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Abstract Background Microbiological studies frequently involve exchanges of strains between laboratories and/or stock centers. The integrity of exchanged strains is vital for archival reasons and to ensure reproducible experimental results. For at least 50 years, one of the most common means of shipping bacteria was by inoculating bacterial samples in agar stabs. Long-term cultures in stabs exhibit genetic instabilities and one common instability is in rpoS. The sigma factor RpoS accumulates in response to several stresses and in the stationary phase. One consequence of RpoS accumulation is the competition with the vegetative sigma factor σ70. Under nutrient limiting conditions mutations in rpoS or in genes that regulate its expression tend to accumulate. Here, we investigate whether short-term storage and mailing of cultures in stabs results in genetic heterogeneity. Results We found that samples of the E. coli K-12 strain MC4100TF exchanged on three separate occasions by mail between our laboratories became heterogeneous. Reconstruction studies indicated that LB-stabs exhibited mutations previously found in GASP studies in stationary phase LB broth. At least 40% of reconstructed stocks and an equivalent proportion of actually mailed stock contained these mutations. Mutants with low RpoS levels emerged within 7 days of incubation in the stabs. Sequence analysis of ten of these segregants revealed that they harboured each of three different rpoS mutations. These mutants displayed the classical phenotypes of bacteria lacking rpoS. The genetic stability of MC4100TF was also tested in filter disks embedded in glycerol. Under these conditions, GASP mutants emerge only after a 3-week period. We also confirm that the intrinsic high RpoS level in MC4100TF is mainly due to the presence of an IS1 insertion in rssB. Conclusions Given that many E. coli strains contain high RpoS levels similar to MC4100TF, the integrity of such strains during transfers and storage is questionable. Variations in important collections may be due to storage-transfer related issues. These results raise important questions on the integrity of bacterial archives and transferred strains, explain variation like in the ECOR collection between laboratories and indicate a need for the development of better methods of strain transfer.

Abstract Background Sigma factors and the alarmone ppGpp control the allocation of RNA polymerase to promoters under stressful conditions. Both ppGpp and the sigma factor σS (RpoS) are potentially subject to variability across the species Escherichia coli. To find out the extent of strain variation we measured the level of RpoS and ppGpp using 31 E. coli strains from the ECOR collection and one reference K-12 strain. Results Nine ECORs had highly deleterious mutations in rpoS, 12 had RpoS protein up to 7-fold above that of the reference strain MG1655 and the remainder had comparable or lower levels. Strain variation was also evident in ppGpp accumulation under carbon starvation and spoT mutations were present in several low-ppGpp strains. Three relationships between RpoS and ppGpp levels were found: isolates with zero RpoS but various ppGpp levels, strains where RpoS levels were proportional to ppGpp and a third unexpected class in which RpoS was present but not proportional to ppGpp concentration. High-RpoS and high-ppGpp strains accumulated rpoS mutations under nutrient limitation, providing a source of polymorphisms. Conclusions The ppGpp and σS variance means that the expression of genes involved in translation, stress and other traits affected by ppGpp and/or RpoS are likely to be strain-specific and suggest that influential components of regulatory networks are frequently reset by microevolution. Different strains of E. coli have different relationships between ppGpp and RpoS levels and only some exhibit a proportionality between increasing ppGpp and RpoS levels as demonstrated for E. coli K-12.

Beneficial mutations in diversifying glucose-limited Escherichia coli populations are mostly unidentified. The genome of an evolved isolate with multiple differences from that of the ancestor was fully assembled. Remarkably, a single mutation in hfq was responsible for the multiple benefits under glucose limitation through changes in at least five regulation targets. doi: 10.1128/JB.00368-10

The genome of an Escherichia coli MC4100 strain with a [lambda] placMu50 fusion revealed numerous regulatory differences from MG1655, including one that arose during laboratory storage. The 194 mutational differences between MC4100(MuLac) and other K-12 sequences were mostly allocated to specific lineages, indicating the considerable mutational divergence between K-12 strains.

Laboratory strains and natural isolates of Escherichia coli differ in their level of stress resistance due to strain variation in the level of the sigma factor [[sigma].sup.S] (or RpoS), the transcriptional master controller of the general stress response. We found that the high level of RpoS in one laboratory strain (MC4100) was partially dependent on an elevated basal level of ppGpp, an alarmone responding to stress and starvation. The elevated ppGpp was caused by two mutations in spoT, a gene associated with ppGpp synthesis and degradation. The nature of the spoT allele influenced the level of ppGpp in both MC4100 and another commonly used K-12 strain, MG1655. Introduction of the spoT mutation into MG1655 also resulted in an increased level of RpoS, but the amount of RpoS was lower in MG1655 than in MC4100 with either the wild-type or mutant spoT allele. In both MC4100 and MG1655, high ppGpp concentration increased RpoS levels, which in turn reduced growth with poor carbon sources like acetate. The growth inhibition resulting from elevated ppGpp was relieved by rpoS mutations. The extent of the growth inhibition by ppGpp, as well as the magnitude of the relief by rpoS mutations, differed between MG1655 and MC4100. These results together suggest that spoT mutations represent one of several polymorphisms influencing the strain variation of RpoS levels. Stress resistance was higher in strains with the spoT mutation, which is consistent with the conclusion that microevolution affecting either or both ppGpp and RpoS can reset the balance between self-protection and nutritional capability, the SPANC balance, in individual strains of E. coli.

The energetic efficiency of nutrient uptake and conversion into biomass is a key factor in the ecological behavior of microorganisms. The constraints shaping the metabolic rate-yield trade-off in bacteria are not well understood. To examine whether metabolic rate-yield settings and physiological strategies evolve toward a particular optimum in a constant environment, we studied multiple Escherichia coli isolates evolving in a glucose-limited chemostat population. A major divergence in transport and metabolic strategies was observed, and the isolates included inefficient rate strategists (polluters or cheaters) and yield strategists (conservationists), as well as various hybrid rate-yield strategists and alternative ecotypes (dropouts). Sugar transport assays, strain comparisons based on metabolomics, and Biolog profiling revealed variance to the point of individuality within an evolving population. Only 68 of 177 metabolites assayed were not affected in 10 clonally related strains. The parallel enrichment of rate and yield strategists and the divergence in metabolic phylogenies indicate that bacteria do not converge on a particular rate-yield balance or unique evolutionary solutions. Redundancies in transport and metabolic pathways are proposed to have laid the framework for the multiplicity of bacterial adaptations.

Polymorphisms in rpoS are common in Escherichia coli. rpoS status influences a trade-off between nutrition and stress resistance and hence fitness across different environments. To analyze the selective pressures acting on rpoS, measurement of glucose transport rates in rpo[S.sup.+] and rpoS bacteria was used to estimate the role of [F.sub.nc], the fitness gain due to improved nutrient uptake, in the emergence of rpoS mutations in nutrient-limited chemostat cultures. Chemostats with set atmospheres, temperatures, pH's, antibiotics, and levels of osmotic stress were followed. [F.sub.nc] was reduced under anaerobiosis, high osmolarity, and with chloramphenicol, consistent with a reduced rate of rpoS enrichment in these conditions. [F.sub.nc] remained high, however, with alkaline pH and low temperature but rpoS sweeps were diminished. Under these conditions, [F.sub.sp], the fitness reduction due to lowered stress protection, became significant. We also estimated whether the fitness need for the gene was related to its regulation. No consistent pattern emerged between the level of RpoS and the loss of rpoS function in particular environments. This dissection allows an unprecedented view of the genotype-by-environment interactions controlling a mutational sweep and shows that both [F.sub.nc] and [F.sub.sp] are influenced by individual stresses and that additional factors contribute to selection pressure in some environments.

There are few existing indications that strain variation in prokaryotic gene regulation is common or has evolutionary advantage. In this study, we report on isolates of Escherichia coli with distinct ratios of sigma factors (RpoD, [[sigma].sup.D], or [[sigma].sup.70] and RpoS or [[sigma].sup.s]) that affect transcription initiated by RNA polymerase. Both laboratory E. coli K-12 lineages and nondomesticated isolates exhibit strain-specific endogenous levels of RpoS protein. We demonstrate that variation in genome usage underpins intraspecific variability in transcription patterns, resistance to external stresses, and the choice of beneficial mutations under nutrient limitation. Most unexpectedly, RpoS also controlled strain variation with respect to the metabolic capability of bacteria with more than a dozen carbon sources. Strains with higher [[sigma].sup.s] levels were more resistant to external stress but metabolized fewer substrates and poorly competed for low concentrations of nutrients. On the other hand, strains with lower [[sigma].sup.s] levels had broader nutritional capabilities and better competitive ability with low nutrient concentrations but low resistance to external stress. In other words, RpoS influenced both r and K strategist functions of bacteria simultaneously. The evolutionary principle driving strain variation is proposed to be a conceptually novel trade-off that we term SPANC (for 'self-preservation and nutritional competence'). The availability of multiple SPANC settings potentially broadens the niche occupied by a species consisting of individuals with narrow specialization and reveals an evolutionary advantage offered by polymorphic regulation. Regulatory diversity is likely to be a significant contributor to complexity in a bacterial world in which multiple sigma factors are a universal feature.

The kinetics of mutator sweeps was followed in two independent populations of Escherichia coli grown for up to 350 generations in glucose-limited continuous culture. A rapid elevation of mutation rates was observed in both populations within 120-150 generations, as was apparent from major increases in the proportion of the populations with unselected mutations in fhuA. The increase in mutation rates was due to sweeps by mutY mutators. In both cultures, the enrichment of mutators resulted from hitchhiking with identified beneficial mutations increasing fitness under glucose limitation; mutY hitchhiked with mgl mutations in one culture and ptsG in the other. In both cases, mutators were enriched to constitute close to 100% of the population before a periodic selection event reduced the frequency of unselected mutations and mutators in the cultures. The high proportion of mutators persisted for 150 generations in one population but began to be eliminated within 50 generations in the other. The persistence of mutator, as well as experimental data showing that mutY bacteria were as fit as near-isogenic [mutY.sup.+] bacteria in competition experiments, suggest that mutator load by deleterious mutations did not explain the rapidly diminishing proportion of mutators in the populations. The nonmutators sweeping out mutators were also unlikely to have arisen by reversion or antimutator mutations; the mutY mutations were major deletions in each case and the bacteria sweeping out mutators contained intact mutY. By following mgl allele frequencies in one population, we discovered that mutators were outcompeted by bacteria that had rare mgl mutations previously as well as additional beneficial mutation (s). The pattern of appearance of mutY, but not its elimination, conforms to current models of mutator sweeps in bacterial populations. A mutator with a narrow mutational spectrum like mutY may be lost if the requirement for beneficial mutations is for changes other than GC [right arrow] TA transversions. Alternatively, epistatic interactions between mutator mutation and beneficial mutations need to be postulated to explain mutator elimination.

Research shows that the N-terminal domain, DT1, of the transcription activator MalT interacts with MalY, an enzyme with cystathionase activity, but not with MalK, the ATP-hydrolyzing subunit of the ABC transporter. Further, the binding site for Aes, an acetyl esterase, is also localized to the same terminus encompassing DT1 and DT2.

Research shows that wild-type bacteria are quickly displaced by rpoS mutants in both glucose- and nitrogen-limited populations due to competition for the core RNA polymerase. Data indicate that glucose limitation leads to induction of transporter genes in the rpoS mutant, conferring a selective advantage to bacteria in the absence of RpoS sigma factor.

Results show that mutY gene inactivation increases mutation rates of the gene under nutrient limitation, which lead to the most common occurrence of G:C-to-T:A transversions in Escherichia coli. MutY-dependent DNA repair is repressed by the mutY gene mutation.

Research has been conducted on the porin regulation. The role of the OmpR phosphorylation in porin regulation has been tested, and the evidence of the AcP, cAMP and RpoS in porin gene expression importance has been provided.

Research reveals that glucose-limited chemostat cultures of Escherichia coli exhibit substitutions in the glucose-specific enzyme II. Data indicate that the enzyme's conformation determining the sugar substrate accessibility and its transcriptional signal transduction are linked.

Results demonstrate that the metabolome properties of bacteria from high density cultures were significantly different from those of low-density cultures. Data further indicate that regulation of ompf porin transcription is not affected by the density of the cultures.

Mutational adaptations leading to improved glucose transport were followed with Escherichia coli K-12 growing in glucose-limited continuous cultures. When populations were oxygen limited as well as glucose limited, all bacteria within 280 generations contained mutations in a single codon of the ptsG gene. V12F and V12G replacements in the enzyme [IIBC.sup.Glc] component of the glucose phosphotransferase system were responsible for improved transport. In stark contrast, ptsG mutations were uncommon in fully aerobic glucose-limited cultures, in which polygenic mutations in mgl, mlc, and malT (regulating an alternate high-affinity Mgl/LamB uptake pathway) spread through the adapted population. Hence the same organism adapted to the same selection (glucose limitation) by different evolutionary pathways depending on a secondary environmental factor. The clonal diversity in the adapted populations was also significantly different. The PtsG V12F substitution under [O.sub.2] limitation contributed to a universal 'winner clone' whereas polygenic, multiallelic changes led to considerable polymorphism in aerobic cultures. Why the difference in adaptive outcomes? E. coli physiology prevented scavenging by the LamB/Mgl system under [O.sub.2] limitation; hence, ptsG mutations provided the only adaptive pathway. But ptsG mutations in aerobic cultures are overtaken by mgl, mlc, and malT adaptations with better glucose-scavenging ability. Indeed, when an mg/A::Tn 10 mutant with an inactivated Mgl/LamB pathway was introduced into two independent aerobic chemostats, adaptation of the [Mgl.sup.-] strain involved the identical ptsG mutation found under [O.sub.2]-limited conditions with wild-type or [Mgl.sup.-] bacteria.

Escherichia coli growing on glucose in minimal medium regulates its metabolite pools in response to environmental conditions. The extent of pool modifications was examined through two-dimensional thin-layer chromatography of all 14C-glucose labelled compounds extracted from bacteria. Findings suggested that total metabolite pool analysis provides a method for determining the unique aspects of cellular metabolism and global regulation.

Porin proteins regulate the permeability of polar solutes across the outer membrane of gram-negative bacteria such as Escherichia coli. The best known input regarding the regulation of porin levels involves EnvZ and OmpR which together regulate ompF and ompC gene expression. Chemostat cultures with glucose or nitrogen limitation were used at different growth rates to study the control of major porins ompF and ompC under nutrient limitation. Three investigative approaches to study the chemostat cultures found a consistent and complex pattern of regulation especially of ompF porin levels.

cAMP assay and measurement of expression of cAMP-regulated genes were conducted to determine the relationship between external glucose concentration and cAMP levels inside Escherichi coli. Results reveal that E. coli controls its cAMP levels through two or more transitions influenced by external glucose concentration and does not respond to decreasing saturation of glucose transport. Furthermore, results present implications for models of phosphotransferase-mediated regulation of adenylate cyclase.

The sucrase, encoded by the surA gene of Bacillus stearothermophilus, shows a sequence similarity with sucrose hydrolising enzymes of B. subtilis, Klebsiella pneumoniae and Vibrio alginolyticus. The enzyme's sucrose box, a sequence of nine amino acids, is important in the catalysis of sucrose into glucose and fructose. SurA shows a high degree of stability at room temperature, with an optimum temperature of 55 degree celsius for sucrose hydrolysis. The active form of the enzyme is a dimer and it is unable to operate during sucrose phosphate cleavage.

The expressions of the genes treA and osmY and the quantity of RpoS protein in a glucose-deficient continuous culture of Escherichia coli were studied. Findings indicated that E. coli utilizes a protective stationary-phase behavior when there is not enough glucose to saturate scavenger transporters controlled by cAMP plus endoinducers. The high-level induction of protective functions also elucidated the maintenance energy needed for bacterial growth at low dilution levels.

Endogenous inducer synthesis under conditions of reduced nutrient availability represents a natural adaptation to increase the ability of Escherichia coli for scavenging glucose. Exogenous induction exhibits no influence on bacterial growth with limiting carbohydrates. Endogenous galactose and cyclic AMP pools are enough for high expression of mgl and gal genes. A functional galETK operon is essential for limitation-induced induction of mgl. Mgl/gal induction exhibits a better correlation with inducer availability than with cAMP concentrations. Internal inducer synthesis is minimum during oligotrophic conditions.

LamB protein is the general glycoporin in the Escherichia coli outer membrane. Contrary to an earlier finding, the LamB- phenotype exhibited reduced competitive abilities when cultured together with LamB+ bacteria in conditions with carbohydrate concentration as the limiting factor. Further, LamB was demonstrated to have a more extensive sugar transportfunction than mere maltosaccharide uptake.

Combinatorial mutagenesis was applied to residues 40 through 60 of LamB protein (maltoporin) of Escherichia coli in order to identify amino acids important in the structureand function of LamB. In this strategy the mutagenic oligonucleotides were forced to replace a nonsense codon in the template to ensure that functional sequences result from the oligonucleotides. Residues 41 to 43 accepted few neutral substitutions, whereas residues 55 to 57 were highly flexible in accepting substitutions. The results suggest that residues 41 to 43 of LamB play an important role in the process of outer membrane localization.