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The trade-off relationship between antibiotic exclusion and nutrient access across the Gram-negative outer membrane is determined by structural constraints in porin channels. The precise nutritional cost of exclusion is unknown for different antibiotics, as are the shapes of the nutrition-susceptibility trade-off. Using a library of 10 engineered isogenic Escherichia coli strains with structural modifications of OmpF porin expressed at a constant level, susceptibilities were measured for nine antibiotics and the nutritional fitness costs estimated by competitions in chemostats. Different antibiotics exhibited a remarkably varied range of geometries in the nutrition-susceptibility trade-off, including convex, concave and sigmoidal trade-off shapes. The trade-off patterns predict the possibility of adaptations in contributing to antibiotic resistance; exclusion of amoxicillin or trimethoprim in ompF mutants can occur with little loss of fitness whereas kanamycin and streptomycin exclusion has a high cost. Some individual OmpF changes even allow positive correlations (trade-ups), resulting in increased fitness and decreased susceptibility specifically to cephalexin or ciprofloxacin. The surprising plasticity of the nutrition-exclusion relationship means that there are no generalisable rules that apply to decreasing susceptibility for all antibiotics. The protein changes are exquisitely specific in determining nutritional fitness and adaptive outcomes in a structural constraint trade-off.

The trade-off relationship between antibiotic exclusion and nutrient access across the Gram-negative outer membrane is determined by structural constraints in porin channels. The precise nutritional cost of exclusion is unknown for different antibiotics, as are the shapes of the nutrition-susceptibility trade-off. Using a library of 10 engineered isogenic Escherichia colistrains with structural modifications of OmpF porin expressed at a constant level, susceptibilities were measured for nine antibiotics and the nutritional fitness costs estimated by competitions in chemostats. Different antibiotics exhibited a remarkably varied range of geometries in the nutrition-susceptibility trade-off, including convex, concave and sigmoidal trade-off shapes. The trade-off patterns predict the possibility of adaptations in contributing to antibiotic resistance; exclusion of amoxicillin or trimethoprim in ompFmutants can occur with little loss of fitness whereas kanamycin and streptomycin exclusion has a high cost. Some individual OmpF changes even allow positive correlations (trade-ups), resulting in increased fitness and decreased susceptibility specifically to cephalexin or ciprofloxacin. The surprising plasticity of the nutrition-exclusion relationship means that there are no generalisable rules that apply to decreasing susceptibility for all antibiotics. The protein changes are exquisitely specific in determining nutritional fitness and adaptive outcomes in a structural constraint trade-off.

The genomic richness and intra-species heterogeneity of the prokaryotic world is suggestive of extensive biochemical diversity. In this study, metabolomic profiling permitted a phylogenetic assessment of metabolic diversification amongst environmental, medical and laboratory strains of Escherichia coli. Strikingly, no two E. coliisolates exhibited the same metabolite pool profile. Only 27% of detected metabolite spots in 2-dimensional high-performance thin layer chromatography (2DHPTLC) were found in all strains, indicating that a relatively small core of metabolism is conserved across a species. The population structure determined using metabolomics exhibited clustering of strains in parallel to genetic relatedness, as established by multi-locus DNA sequencing. On the other hand, metabolome patterns did not cluster in parallel with the pathogenicity or environmental origins of strains, but some unique spots were found in most bacteria. These results suggest that great metabolic diversity, to the point of individuality, is likely to be characteristic of a bacterial species. Furthermore, the high resolving power of 2DHPTLC metabolite fingerprinting provides an economic and powerful means of using metabolomics for the analysis of evolutionary relationships and the precise typing of organisms.

The disaccharide lactose has no specific diffusion pathway across the outer membrane of Escherichia coli. At least three classes of spontaneous mutation affecting outer membrane permeability arose with each of three independent E. colipopulations adapting to prolonged lactose limitation in chemostats. Both structural and regulatory mutations affecting OmpF porin predominated in isolates after 210–280 generations of culture. Six types of ompFmutation were found, including in‐frame deletions and substitutions at Arg82 and Asp113, all affecting the channel constriction residues of OmpF. Isolates had increased susceptibility to antibiotics and were affected in the quantity of OmpF, LamB and OmpA proteins. A minimum of three or four mutations was evident in all isolates after 280 generations in a lactose‐limited environment, in addition to lacmutations defined in previous studies.

Growth with suboptimal nutrient levels elicits adaptations not observed with either starving (resting) or unstressed bacteria. The hunger response results in patterns of gene expression optimising scavenging capabilities through novel control mechanisms. In Escherichia coli, recent results indicate that outer membrane permeability (porin and glycoporin regulation) as well as transport involving the phosphotransferase system and ABC-type high affinity transporters change under glucose limitation. Many other adaptations in expression and metabolic capabilities at subsaturating growth rates are still poorly understood, even in E. coli.

The starch polysaccharides amylose and amylopectin are not utilized by Escherichia coli, but are bound by the bacteria. The following evidence supports the view that the outer membrane λ receptor protein, a component of the maltose/ maltodextrin transport system is responsible for the binding. (i) Amylose and amylopectin both inhibit the transport of maltose into E. coli. (ii) Both polysaccharides prevent binding of non-utilizable maltodextrins by the intact bacterium, a process previously shown to be dependent on components of the maltose transport system (T. Ferenci, Eur. J. Biochem., in press). (iii) A fluorescent amylopectin derivative, O-(fluoresceinyl thiocarbamoyl)-amylopectin, has been synthesized and shown to bind to E. coliin a reversible, saturable manner. Binding of O-(fluoresceinyl thiocarbamoyl)-amylopectin is absent in mutants lacking the λ receptor, but mutations in any of the other components of the maltose transport system do not affect binding as long as λ receptor is present. (iv) Using the inhibition of λ receptor-dependent O-(fluoresceinyl thiocarbamoyl)-amylopectin binding as an assay, the affinities of the λ receptor for maltodextrins and other sugars have been estimated. The affinity for dextrins increases with increasing degree of polymerization (Kdfor maltose, 14 mM; for maltotetraose, 0.3 mM; for maltodecaose, 0.075 mM). Maltose and some other di- and trisaccharides are inhibitory to amylopectin binding, but only at concentrations above 1 mM.

Members of the LamB family of sugar-selective porins (glyco-porins) are β-barrel proteins in the outer membrane of Gramnegative bacteria. To study the determinants of structure and sugar selectivity, 68 non-identical single amino acid substitutions were introduced into the stretch of sequence consisting of residues 106 through 125 in Escherichia coli LamB. This region includes all bar one residue of the channel constriction loop L3 and extends into the transmembrane β6 strand in the LamB structure. Mutants were assayed for dextrin utilization, starch binding, λ binding, monoclonal antibody binding and for qualitative changes in protein expression. The importance of the L3 amino acids was emphasized by the observation that only four residues permitted a majority of neutral substitutions. Changes to the channel constriction zone strongly affected sugar binding yet no single amino acid change of residues exposed to the channel lumen caused a complete defect in maltodextrin utilization (i.e. were still Dex). Substitutions in the L3 loop did not affect phage λ binding, except one change at residue 122, nor changed recognition by anti-LamB antibodies specific for surface epitopes, consistent with the lack of a role of L3 residues in surface receptor function. In marked contrast, four substitutions in transmembrane strand β5 resulted in a Dex phenotype and gross changes in protein properties, indicating the significance of β5 in the architecture of LamB.

The involvement of lipopolysaccharide and outer membrane proteins in the binding ofEscherichia coli to cellulose was investigated. Cellulose binding was assayed in defined strains with or without O-antigenic polysaccharide and in mutants with defects in lipopolysaccharide core synthesis. Binding was also tested in strains lacking major outer membrane proteins. Optimal cellulose binding was exhibited by rough strains and was reduced to various extents in the presence of different O-antigens. Core defects also reduced but did not abolish binding to cellulose. Reduced binding was also found in mutants lacking OmpC protein, but OmpC/OmpA double mutants orompB mutants lacking OmpC and OmpF were not affected. Mutants with reduced cellulose binding were also isolated directly through selection of nonbinding populations after chromatography on cellulose columns. Each of the independent isolates derived fromE. coli K12 with reduced cellulose binding had multiple mutations, with additional phenotypic changes such as phage resistance, increased sensitivity to bile salts, or altered patterns of outer membrane proteins. These results suggest that no single receptor that could be altered by mutation was responsible for the binding ofE. coli to cellulose. Rather, the nonspecific binding of cellulose was more likely to be due to interaction with, or the combined activity of, several integral outer membrane components that could be masked by O-antigen.

malB+malQstrains accumulate maltose via the maltose-binding-protein-dependent transport system but are unable to metabolize it. Nevertheless, some of the maltose is modified after entering the cell. This newly formed compound exhibited a higher Rfvalue than did maltose upon thin-layer and paper chromatography with the usual sugar-separating solvents. Treatment of this compound with acid and alkali reformed maltose. The identity of this compound with acetylmaltose was derived from mass spectrometry. Nuclear magnetic resonance spectra of the compound confirmed the presence of the acetyl group but did not allow its precise location on the maltose moiety. However, linkage to the 1-position of maltose could be excluded. Analysis of the mass spectra indicated that the nonreducing end of maltose was acetylated. Other substrates of the maltose transport system, such as maltotetraose, maltopentaose, and maltohexaose, were also modified after accumulation into the cell. Several products were formed; the heterogeneity of these products was probably caused by different degrees of acetylation. The enzymatic activity responsible for maltose and maltodextrin acetylation is unknown. However, it is clear that the lacA-dependent thiogalactoside transacetylase was not necessary for the acetylation of maltose. Strains that accumulate maltose via a bypass of the normal malB-dependent transport system also acetylated maltose even in the absence of any malBgene products. Thus, the acetylating activity was not connected to the malBsystem. Acetylmaltose as well as acetylated maltodextrins was excreted into the medium. Acetylmaltose is not a substrate of the maltose transport system. Thus, maltose acetylation may be an effective detoxification mechanism.

Bacterial populations in clinical and laboratory settings contain a significant proportion of mutants with elevated mutation rates (mutators). Mutators have a particular advantage when multiple beneficial mutations are needed for fitness, as in antibiotic resistance. Nevertheless, high mutation rates potentially lead to increasing numbers of deleterious mutations and subsequently to the decreased fitness of mutators. To test how fitness changed with mutation accumulation, genome sequencing and fitness assays of nine Escherichia coli mutYmutators were undertaken in an evolving chemostat population at three time points. Unexpectedly, the fitness in members of the mutator subpopulation became constant despite a growing number of mutations over time. To test if the accumulated mutations affected fitness, we replaced each of the known beneficial mutations with wild-type alleles in a mutator isolate. We found that the other 25 accumulated mutations were not deleterious. Our results suggest that isolates with deleterious mutations are eliminated by competition in a continuous culture, leaving mutators with mostly neutral mutations. Interestingly, the mutator–non-mutator balance in the population reversed after the fitness plateau of mutators was reached, suggesting that the mutator–non-mutator ratio in populations has more to do with competition between members of the population than the accumulation of deleterious mutations.

Evolutionary divergence is common within bacterial species and populations, even during a single bacterial infection. We use large-scale genomic and phenotypic analysis to identify the extent of diversification in controlled experimental populations and apply these data to differentiate between several potential mechanisms of evolutionary divergence.

The disaccharide lactose has no specific diffusion pathway across the outer membrane of Escherichia coli. At least three classes of spontaneous mutation affecting outer membrane permeability arose with each of three independent E. coli populations adapting to prolonged lactose limitation in chemostats. Both structural and regulatory mutations affecting OmpF porin predominated in isolates after 210–280 generations of culture. Six types of ompF mutation were found, including in-frame deletions and substitutions at Arg82 and Asp113, all affecting the channel constriction residues of OmpF. Isolates had increased susceptibility to antibiotics and were affected in the quantity of OmpF, LamB and OmpA proteins. A minimum of three or four mutations was evident in all isolates after 280 generations in a lactose-limited environment, in addition to lac mutations defined in previous studies.

Site-directed mutagenesis of the lamB gene was used to introduce individual cysteine substitutions at 20 sites in two regions (surface loops L7 and L8) of LamB protein significant in antibody recognition. Characterisation of cysteine mutants involved immunoblotting with three surface-specific monoclonal antibodies (mAb72, mAb302, mAb347) before and after incubation with thiol-specific reagents. In contrast to an earlier study that showed no amino acid changes affecting recognition by all three antibodies, changes at six amino acids were found to influence a common core epitope. These core sites included one residue (T336) in the predicted loop L7 containing amino acids 329–342 and four (Y379, N387, N389, K392, F398) in the large surface loop involving residues 370–412. Individual antibodies made additional but distinct contacts within the two studied regions, with mAb347 binding the most different and affected by seven substitutions in the 328–338 regions. The lamB mutants were also tested for phage λ receptor activity and starch binding before and after thiol modification and were useful in extending previous maps of these ligand binding sites.

Maltoporin in the outer membrane of Escherichia coli contains two cysteine residues, at positions 22 and 38 in the primary sequence. The role of these residues in determining structural stability, and their contributions to the maltoporin binding sites for maltodextrins and bacteriophage λ, was investigated. Site-directed mutagenesis was used to alter each of these residues to a serine. A double mutant lacking both cysteines was also isolated. None of the substitutions affected maltodextrin binding or the binding of phage λ, suggesting the variant proteins retain a native binding-site conformation. The mutants were assembled at wild-type levels into the outer membrane as maltoporin trimers but the temperature-stability of the trimer > monomer dissociation was slightly reduced in the presence of the Cys 38 substitution. However, it is unlikely that the stability of trimers was due to disulfide linkages between subunits since the native trimers are stable under highly reducing conditions in the presence of SDS; more likely the Cys > Ser substitutions slightly perturb intra- or inter-subunit hydrophobic interactions in regions predicted to span across the outer membrane.

Escherichia coli K-12 populations containing up to 3 · 109 bacteria were reversibly retained by columns containing fibrous cellulose. Using chromatographic retention as an assay of attachment to cellulose, the factors affecting bacterial binding were investigated. Minimal binding was observed in the absence of ions or at high pH. Optimal binding was observed in the presence of low concentrations of divalent cations at any pH or at pH below 4 in the absence of divalent cations. Factors found not to significantly affect bacterial retention by cellulose included the culture conditions of the bacteria, the presence of surface appendages, the presence of various sugars during chromatography or variations in elution temperature between 0 and 70°C. These results suggest that mechanisms not dependent on appendages contribute to the adhesion of a bacterium to an inert surface.