Numerous microbes inhabit the mammalian intestinal track and strongly impact host physiology; however, our understanding of this ecosystem remains limited owing to the high complexity of the microbial community and the presence of numerous non-culturable microbes. Segmented filamentous bacteria (SFBs), which are clostridia-related Gram-positive bacteria, are among such non-culturable populations and are well known for their unique morphology and tight attachment to intestinal epithelial cells. Recent studies have revealed that SFBs play crucial roles in the post-natal maturation of gut immune function, especially the induction of Th17 lymphocytes. Here, we report the complete genome sequence of mouse SFBs. The genome, which comprises a single circular chromosome of 1 620 005 bp, lacks genes for the biosynthesis of almost all amino acids, vitamins/cofactors and nucleotides, but contains a full set of genes for sporulation/germination and, unexpectedly, for chemotaxis/flagella-based motility. These findings suggest a triphasic lifestyle of the SFB, which comprises two types of vegetative (swimming and epicellular parasitic) phases and a dormant (spore) phase. Furthermore, SFBs encode four types of flagellin, three of which are recognized by Toll-like receptor 5 and could elicit the innate immune response. Our results reveal the non-culturability, lifestyle and immunostimulation mechanisms of SFBs and provide a genetic basis for the future development of the SFB cultivation and gene-manipulation techniques.

The clinical importance of nondiphtherial Corynebacterium, a ubiquitous member of the normal human microflora of the skin and mucous membrane, for ocular surface infections has been recognized recently. We performed an antimicrobial susceptibility test with Etest strips for three fluoroquinolones (ciprofloxacin, norfloxacin, and levofloxacin) and a taxonomic analysis on 21 isolates of Corynebacterium from ophthalmic samples. Of these, 16 isolates were identified as C. macginleyi at the species level on the basis of 16S rRNA gene sequence comparisons. The remaining five isolates were determined to be C. mastitidis (four) or C. accolens (one). Eleven of the C. macginleyi isolates showed high levels of resistance to all of the fluoroquinolones tested, and one isolate was resistant to norfloxacin alone. An analysis of the amplified quinolone-resistance-determining regions of the gyrA genes revealed that a single amino acid substitution in position 83 of the gyrA product was sufficient to generate the norfloxacin resistance phenotype, and double mutations leading to amino acid changes in positions 83 and 87 were necessary for high-level resistance against the other fluoroquinolones. We conducted the first example of multilocus sequence typing (MLST) analysis on C. macginleyi. The MLST analysis grouped the majority of C. macginleyi isolates into a single lineage, and another molecular strain typing by random amplified polymorphic DNA fragment patterns supported the finding, indicating that a particular lineage of C. macginleyi is dominant on the human ocular surface. This type of population might be particularly adaptable to the milieu on the human ocular surface.

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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.

This study describes refined electroporation parameters for efficient transformation of Bacteroides fragilis by plasmids prepared from laboratory strains of Escherichia coli. Development of the method used included determination of the optimal growth conditions for competent cell preparation, selectable antimicrobial resistance markers, electric field strength, and postpulse incubation time. Of the four E. coli-Bacteroides shuttle plasmids tested (pVAL-1, pVAL-2, pNLY1, and pLYL05), pLYL05 containing the cefoxitin resistance marker was found to be the most suitable for B. fragilis transformation, and it generated 2-to 900-fold more transformants (about 10 4 transformants per g pLYL05 DNA) than the other plasmids. Numerous microbes inhabit the human intestine, and the number of microbial cells can be nearly 10 11 cells per g of feces. The colon is the most densely populated environment in the human body, and the genus Bacteroides, which contains Gramnegative, obligate anaerobes, is one of the most abundant genera in the intestinal microbiota. In the clinical setting, Bacteroides strains are considered opportunistic pathogens that can occasionally cause infections, such as peritoneal abscesses, appendicitis, and septicemia (3,4,23). In the genus Bacteroides, B. fragilis is considered the most virulent species, and its capsular polysaccharides are especially linked to its pathogenesis (8, 13, 22). However, recent reports have highlighted the symbiotic properties of B. fragilis since the capsular polysaccharide displayed by this species can modify the human immune system (21). Another example of a host-Bacteroides interaction is a B. thetaiotaomicron interaction; this species can repress host inflammatory responses by promoting the binding of peroxisome proliferator-activated receptor gamma (PPAR-␥) to the NF-b subunit RelA (11). B. thetaiotaomicron is also known to induce production of antimicrobial peptides from Paneth cells in host intestinal epithelia (10).The recent accumulation of genomic information about several Bacteroides species (2) has enabled us to search for the genes responsible for the symbiotic properties of intestinal Bacteroides described above. However, screening for genes involved in host-Bacteroides symbioses requires development of a suitable, simple, and efficient genetic manipulation system. Although transconjugation by filter mating can be performed with Bacteroides species (19, 24 ), this technique is labor-intensive and time-consuming. Electroporation is a simple and reproducible procedure that is widely used for introduction of foreign nucleic acids into various bacteria. While Smith et al. previously described an electroporation protocol for Bacteroides species that could transform Bacteroides with plasmids from homologous hosts, plasmids derived from Escherichia coli laboratory strains could be transformed into Bacteroides only with difficulty (20). Recently, Patrick et al. described an efficient electrotransformation method for B. fragilis using purified T7 antirestriction protein Ocr...