Identification of the Site-Specific DNA Invertase Responsible for the Phase Variation of SusC/SusD Family Outer Membrane Proteins in Bacteroides fragilis

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 AGTYYYN₄GDACT. 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.The human gut harbors an abundant and diverse microbiota. Bacteroides is one of the most abundant genera of human gut microflora (10, 17, 20), and the biological activities of Bacteroides species are deeply integrated into human physiology through nutrient degradation, the production of short-chain fatty acids, or immunomodulatory molecules (11-14, 24). Recent genomic analyses of Bacteroides have revealed that the bacteria possess redundant abilities not only to bind and degrade otherwise indigestible dietary polysaccharides but also to produce vast arrays of capsular polysaccharide (5, 19, 38, 39). These functional redundancies have been established by the extensive duplication of various genes that encode molecules such as glycosylhydrolases, glycosyltransferases, and outer membrane proteins of the SusC/SusD family (starch utilization system) known to be involved in polysaccharide recognition and transport (7, 27, 28, 30). It has been assumed that these functional redundancies of Bacteroides contribute to the stability of the gut ecosystem (3, 21, 23, 32, 39).Another characteristic feature common in Bacteroides species is that the expression of some of the genes is altered in an on-off manner by reversible DNA inversions at gene promoters or within the protein-coding regions (5, 9, 19, 38, 39). These phase-variable phenotypes are associated with surface architectures such as capsular polysaccharides and SusC/SusD family proteins (5, 6, 16, 19). Our previous genomic analyses of Bacteroides fragilis strain YCH46 revealed that it contained as many as 31 invertible regions in its chromosome (19). These invertible regions can be grouped into six classes according to the internal motif sequences within inverted repeat sequences (IRs) (Table 15). B. fragilis is unique in that this anaerobe possesses not only locally acting DNA invertases but also globally acting DNA invertases that mediate DNA inversions at distant loci (8, 29). It has been reported that B. fragilis possesses at least two types of master DNA invertase that regulate DNA inversions at multiple loci simultaneously (8, 29). One is Mpi, an Ssr that mediates the on-off switching of 13 promoter regions (corresponding to class I regions in B. fragilis strain YCH46), including seven promoter regions for capsular polysaccharide biosynthesis in B. fragilis strain NCTC9343 (8). The other master DNA invertase is Tsr19, a Tsr that regulates DNA inversions at two distantly located promoter regions (corresponding to class IV regions in B. fragilis strain YCH46) associated with the large encapsulation phenotype (6, 26, 29). The invertible regions contain specific consensus motifs within the IRs corresponding to each DNA invertase and constitute a regulatory unit. We designated this type of regulatory unit as an “inverlon,” which consists of at least two invertible regions controlled by a single master DNA invertase.

TABLE 1.

Classification of the invertible regions in B. fragilis strain YCH46 based on internal motif sequences within IRs

Classa	No.	Consensus motif sequencesb	Master DNA invertase genec	Regulated genes	Source or reference(s)
I	14	ARACGTWCGT	BF2765 (mpi)	Capsular polysaccharide biosynthesis genes	8
II	10	AGTTC{N5}GAACT	BF0667	susC/susD paralogs	This study
III	3	GTTAC{N7}GTAAC	BF3038, BF4033, BF4283	Putative outer membrane protein genes	36
IV	2	TACTTANTAGGTAANAGAA	BF2766	Extracellular polysacharide biosynthesis genes	6, 26, 29
V	1	TCTGCAAAGNCTTTGCAGA	BF0667	susC/susD paralogs	This study
VI	1	ACTAAGTTCTATCGG	BF0667	susC/susD paralogs	This study

Open in a separate window^aOur previous classification of the invertible regions identified in B. fragilis strain YCH46 genome (19).^bConsensus motif sequences found within IRs are shown. R = A or G, W = A or T, and N = A, G, C, or T.^cThe gene identifications in B. fragilis strain YCH46 genome are shown.Our previous studies indicated that an additional inverlon other than the Mpi- and Tsr19-regulated inverlons is present in B. fragilis, based on the finding that at least 10 invertible regions (corresponding to class II regions in B. fragilis strain YCH46) contain a particular consensus motif sequence (AAGTTCN₅GAACTT) within their IRs (19) but do not appear to colocalize with a DNA invertase gene. The majority of the class II regions were associated with the selective switching of a particular set of susC/susD family genes. Since the SusC/SusD family of outer membrane proteins play an important role in polysaccharide utilization by Bacteroides (3, 23, 32), the inverlon associated with the phase variation of SusC/SusD family proteins would likely be involved in the survival of this anaerobe in the distal gut.In the present study, we sought to identify the DNA invertase regulating the additional inverlon in B. fragilis. Our results indicated that the Tsr encoded by BF0667 is a master DNA invertase for this inverlon (designated the Tsr0667-inverlon) in B. fragilis.