Mpi recombinase globally modulates the surface architecture of a human commensal bacterium Michael J. Coyne, Katja G. Weinacht, Corinna M. Krinos, and Laurie E. Comstock* Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115 Edited by Jeffrey I. Gordon, Washington University School of Medicine, St. Louis, MO, and approved July 8, 2003 (received for review May 4, 2003) The mammalian gut represents a complex and diverse ecosystem, extraintestinal sites, and its virulence is linked to production of consisting of unique interactions between the host and microbial its capsular polysaccharides (7, 8), elucidation of the factor(s) residents. Bacterial surfaces serve as an interface that promotes that governs these DNA inversions is paramount to understand- and responds to this dynamic exchange, a process essential to the ing the complex interplay between the host and this microbe biology of both symbionts. The human intestinal microorganism, during both health and disease. Bacteroides fragilis, is able to extensively modulate its surface. Factors that mediate DNA inversions, namely the DNA Analysis of the B. fragilis genomic sequence, together with genetic invertases, segregate into two distinct families: the tyrosine conservation analyses, cross-species cloning experiments, and mu- site-specific recombinases (Tsrs) (also known as the lambda tational studies, revealed that this organism utilizes an endoge- integrase family) and the serine site-specific recombinases (Ssr) nous DNA inversion factor to globally modulate the expression of (recently reviewed in ref. 9). These two families are evolution- its surface structures. This DNA invertase is necessary for the arily and mechanistically distinct, and both contain members inversion of at least 13 regions located throughout the genome, with diverse functions including transposases, integrases, and including the promoter regions for seven of the capsular polysac- DNA invertases. DNA invertases of the Ssr family are typically charide biosynthesis loci, an accessory polysaccharide biosynthesis encoded by elements imported to bacteria from a phage (10–12) locus, and five other regions containing consensus promoter se- or plasmid (13) and act locally on the imported element. Many quences. Bacterial DNA invertases of the serine site-specific recom- DNA invertases of the Tsr family have been shown to regulate binase family are typically encoded by imported elements such as phase variation of fimbriae by inverting a single area of the phage and plasmids, and act locally on a single region of the chromosome adjacent to the Tsr gene (14–16). imported element. In contrast, the conservation and unique global In this study, we demonstrate that the inversions of all seven regulatory nature of the process in B. fragilis suggest an evolu- invertible polysaccharide promoter regions of B. fragilis are tionarily ancient mechanism for surface adaptation to the changing mediated by a single member of the Ssr family, which we have intestinal milieu during commensalism. designated Mpi. We show that in addition to these seven promoters, Mpi is also involved in inversion of six other pro- onsiderable progress has been made in elucidating the moter regions that control the transcription of uncharacterized Cmechanisms used by pathogenic bacteria to cause disease. In products. To our knowledge, this is the first report of a DNA contrast, relatively little is known about how the multitude of invertase acting globally to modulate the expression of multiple bacteria that inhabit the human intestinal tract establish suc- surface molecules encoded by regions distributed throughout a cessful commensal and symbiotic relationships with the host. bacterial genome. The unique global regulatory nature and Bacteria that reside in a mammalian host over its lifetime must conservation of Mpi in the species suggest that its regulation of have mechanisms that allow them to interact with the host in a bacterial surface variability is ancient, conferring a selective dynamic and responsive manner. Host mucosal surfaces have advantage to the microbe during its coevolution with the mam- been shown to respond to the presence of their endogenous malian host. microflora (1). The surfaces of commensal bacteria also have a Materials and Methods tremendous capacity for adapting to the changing environment Analysis of Gene Conservation. of the host. Bacteroides thetaiotaomicron, a numerically abundant Southern blot analysis was used to member of the intestinal microbiota, is able to extensively determine the conservation of the tsr and ssr genes within the regulate the expression of surface proteins involved in nutrient species B. fragilis. An internal portion of each of the 30 site- utilization in response to environmental stimuli (2–5). specific recombinase genes was PCR amplified by using the primers listed in Table 1, which is published as supporting The ability of some Bacteroides species to vary their surface information on the PNAS web site, www.pnas.org. The purified architecture also includes molecules not involved in nutrient PCR products were labeled by using the ECL Direct Labeling kit acquisition. The human commensal microorganism Bacteroides (Amersham Biosciences). Chromosomal DNA from 44 different fragilis is able to extensively change its surface by producing at B. fragilis strains was digested with EcoRI, and fragments were least eight distinct capsular polysaccharides (PSA–H), each of separated by agarose gel electrophoresis and transferred to which undergoes a reversible ON–OFF phenotype known as nylon. Blots containing chromosomal DNA from at least 12 phase variation. Phase variation of seven of these polysaccha- strains were probed with each of the labeled probes. If the probe rides is controlled by DNA inversions of the promoter regions of hybridized under high stringency conditions with at least 95% of their biosynthesis loci, placing them in the correct or incorrect the B. fragilis strains tested, the gene was deemed conserved. orientation for transcription of the downstream polysaccharide biosynthesis genes (6). An analogous system for the generation Plasmid Constructs. Plasmids containing regions encompassing of surface diversity may also exist in B. thetaiotaomicron, whose each of the seven invertible polysaccharide promoter regions genome was recently shown to contain seven polysaccharide biosynthesis loci, many of which are likely regulated by DNA inversions (5). In the intestinal ecosystem, the ability of these This paper was submitted directly (Track II) to the PNAS office. organisms to vary the expression of such a large array of surface Abbreviations: EtBr, ethidium bromide; IR, inverted repeat; MCR, Mpi-controlled region; polysaccharides may allow them to camouflage themselves from Ssr, serine site-specific recombinase; Tsr, tyrosine site-specific recombinase. the host and other members of the intestinal microbiota. Because *To whom correspondence should be addressed. E-mail: lcomstock@channing. B. fragilis is one of the most clinically important anaerobes in harvard.edu. 10446–10451 ͉ PNAS ͉ September 2, 2003 ͉ vol. 100 ͉ no. 18 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1832655100 Downloaded by guest on September 28, 2021 were constructed as follows: each of these regions and some were used to further query the B. fragilis database. In all, 28 flanking DNA was PCR amplified by using the primers listed in distinct members of the Tsr family were detected and designated Table 2, which is published as supporting information on the Tsr1–Tsr28 (Fig. 5, which is published as supporting information PNAS web site. These PCR products were cloned into the PstI on the PNAS web site). site of the Escherichia coli–Bacteroides shuttle vector pFD340 A similar set of analyses was used to detect members of the Ssr (17) and screened so that the forward primer (VarX-F) of each family. The B. fragilis genome was searched by using the PCR produced a product with plasmid-borne primer C7. Sub- pfam00239 motif, a 139-aa consensus sequence present in mem- sequently, individual site-specific recombinase genes were am- bers of the Ssr family (22). Three products similar to site-specific plified by using the primers listed in Table 2, cloned into the recombinases were retrieved and designated Ssr1–Ssr3. The sizes BamHI site of these plasmids, and screened for the proper and protein sequences of Ssr2 and Ssr3 identified them as orientation so that transcription was driven by the characterized members of the resolvase͞invertase branch of the Ssr family. In plasmid-borne promoter (Figs. 1A and 3A). Plasmids were contrast, Ssr1 is larger (585 aa) and segregates into the large introduced into B. fragilis 9343 (National Collection of Type serine recombinase branch, whose members mediate integration Cultures) or Bacteroides vulgatus 8482 (American Type Culture (23), excision (24), and transposition (25) events, but not DNA Collection) by mobilization from E. coli with the conjugal helper inversion. Therefore, of these three Ssr products, only Ssr2 and plasmid RK231. Ssr3 were further considered as candidate DNA invertases. Construction of ⌬mpi (⌬ssr2) Mutants. ⌬mpi mutants were con- To narrow the list of candidates that may be involved in the structed so that 534 bp of the 591-bp mpi gene was removed. inversions of the polysaccharide promoter regions, we took DNA segments upstream and downstream of the region to be advantage of the fact that these invertible regions are contained deleted