title: An Escherichia coli MG1655 lipopolysaccharide deep-rough core mutant grows and survives in mouse cecal mucus but fails to colonize the mouse large intestine.
abstract: The ability of E. coli strains to colonize the mouse large intestine has been correlated with their ability to grow in cecal and colonic mucus. In the present study, an E. coli MG1655 strain was mutagenized with a mini-Tn5 Km (kanamycin) transposon, and mutants were tested for the ability to grow on agar plates with mouse cecal mucus as the sole source of carbon and nitrogen. One mutant, designated MD42 (for mucus defective), grew poorly on cecal-mucus agar plates but grew well on Luria agar plates and on glucose minimal-agar plates. Sequencing revealed that the insertion in MD42 was in the waaQ gene, which is involved in lipopolysaccharide (LPS) core biosynthesis. Like "deep-rough" E. coli mutants, MD42 was hypersensitive to sodium dodecyl sulfate (SDS), bile salts, and the hydrophobic antibiotic novobiocin. Furthermore, its LPS core oligosaccharide was truncated, like that of a deep-rough mutant. MD42 initially grew in the large intestines of streptomycin-treated mice but then failed to colonize (<10(2) CFU per g of feces), whereas its parent colonized at levels between 10(7) and 10(8) CFU per g of feces. When mouse cecal mucosal sections were hybridized with an E. coli-specific rRNA probe, MD42 was observed in cecal mucus as clumps 24 h postfeeding, whereas its parent was present almost exclusively as single cells, suggesting that clumping may play a role in preventing MD42 colonization. Surprisingly, MD42 grew nearly as well as its parent during growth in undiluted, highly viscous cecal mucus isolated directly from the mouse cecum and, like its parent, survived well after reaching stationary phase, suggesting that there are no antimicrobials in mucus that prevent MD42 colonization. After mini-mariner transposon mutagenesis, an SDS-resistant suppressor mutant of MD42 was isolated. The mini-mariner insertion was shown to be in the bipA gene, a known regulator of E. coli surface components. When grown in Luria broth, the LPS core of the suppressor mutant remained truncated; however, the LPS core was not truncated when the suppressor mutant was grown in the presence of SDS. Moreover, when the suppressor mutant was grown in the presence of SDS and fed to mice, it colonized the mouse large intestine. Collectively, the data presented here suggest that BipA may play a role in E. coli MG1655 LPS core biosynthesis and that because MD42 forms clumps in intestinal mucus, it is unable to colonize the mouse large intestine.
authors.affiliation: Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA.
authors.forename: Annette K
authors.initials: AK
authors.lastname: Møller
authors.forename: Mary P
authors.initials: MP
authors.lastname: Leatham
authors.forename: Tyrrell
authors.initials: T
authors.lastname: Conway
authors.forename: Piet J M
authors.initials: PJ
authors.lastname: Nuijten
authors.forename: Louise A M
authors.initials: LA
authors.lastname: de Haan
authors.forename: Karen A
authors.initials: KA
authors.lastname: Krogfelt
authors.forename: Paul S
authors.initials: PS
authors.lastname: Cohen
mesh_terms: D000818:Animals; D002432:Cecum / Q000382:microbiology*; D003470:Culture Media; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D007413:Intestinal Mucosa / Q000382:microbiology*; D007420:Intestine, Large / Q000382:microbiology*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008297:Male; D051379:Mice; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D010750:Phosphoproteins; D016695:Glycosyltransferases; C409702:WaaQ protein, E coli; D020558:GTP Phosphohydrolases; C408370:typA protein, E coli