Genomic analysis of Bacteroides fragilis reveals extensive DNA inversions regulating cell surface adaptation

Abstract

Bacteroides are predominant human colonic commensals, but the principal pathogenic species, Bacteroides fragilis (BF), lives closely associated with the mucosal surface, whereas a second major species, Bacteroides thetaiotaomicron (BT), concentrates within the colon. We find corresponding differences in their genomes, based on determination of the genome sequence of BF and comparative analysis with BT. Both species have acquired two mechanisms that contribute to their dominance among the colonic microbiota: an exceptional capability to use a wide range of dietary polysaccharides by gene amplification and the capacity to create variable surface antigenicities by multiple DNA inversion systems. However, the gene amplification for polysaccharide assimilation is more developed in BT, in keeping with its internal localization. In contrast, external antigenic structures can be changed more systematically in BF. Thereby, at the mucosal surface, where microbes encounter continuous attack by host defenses, BF evasion of the immune system is favored, and its colonization and infectious potential are increased.

Fig. 1.

Circular maps of the chromosome and the pBFY46 plasmid of BF strain YCH46. Each circle represents (from inside out): G+C content, GC skew, rRNA operon (all consisted of 16S-23S-5S rRNA genes), tRNA genes, conjugative (blue bars) and mobilizable (green bars) transposons, capsular PS loci, SusC (blue bars) and SusD (red bars) families of outer membrane proteins, invertible regions, predicted genes transcribed into the counterclockwise direction, and those into clockwise direction. Classification of the invertible regions based on the motif sequence within IRs is indicated by the color of triangles. The inner and outer circles of the plasmid represent predicted genes transcribed into the counterclockwise direction and those into clockwise direction, respectively. All of the predicted genes are colored according to the Clusters of Orthologous Groups of Proteins functional classification (13).

Fig. 2.

Functional classification of DNA inversions identified in Bacteroides genomes. DNA inversions control the expressions of genes downstream or between the IRs (indicated by red arrowheads) in several ways. Locations and directions of each promoter are indicated by open triangles. Type I inversions mediate the on-off switching of the downstream genes monodirectionally (1-a and 1-d) or bidirectionally (1-b and 1-c) by changing the promoter orientation. In the cases of 1-c and 1-d, a small ORF encoded in the invertible segment fuses to the downstream gene by DNA inversion to create an N-terminal extension. In many cases, signal sequences are added to the downstream genes. Type 2 mediates the formation of two types of hybrid proteins with different C-terminal sequences. Type 3 modulates the operon structure. Type 4 is a shufflon-like multiple inversion system. In this case, segments flanked by IRs are inverted independently or in groups to deliver the promoter to either of the gene cassettes. The numbers of each type of invertible region in the chromosome of BF strain YCH46 are shown in parentheses.

Fig. 3.

A shufflon-like DNA inversion system in BF regulates variable expression patterns of SusC/D family proteins. (A) The gene organization of SusC/D cluster 1 and the locations of PCR primers used to detect DNA inversions are shown. Genes for SusC, SusD, antisigma factor, extracytoplasmic function-type sigma factor, and hypothetical proteins are indicated by blue, light green, green, yellow, and light blue arrows, respectively. The position of a Bacteroides consensus promoter sequence (41) is indicated by an open triangle. Circles represent DNA inversions at each IR (IR-1, IR-2, and IR-3). (B) Detection of the possible inversion patterns in SusC/D cluster 1 by PCR. PCR products obtained by various combinations of primers were analyzed by agarose gel electrophoresis. Primer pairs used for each amplification are indicated above each lane. Details, including predicted size for each product, are available in Fig. 5.

Fig. 4.

A hypothetical model for the hierarchical control of DNA inversions in the BF genome. Genes for a globally acting site-specific recombinase, Mpi (BF2765), and a tyrosine-type site-specific recombinase (BF2766) are indicated by red and pink arrows, respectively. IRs are indicated by purple or red arrowheads, and promoter sequences are indicated by open triangles. The BF2766 recombinase mediates the inversion of the downstream promoter-containing segment, and thus regulates the selective expression of the accessory PS locus and the mpi gene. The expressed Mpi recombinase in turn regulates the on-off switching of seven PS loci and seven additional loci that are scattered on the BF genomes. Genes for polysaccharide biosynthesis and transport are indicated by green arrows. UpxYgene homologs, UpxZ homologs, outer membrane lipoproteins, an outer membrane protein, an electron transport protein, a polysaccharide deacetylase, and hypothetical proteins are indicated by light green, yellow, purple, blue, orange, brown, and light blue arrows, respectively.