anti-z66 antibody is stained blue (DAPI nuclear stain) and the z66 flagellar antigen is stained green. Scale is estimated by magnification. B. Image of 404ty-fliC(d) (pre-phase change) post incubation with an anti-d flagellar antibody (red) and nuclear stained with DAPI (blue). C. Confocal microscopy image of 404tya-fliC(d) (post-phase change). Bacterial cell is stained as A. The magnification has been decreased with respect to A to demonstrate that S. Typhi cells expressing the z66 antigen could not be detected after undergoing phase change. D. Image of 404tya-fliC(d) (post-phase change), the bacterial cell is stained as in C.

Fig. 3

Measuring the stability of pBSSB2. Graph demonstrating the stability of pBSSB2 compared with a number of other plasmids. The approximate number of bacterial generations calculated from the initial inoculum at each subculture is represented on the x-axis. The log₁₀ of the average cfu ml⁻¹ at stationary phase for the various experimental strains calculated by serial dilution is represented on the y-axis. The cfu ml⁻¹ was calculated by growth when diluted strains were incubated on LB media supplemented with the appropriate antibiotic. The cfu ml⁻¹ was also calculated on none selective media to ensure normal culture conditions. Strains and plasmids compared; Solid line with diamond, E. coli pBSSB2 (kanamycin); Solid line with star, S. Typhi pBSSB2 (kanamycin); Broken line with cross, E. coli RSF1010 (streptomycin); Broken line with triangle, E. coli pFOS1. (chloramphenicol); Broken line with circle, E. coli pBR322 (ampcillin); Broken line with square, E. coli pUC18 (ampcillin).

Fig. 4

Molecular basis of fljB^z66 to fliC phase change. A. Agarose gel electrophoresis of alkaline lysis plasmid preparation from S. Typhi strains pre- and post-phase switch. Lane 1, S. Typhi 403ty-fliC(j) (j); Lane 2, 403tya; Lane 3, 403tyb; Lane 4, 403tyc; Lane 5, 403tyd; Lane 6, S. Typhi 404ty-fliC(d); Lane 7, 404tya; Lane 8, 404tyb; Lane 4, 404tyc; Lane 5, 404tyd. Sizes are estimated with respect to pBSSB1 (27, 037 kbp) isolated from S. Typhi 403ty(j) and S. Typhi 404ty-fliC(d). B. Agarose gel of PCR amplicons produced to identify the nature of the deletion produced post phase change in strains derived from S. Typhi 403ty-fliC(j). The target for the PCR primers are; (i) fliC, primers fliC_F/R; (ii) fljB^z66, primers z66flag_F/R; (iii) terminal inverted repeat (tir), primers tir_a, tir_d and tir_e. Each panel represents template genomic DNA from: 1, S. Typhi 403ty-fliC(j); 2, 403tya; 3, 403tyb; 4, 403tyc and 5, 403tyd. Sizes are compared with migration of Hyperladder I (Bioline). C. PCR amplifications as B using genomic template DNA from: 1, S. Typhi 404ty -fliC(d); 2, 404tya; 3, 404tyb; 4, 404tyc and 5, 404tyd.

Fig. 5

Gene map alignment of pBSSB1 and pBSSB3. The map of pBSSB1(upper) is manipulated from Baker et al. (2007) and depicts the 33 open reading frames encoded on the element. Predicted coding sequences with no similarities to other coding sequences in database searches are coloured green, while those with predicted functions are coloured red. Previously sequenced genes including fljB^z66 and fljA^z66 are coloured yellow and the tir are labelled blue. The targeted locations for primers used in Fig. 4B are demonstrated by; b, fljB^z66 and the left and right tir, cL and cR respectively. The shaded region between pBSSB1 and pBSSB3 demonstrates identical sequence. The palindromic terminus sequence (as shown in Fig. 7) are labelled A and B. The asterisk distinguishes the location of the addition 120 bp at the left terminus.

Fig. 7

Palindromic sequences at the termini of pBSSB3. A. Sequence alignment of the fljB^z66 gene from pBSSB1 and the truncated fljB^z66 gene from pBSSB3. Numbers correspond to the nucleotide position within the fljB^z66 gene. The palindromic sequence at the termini is highlighted; arrows correspond to the direction of the repeated sequence. B. Repeat sequence analysis of 1–500 bp and 900–1100 bp of the tir of pBSSB3, generated by blastN repeat finder. Repeat sequences (over eight nucleotides) are highlighted and numbered correspondingly. Arrows demonstrate the direction of the repeated sequence.

Fig. 6

Sothern blotting investigation of truncated linear plasmid. A. Analysis of tir region of pBSSB1 and derivatives using labelled DNA prepared from a PCR amplicon (primers tir_f and tir_g) generated with template DNA from S. Typhi 404ty-fliC(d) as probe against genomic DNA digested with EcoRV. Lanes: 1, S. Typhi 403ty-fliC(j); 2, 403tya; 3, 403tyb; 4, 403tyc; 5, 403tyd; 6, S. Typhi 404ty-fliC(d); 7, 404tya; 8, 404tyb; 9, 404tyc and 10, 404tyd. Sizes estimated to migration of Hyperladder I (Bioline). B. Analysis of new right terminus of the truncated plasmids. Genomic DNA digested with SacI and probed with a PCR amplicon specific for the 5′ region of the fljB^z66 gene generated with template DNA from S. Typhi 404ty-fliC(d). Lanes as Fig. 5A.