D-Gluconate which is primarily catabolized via the Entner-Doudoroff (ED) pathway, has been implicated as being important for colonization of the streptomycin-treated mouse large intestine by Escherichia coli MG1655, a human commensal strain. In the present study, we report that an MG1655 ⌬edd mutant defective in the ED pathway grows poorly not only on gluconate as a sole carbon source but on a number of other sugars previously implicated as being important for colonization, including L-fucose, D-gluconate, D-glucuronate, N-acetyl-D-glucosamine, D-mannose, and D-ribose. Furthermore, we show that the mouse intestine selects mutants of MG1655 ⌬edd and wild-type MG1655 that have improved mouse intestinecolonizing ability and grow 15 to 30% faster on the aforementioned sugars. The mutants of MG1655 ⌬edd and wild-type MG1655 selected by the intestine are shown to be nonmotile and to have deletions in the flhDC operon, which encodes the master regulator of flagellar biosynthesis. Finally, we show that ⌬flhDC mutants of wild-type MG1655 and MG1655 ⌬edd constructed in the laboratory act identically to those selected by the intestine; i.e., they grow better than their respective parents on sugars as sole carbon sources and are better colonizers of the mouse intestine.

Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD 4 C 2 . Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD 4 C 2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.Intestinal colonization is defined as the indefinite persistence of a bacterial population in the intestine of an animal in stable numbers without repeated introduction of the bacterium into the animal. Commensal Escherichia coli strains colonize the mouse intestine by growing in the intestinal mucus layer which covers the epithelium (18,24,25,28,38,39,43). The intestinal mucus layer is constantly being synthesized, degraded by indigenous bacteria, and sloughed into feces (15,27). Colonization requires bacteria to penetrate the mucus layer, compete for nutrients with the indigenous flora, and divide at a rate that is at least equal to the washout rate caused by sloughing of the mucus layer into feces (7, 16); however, the role of motility in mucosal colonization is complex and appears to differ in different microorganisms (12).Motility and chemotaxis in E. coli are controlled by a master regulator encoded by flhD and flhC, which comprise the flhDC operon. Together, FlhD and FlhC form the FlhD 4 C 2 complex (20, 44), which activates transcription of class II flagellar genes that encode components of the flagellar basal body and export machinery (30). One of the class II flagellar genes, fliA, encodes an RNA polymerase sigma factor, 28 , which switches on expression of the class III genes coding for the cell-distal structural components of the flagellum (30). The sequenced E. coli MG1655 strain has an IS1 element in the regulatory region of the flhDC promoter (3), which increases the expression of the flhDC operon and mak...

Previously we showed that the tricarboxylic acid (TCA) cycle operates as a full cycle during Salmonella enterica serovar Typhimurium SR-11 peroral infection of BALB/c mice (M. Tchawa Yimga et al., Infect. Immun. 74:1130-1140, 2006). The evidence was that a ⌬sucCD mutant (succinyl coenzyme A [succinyl-CoA] synthetase), which prevents the conversion of succinyl-CoA to succinate, and a ⌬sdhCDA mutant (succinate dehydrogenase), which blocks the conversion of succinate to fumarate, were both attenuated, whereas an SR-11 ⌬aspA mutant (aspartase) and an SR-11 ⌬frdABCD mutant (fumarate reductase), deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent. In the present study, evidence is presented that a serovar Typhimurium SR-11 ⌬frdABCD ⌬sdhCDA double mutant is avirulent in BALB/c mice and protective against subsequent infection with the virulent serovar Typhimurium SR-11 wild-type strain via the peroral route and is highly attenuated via the intraperitoneal route. These results suggest that fumarate reductase, which normally runs in the reductive pathway in the opposite direction of succinate dehydrogenase, can replace it during infection by running in the same direction as succinate dehydrogenase in order to run a full TCA cycle in an SR-11 ⌬sdhCDA mutant. The data also suggest that the conversion of succinate to fumarate plays a key role in serovar Typhimurium virulence. Moreover, the data raise the possibility that S. enterica ⌬frdABCD ⌬sdhCDA double mutants and ⌬frdABCD ⌬sdhCDA double mutants of other intracellular bacterial pathogens with complete TCA cycles may prove to be effective live vaccine strains for animals and humans.

Seventeen bacterial strains previously identified as Vibrio harveyi or V. carchariae (Grimes et al. 1984) and the type strains of V. harveyi, V. carchariae and V. campbellii were analyzed by 16S ribosomal DNA (rDNA) sequencing. Four clusters were identified in a phylogenetic analysis performed by comparing a 746 base pair fragment of the 16S rDNA and previously published sequences of other closely related Vibrio species. The type strains of V. harveyi and V. carchariae and about half of the strains identified as V. harveyi or V. carchariae formed a single, wellsupported cluster designed as 'bona fide' V. harveyi/carchariae. A second more heterogeneous cluster included most other strains and the V. campbellii type strain. Two remaining strains are shown to be more closely related to V. rumoiensis and V. mediterranei. 16S rDNA sequencing has confirmed the homogeneity and synonymy of V. harveyi and V. carchariae. Analysis of API20E biochemical profiles revealed that they are insufficient by themselves to differentiate V. harveyi and V. campbellii strains. 16S rDNA sequencing, however, can be used in conjunction with biochemical techniques to provide a reliable method of distinguishing V. harveyi from other closely related species.KEY WORDS: Vibrio harveyi · Vibrio carchariae · Vibrio campbellii · Vibrio trachuri · Ribosomal DNA · Biochemical characteristics · Diagnostic · API20E Resale or republication not permitted without written consent of the publisherDis Aquat Org 52: [39][40][41][42][43][44][45][46] 2002 netic relationship, based on 16S rDNA sequences, of the type strains of V. harveyi and V. carchariae, 17 putative V. harveyi and V. carchariae strains (Yii et al. 1997, Pedersen et al. 1998, Soffientino et al. 1999, and published 16S rDNA sequences of V. harveyi, V. carchariae and other closely related Vibrio spp. (Valle et al. 1990, Ruimy et al. 1994, Yumoto et al. 1999. Also, we compared 16S rDNA sequencing data to biochemical profiles. The implications for bacterial classification and diagnosis are discussed. MATERIALS AND METHODSBacterial strains. Bacterial strains are listed in Table 1. The strains designated VIB 286, 295, 350, 394, 400, 403, 411, 570, 573, 645, 651, 654, 655, 656, 657, 660, and 697 belong to a collection maintained by Dr. Dawn Austin of Heriot-Watt University (Edinburgh, UK) (Pedersen et al. 1998). Also included in this study are a strain of Vibrio carchariae from grouper in Taiwan (Yii et al. 1997), designated here as V. carchariae grouper, a strain from summer flounder in the USA (Soffientino et al. 1999), designated here as V. carchariae flounder, and the type strains ( T ) of V. harveyi (American Type Culture Collection, ATCC 14126 T ) and V. campbellii (ATCC 25920 T ). VIB 286 is the designation given to the V. carchariae type strain in Pedersen et al. (1998).Throughout the text, the names VIB 286 and V. carchariae type strain are used interchangeably and refer to the same strain. VIB 295 is the designation given to the V. harveyi type strain by Pedersen et al. (1998...

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