An Escherichia coli Nissle 1917 Missense Mutant Colonizes the Streptomycin-Treated Mouse Intestine Better than the Wild Type but Is Not a Better Probiotic

Abstract

f Previously we reported that the streptomycin-treated mouse intestine selected for two different Escherichia coli MG1655 mutants with improved colonizing ability: nonmotile E. coli MG1655 flhDC deletion mutants that grew 15% faster in vitro in mouse cecal mucus and motile E. coli MG1655 envZ missense mutants that grew slower in vitro in mouse cecal mucus yet were able to cocolonize with the faster-growing flhDC mutants. The E. coli MG1655 envZ gene encodes a histidine kinase that is a member of the envZ-ompR two-component signal transduction system, which regulates outer membrane protein profiles. In the present investigation, the envZ P41L gene was transferred from the intestinally selected E. coli MG1655 mutant to E. coli Nissle 1917, a human probiotic strain used to treat gastrointestinal infections. Both the E. coli MG1655 and E. coli Nissle 1917 strains containing envZ P41L produced more phosphorylated OmpR than their parents. The E. coli Nissle 1917 strain containing envZ P41L also became more resistant to bile salts and colicin V and grew 50% slower in vitro in mucus and 15% to 30% slower on several sugars present in mucus, yet it was a 10-fold better colonizer than E. coli Nissle 1917. However, E. coli Nissle 1917 envZ P41L was not better at preventing colonization by enterohemorrhagic E. coli EDL933. The data can be explained according to our "restaurant" hypothesis for commensal E. coli strains, i.e., that they colonize the intestine as sessile members of mixed biofilms, obtaining the sugars they need for growth locally, but compete for sugars with invading E. coli pathogens planktonically. P reviously we reported that when streptomycin-treated mice are fed wild-type Escherichia coli MG1655, the intestine selects for nonmotile flhDC deletion mutants (1, 2) and envZ missense mutants (3), both of which are better colonizers than the wild type. The flhDC mutants have deletions of various sizes, beginning downstream of an IS1 element in the flhDC regulatory region and extending into or beyond the flhDC structural genes (1, 2). FlhD and FlhC form the FlhD 4 C 2 complex (4), which activates transcription of class II flagellar genes that encode components of the flagellar basal body and export machinery (5). The IS1 element immediately upstream of the E. coli MG1655 flhDC promoter increases expression of the flhDC operon and makes E. coli MG1655 hypermotile (1, 6). One of the better-colonizing nonmotile flhDC deletion mutants, E. coli MG1655 ⌬flhD, grew 15% faster in vitro in mouse cecal mucus and 15% to 30% faster on several sugars present in cecal mucus than E. coli MG1655 (1), presumably explaining its better colonizing ability. Additional studies suggested that the E. coli MG1655 flhDC operon deletion mutants utilize sugars better than their parent at least in part because a number of metabolic genes are repressed by the FlhD 4 C 2 regulatory complex, including gltA (citrate synthase), sdhCDAB (succinate dehydrogenase), mdh (malate dehydrogenase), mglBAC (galactose transport), and a large number of sugar...

Cytoplasmic sensing by the inner membrane histidine kinase EnvZ

“…This concentration of OmpR ~ P was much higher than what was observed at high osmolality, which is considered to be a highly activating state. Mutants P148S (TM2) and V33E (TM1) behaved similarly (Adediran et al, 2014). At this high OmpR ~ P level, OmpR would be expected to exert pleiotropic effects, i.e., regulating genes normally outside of its repertoire.…”

“…Yet, the level of OmpR ~ P in the wildtype strain at high osmolality was barely detectible (Fig. 7) (Adediran et al, 2014), a condition that is considered to be highly activating for OmpR. If there is 3.5 μM OmpR in the cell, why is there so little of it phosphorylated?…”

“…Bacterial culture conditions, β-galactosidase assays preparation of outer membranes and separation on urea SDS PAGE and phos-tag labeling were as previously described (Adediran et al, 2014;.…”

“…For example, envZ mutations were created by hydroxylamine (Tokishita and Mizuno, 1994) or nitrosoguanadine mutagenesis (Russo et al, 1993) or by UV irradiation (Hsing et al, 1998). Another study isolated missense mutations in envZ that were better able than wildtype E. coli MG1655 to colonize the streptomycin-treated mouse (Adediran et al, 2014;Leatham-Jensen et al, 2012). These envZ missense mutations were located in TM1 and TM2, and were more resistant to bile salts and colicin V compared to E. coli MG1655.…”

“…These envZ missense mutations were located in TM1 and TM2, and were more resistant to bile salts and colicin V compared to E. coli MG1655. The phenotype of the EnvZ mutants was consistent with the presence of higher OmpR ~ P levels (Adediran et al, 2014). OmpR ~ P was isolated from these strains and separated on SDS-PAGE after treatment with the Phos-tag reagent (see Fig.…”