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. 2009 Oct;191(19):6003-11.
doi: 10.1128/JB.00687-09. Epub 2009 Jul 31.

Identification of the site-specific DNA invertase responsible for the phase variation of SusC/SusD family outer membrane proteins in Bacteroides fragilis

Haruyuki Nakayama-Imaohji  1 , Hideki Hirakawa, Minoru Ichimura, Shin Wakimoto, Satoru Kuhara, Tetsuya Hayashi, Tomomi Kuwahara
Affiliations

Identification of the site-specific DNA invertase responsible for the phase variation of SusC/SusD family outer membrane proteins in Bacteroides fragilis

Haruyuki Nakayama-Imaohji et al. J Bacteriol. 2009 Oct.

Abstract

The human gut microbe Bacteroides fragilis can alter the expression of its surface molecules, such as capsular polysaccharides and SusC/SusD family outer membrane proteins, through reversible DNA inversions. We demonstrate here that DNA inversions at 12 invertible regions, including three gene clusters for SusC/SusD family proteins, were controlled by a single tyrosine site-specific recombinase (Tsr0667) encoded by BF0667 in B. fragilis strain YCH46. Genetic disruption of BF0667 diminished or attenuated shufflon-type DNA inversions at all three susC/susD genes clusters, as well as simple DNA inversions at nine other loci, most of which colocalized with susC/susD family genes. The inverted repeat sequences found within the Tsr0667-regulated invertible regions shared the consensus motif sequence AGTYYYN(4)GDACT. Tsr0667 specifically mediated the DNA inversions of 10 of the 12 regions, even under an Escherichia coli background when the invertible regions were exposed to BF0667 in E. coli cells. Thus, Tsr0667 is an additional globally acting DNA invertase in B. fragilis, which probably involves the selective expression of SusC/SusD family outer membrane proteins.

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Figures

FIG. 1.
FIG. 1.
Genetic structures of invertible regions previously classified as class II regions in B. fragilis strain YCH46 (19). The open reading frames present in each locus are indicated by arrows that are differentiated according to function. IRs are indicated by arrowheads. Bent arrows show the positions and orientations of Bacteroides consensus promoter sequences (1) basing on the final genome sequence data of B. fragilis strain YCH46 (GenBank accession no. AP006841). Dotted circles represent the DNA inversions via for each IR pair. In the cases of class II-1, -2, and -6 regions, the outcomes of DNA inversions are shown on the right side of horizontal line with arrowheads. DNA inversions in class II-1 region mediate the formation of two types of hybrid proteins with different C-terminal sequences. A small open reading frame encoded in the invertible segments in class II-2 and -6 regions fuses to the downstream or upstream gene by DNA inversion resulting in an N-terminal extension.
FIG. 2.
FIG. 2.
Clustering analysis of Tsr amino acid sequences from B. fragilis strains YCH46 (red) and NCTC9343 (blue) and B. thetaiotaomicron strain VPI-5482 (green). The deduced amino acid sequences of Tsr were aligned and compared by using the CLUSTAL W program (34), and the unrooted trees were drawn by using MEGA-4 software (33). The outermost circle indicates the three major Tsr families (clusters 1 to 3). The second circle indicates the Tsr-encoding genes that mediate DNA inversion at the regions indicated. New classes indicate the putative invertible regions found in the present study. Tsr proteins of B. fragilis strain YCH46 that are conserved between B. fragilis strain NCTC9343 and B. thetaiotaomicron strain VPI-5482, and those conserved between B. fragilis strains only are labeled by yellow and blue circles, respectively. The B. fragilis strain YCH46 Tsr-encoding genes that were disrupted in the present study are indicated by asterisks. A bar indicates the phylogenetic distance.
FIG. 3.
FIG. 3.
PCR assay for DNA inversions in class II regions of wild-type (WT) and BF0667-deletion mutant (TSRM0667) strains. TSRM0667 strain complemented with plasmid-borne BF0667 in trans, TSRM0667(pVAL0667), was also analyzed. The results of a 25-cycle amplification are shown. (A) Orientation-specific primer sets used to detect the DNA inversions. (B) Agarose gel electrophoresis of the amplification products from the class II regions as indicated above each lane. F and R show the PCR protocol indicated in panel A. (C) Genetic structure of the B. fragilis strain YCH46 susC/susD gene cluster (class II-3). Genes for SusC and SusD are indicated by black and gray arrows, respectively. Arrowheads indicate IRs. (D) Shufflon-type multiple DNA inversions in the class II-3 susC/susD gene cluster. The annealing position and orientation of each PCR primer used to detect DNA inversions is indicated below the schematic representation of the susC/susD cluster in panel C. Three types of DNA inversions are shown in panel C. Inversions 1+2, 2+3, and 1+3 indicate the combinational DNA inversions of inversion 1 and inversion 2, inversion 2 and inversion 3, and inversion 1 and inversion 3, respectively. The primer pairs used are shown above the lanes, and DNA inversions to be detected are indicated above the primer pairs. For example, the primers 2F and 2R face to each another in a distance that generate 513-bp PCR product in size when the combinational DNA inversions (inversion 1+2) occur. M, 100-bp ladder molecular size markers.
FIG. 4.
FIG. 4.
Involvement of Tsr0667 in shufflon-type multiple DNA inversions in the two distantly located susC/susD clusters in B. fragilis. Genetic structures of the class V susC/susD cluster (A) and the class VI susC/susD cluster (C) are shown. The results of 25-cycle PCR assays to detect DNA inversions are shown in panels B and D, respectively. Annealing positions and orientations of the PCR primers used to detect DNA inversions are indicated by bent arrows and IRs are indicated by arrowheads in panels A and C. Genes for SusC and SusD are indicated by black and gray arrows, respectively. The primer pairs used are shown above the lanes. DNA inversions to be detected are indicated above the primer pairs. M, 100-bp ladder molecular size markers.
FIG. 5.
FIG. 5.
Alignment of IRs within the invertible regions of the Tsr0667-inverlon. IR sequences are underlined. Conserved nucleotides are shadowed and indicated by boldface letters. The consensus motif sequence is shown below the alignments (Y = C or T; D = A, G, or T; and N = A, G, C, or T). IR-L and IR-R indicate the IRs at the left and right junctions of each invertible region, respectively.
FIG. 6.
FIG. 6.
Reconstruction of the B. fragilis Tsr0667-inverlon in an E. coli background. (A) Schematic representation of E. coli strains containing BF0667- or mpi-bearing plasmids and reporter plasmid harboring the substrate region for Tsr0667 (pRep 1 to 12, class II-, V-, and VI-derived invertible DNA fragments) or Mpi (pRep 13, invertible promoter region from capsular polysaccharide biosynthesis locus PS-3). BF0667 and mpi are cloned in both orientations with respect to the lac promoter (plac) of pBluescript KS II. IR indicates the IR sequence. Reporter plasmid-specific (rep-F and rep-R) and region-specific PCR primers (rep-M) for assessment of DNA inversions are shown by bent arrows. (B) PCR to assess DNA inversions on reporter plasmids. Twenty-cycle PCR amplifications were performed. F and R above each lane indicate the primer set as shown in panel A: F, rep-F + repM; and R, repM + rep-R. In the class VI inversion assay, F and R indicate the primer set shown in Fig. 4C: F, A2 + 4F; and R, A2 + 4R. Roman numerals above the lanes indicate the E. coli clone shown in panel A.
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