DksA is a conserved master regulator of stress response in Acinetobacter baumannii.

Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner. Phylogenetically, DksA was highly conserved and well-distributed across Gammaproteobacteria, with 96.6% containing DksA, spanning 88 families. This study lays the groundwork for understanding DksA as a major regulator of general stress response and virulence in this important pathogen.

The Molecular Basis of Acinetobacter baumannii Cadmium Toxicity and Resistance.

Acinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, in soil, and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Metal ion homeostasis in the hospital pathogen Acinetobacter baumannii contributes to pathogenesis; however, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii. Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. We also show that the CzcCBA heavy metal efflux system contributes to cadmium efflux. Collectively, these systems provide A. baumannii with a comprehensive cadmium translocation pathway from the cytoplasm to the periplasm and subsequently the extracellular space. Furthermore, analysis of the A. baumannii metallome under cadmium stress showed zinc depletion, as well as copper enrichment, both of which are likely to influence cellular fitness. Overall, this work provides new knowledge on the role of a broad arsenal of membrane transporters in A. baumannii metal ion homeostasis. IMPORTANCE Cadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, including Acinetobacter baumannii, which is notorious for persisting in harsh environments. Here, we show that A. baumannii utilizes a dedicated cadmium efflux protein in concert with a system that is primarily attuned to zinc efflux to efficiently overcome cadmium stress. The molecular characterization of A. baumannii under cadmium stress revealed how active cadmium efflux plays a key role in preventing the dysregulation of bacterial metal ion homeostasis, which appeared to be a primary means by which cadmium exerts toxicity upon the bacterium.

Splicing factor proline and glutamine rich intron retention, reduced expression and aggregate formation are pathological features of amyotrophic lateral sclerosis.

AIM: Splicing factor proline and glutamine rich (SFPQ) is an RNA-DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case-control cohorts. METHODS: We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA-sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. RESULTS: At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ-positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. CONCLUSIONS: Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.

A shifting mutational landscape in 6 nutritional states: Stress-induced mutagenesis as a series of distinct stress input-mutation output relationships.

Environmental stresses increase genetic variation in bacteria, plants, and human cancer cells. The linkage between various environments and mutational outcomes has not been systematically investigated, however. Here, we established the influence of nutritional stresses commonly found in the biosphere (carbon, phosphate, nitrogen, oxygen, or iron limitation) on both the rate and spectrum of mutations in Escherichia coli. We found that each limitation was associated with a remarkably distinct mutational profile. Overall mutation rates were not always elevated, and nitrogen, iron, and oxygen limitation resulted in major spectral changes but no net increase in rate. Our results thus suggest that stress-induced mutagenesis is a diverse series of stress input-mutation output linkages that is distinct in every condition. Environment-specific spectra resulted in the differential emergence of traits needing particular mutations in these settings. Mutations requiring transpositions were highest under iron and oxygen limitation, whereas base-pair substitutions and indels were highest under phosphate limitation. The unexpected diversity of input-output effects explains some important phenomena in the mutational biases of evolving genomes. The prevalence of bacterial insertion sequence transpositions in the mammalian gut or in anaerobically stored cultures is due to environmentally determined mutation availability. Likewise, the much-discussed genomic bias towards transition base substitutions in evolving genomes can now be explained as an environment-specific output. Altogether, our conclusion is that environments influence genetic variation as well as selection.

Escherichia coli mutation rates and spectra with combinations of environmental limitations.

Micro-organisms often face multiple stresses in natural habitats. Individual stresses are well known to influence mutation rates and the spectra of mutational types, but the extent to which multiple stresses affect the genetic variation in populations is unknown. Here we investigate pair-wise combinations of nutritional stresses in Escherichia coli to determine their effect on mutation rates and mutational types. Environmental interactions modified both the rate and spectrum of mutations in double-limited environments, but the effects were not additive or synergistic relative to single stresses. Generally, bacteria in the mixed environments behaved as if one of the two single-stress stimuli was more dominant and the genetic variation seen with every dual limitation was intermediate between known patterns with individual stresses. The composition of mutational types with double stresses was also intermediate between individual stress patterns. At least with mutations, the single stressor results available are reasonable indicators of stress-induced genetic variation in multifaceted natural habitats. With the influence of 11 conditions available on mutational patterns, we can now also see the clustering of mutational types as a function of these environments.

Natural Escherichia coli isolates rapidly acquire genetic changes upon laboratory domestication.

The adaptation of environmental bacteria to laboratory conditions was analysed through the exploration of genomic changes in four strains of Escherichia coli freshly isolated from their natural habitats and belonging to different taxonomic clusters. Up to 25 mutations were present in all cultures of natural isolates within 10 days of transfer in rich media or with a single growth cycle involving an extended stationary phase. Among numerous individual mutations, two genes were affected in parallel in distinct backgrounds. Mutations in rpoS (encoding sigma factor RpoS), altering a multiplication-survival trade-off in E. coli, were present in isolates derived from all four different ancestors. More surprisingly, two different natural isolates acquired mutations in mutL, affecting DNA mismatch repair, and a third also involved higher mutation rates. The elevated mutation rates in these isolates indicate the danger of increased genetic instability arising from laboratory domestication. Neither rpoS nor mutator mutations were detected in the already-acclimatized MG1655 laboratory strain; only one or no new mutations were present in the laboratory strain under the same culture conditions. Our results indicate rapid adaptation to the laboratory environment. Ancestor-specific responses also arise in the laboratory and mutational events are also sensitive to culture conditions such as extended stationary phase. To maintain natural isolates in a stable state, our data suggest that the transition of strains to the laboratory should minimize culture cycles and extended stationary phase.

Mutational heterogeneity: a key ingredient of bet-hedging and evolutionary divergence?: The broad spectrum of mutations and their flexible frequency in populations provides a source of risk avoidance and alternative evolutionary strategies.

Here, we propose that the heterogeneity of mutational types in populations underpins alternative pathways of evolutionary adaptation. Point mutations, deletions, insertions, transpositions and duplications cause different biological effects and provide distinct adaptive possibilities. Experimental evidence for this notion comes from the mutational origins of adaptive radiations in large, clonal bacterial populations. Independent sympatric lineages with different phenotypes arise from distinct genetic events including gene duplication, different insertion sequence movements and several independent point mutations. The breadth of the mutational spectrum in the ancestral population should be viewed as a form of bet-hedging, reducing the risk of evolutionary dead ends and complementing the phenotypic and epigenetic heterogeneities that improve the survival capabilities of a population. Different mutational events arise from distinct cellular processes and are subject to separate environmental impacts, so the availability of any particular type of mutation may constrain or promote adaptive pathways in populations.

Mutational signatures indicative of environmental stress in bacteria.

Evolutionary innovations are dependent on mutations. Mutation rates are increased by adverse conditions in the laboratory, but there is no evidence that stressful environments that do not directly impact on DNA leave a mutational imprint on extant genomes. Mutational spectra in the laboratory are normally determined with unstressed cells but are unavailable with stressed bacteria. To by-pass problems with viability, selection effects, and growth rate differences due to stressful environments, in this study we used a set of genetically engineered strains to identify the mutational spectrum associated with nutritional stress. The strain set members each had a fixed level of the master regulator protein, RpoS, which controls the general stress response of Escherichia coli. By assessing mutations in cycA gene from 485 cycloserine resistant mutants collected from as many independent cultures with three distinct perceived stress (RpoS) levels, we were able establish a dose-dependent relationship between stress and mutational spectra. The altered mutational patterns included base pair substitutions, single base pair indels, longer indels, and transpositions of different insertion sequences. The mutational spectrum of low-RpoS cells closely matches the genome-wide spectrum previously generated in laboratory environments, while the spectra of high RpoS, high perceived stress cells more closely matches spectra found in comparisons of extant genomes. Our results offer an explanation of the uneven mutational profiles such as the transition-transversion biases observed in extant genomes and provide a framework for assessing the contribution of stress-induced mutagenesis to evolutionary transitions and the mutational emergence of antibiotic resistance and disease states.

The nature of laboratory domestication changes in freshly isolated Escherichia coli strains.

Adaptation of environmental bacteria to laboratory conditions can lead to modification of important traits, what we term domestication. Little is known about the rapidity and reproducibility of domestication changes, the uniformity of these changes within a species or how diverse these are in a single culture. Here, we analysed phenotypic changes in nutrient-rich liquid media or on agar of four Escherichia coli strains newly isolated through minimal steps from different sources. The laboratory-cultured populations showed changes in metabolism, morphotype, fitness and in some phenotypes associated with the sigma factor RpoS. Domestication events and phenotypic diversity started to emerge within 2-3 days in replicate subcultures of the same ancestor. In some strains, increased amino acid usage and higher fitness under nutrient limitation resembled those in mutants with the GASP (growth advantage in stationary phase) phenotype. The domestication changes are not uniform across a species or even within a single domesticated population. However, some parallelism in adaptation within repeat cultures was observed. Differences in the laboratory environment also determine domestication effects, which differ between liquid and solid media or with extended stationary phase. Important lessons for the handling and storage of organisms can be based on these studies.

A case of adaptation through a mutation in a tandem duplication during experimental evolution in Escherichia coli.

BACKGROUND: DNA duplications constitute important precursors for genome variation. Here we analyzed an unequal duplication harboring a beneficial mutation that may provide alternative evolutionary outcomes. RESULTS: We characterized this evolutionary event during experimental evolution for only 100 generations of an Escherichia coli strain under glucose limitation within chemostats. By combining Insertion Sequence based Restriction Length Polymorphism experiments, pulsed field gel electrophoresis and two independent genome re-sequencing experiments, we identified an evolved lineage carrying a 180 kb duplication of the 46' region of the E. coli chromosome. This evolved duplication revealed a heterozygous state, with one copy harboring a 2668 bp deletion that included part of the ogrK gene and both the yegR and yegS genes. By genetically manipulating ancestral and evolved strains, we showed that the single yegS inactivation was sufficient to confer a frequency dependent fitness increase under the chemostat selective conditions in both the ancestor and evolved genetic contexts, implying that the duplication itself was not a direct fitness contributor. Nonetheless, the heterozygous duplicated state was relatively stable in the conditions prevailing during evolution in chemostats, in striking contrast to non selective conditions in which the duplication resolved at high frequency into either its ancestral or deleted copy. CONCLUSIONS: Our results suggest that the duplication state may constitute a second order selection process providing higher evolutionary potential. Moreover, its heterozygous nature may provide differential evolutionary opportunities in alternating environments. Our results also highlighted how careful analyses of whole genome data are needed to identify such complex rearrangements.

The form of a trade-off determines the response to competition.

Understanding how populations and communities respond to competition is a central concern of ecology. A seminal theoretical solution first formalised by Levins (and re-derived in multiple fields) showed that, in theory, the form of a trade-off should determine the outcome of competition. While this has become a central postulate in ecology it has evaded experimental verification, not least because of substantial technical obstacles. We here solve the experimental problems by employing synthetic ecology. We engineer strains of Escherichia coli with fixed resource allocations enabling accurate measurement of trade-off shapes between bacterial survival and multiplication in multiple environments. A mathematical chemostat model predicts different, and experimentally verified, trajectories of gene frequency changes as a function of condition-specific trade-offs. The results support Levins' postulate and demonstrates that otherwise paradoxical alternative outcomes witnessed in subtly different conditions are predictable.

Mutation accumulation and fitness in mutator subpopulations of Escherichia coli.

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 mutY mutators 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.

Nutritional status and psychosocial stimulation associated with cognitive development in preschool children: A cross-sectional study at Western Terai, Nepal.

Quality education at the age of foundation to produce dynamic manpower is a public concern in developing countries including Nepal. Preschool children do not get proper care and support from their parents due to insufficient knowledge of proper feeding habits, nutrition status and methods of psychosocial stimulation, which may affect their proper cognitive development. This study aimed to identify the factors that influence cognitive development in preschool children aged 3-5 years in Rupandehi district of western Terai, Nepal. In this school based cross-sectional survey, a total of 401 preschool children were selected using a multistage random sampling technique. The study was conducted from 4th February to 12th April, 2021 in Rupandehi district of Nepal. Data on the children's socio-economic and demographic status, level of psychosocial stimulation, nutritional status, and stage of cognitive development were collected through scheduled interviews and direct observation. Stepwise regression analysis was performed to determine the predictors of cognitive development in preschool children. A p-value less than 0.05 considered as statistical significance. Of 401 participants, 44.1% had a normal nutritional status based on height for age Z-score (HAZ). Only 1.2% of primary caregivers provided their children with high levels of psychosocial stimulation, and 49.1% of children had a medium level of cognitive development. Furthermore, cognitive development in preschoolers is positively associated with nutritional status based on the height for age z score (ß = 0.280; p<0.0001), psychological stimulation from caregivers (ß = 0.184; p<0.0001), and advantageous castes/ethnicity (ß = 0.190; p<0.0001), but negatively associated with the child's age (ß = - 0.145; p = 0.002) and family type (ß = -0.157; p = 0.001). Nutritional status and psychosocial stimulation appear to be major factors affecting cognitive development of preschoolers. Nutritional promotion strategies, as well as techniques for optimal psychosocial stimulation behavior, may play an important role in enhancing preschoolers' cognitive development.

Cross-protection and cross-feeding between Klebsiella pneumoniae and Acinetobacter baumannii promotes their co-existence.

Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isolated from polymicrobial infections. The infections where these pathogens co-exist can be more severe and recalcitrant to therapy than infections caused by either species alone, however there is a lack of knowledge on their potential synergistic interactions. In this study we characterise the genomes of A. baumannii and K. pneumoniae strains co-isolated from a single human lung infection. We examine various aspects of their interactions through transcriptomic, phenomic and phenotypic assays that form a basis for understanding their effects on antimicrobial resistance and virulence during co-infection. Using co-culturing and analyses of secreted metabolites, we discover the ability of K. pneumoniae to cross-feed A. baumannii by-products of sugar fermentation. Minimum inhibitory concentration testing of mono- and co-cultures reveals the ability for A. baumannii to cross-protect K. pneumoniae against the cephalosporin, cefotaxime. Our study demonstrates distinct syntrophic interactions occur between A. baumannii and K. pneumoniae, helping to elucidate the basis for their co-existence in polymicrobial infections.

Insight into evolution of Bordetella pertussis from comparative genomic analysis: evidence of vaccine-driven selection.

Despite high vaccine coverage, pertussis incidence has increased substantially in recent years in many countries. A significant factor that may be contributing to this increase is adaptation to the vaccine by Bordetella pertussis, the causative agent of pertussis. In this study, we first assessed the genetic diversity of B. pertussis by microarray-based comparative genome sequencing of 10 isolates representing diverse genotypes and different years of isolation. We discovered 171 single nucleotide polymorphisms (SNPs) in a total of 1.4 Mb genome analyzed. The frequency of base changes was estimated as one per 32 kb per isolate, confirming that B. pertussis is one of the least variable bacterial pathogens. We then analyzed an international collection of 316 B. pertussis isolates using a subset of 65 of the SNPs and identified 42 distinct SNP profiles (SPs). Phylogenetic analysis grouped the SPs into six clusters. The majority of recent isolates belonged to clusters I-IV and were descendants of a single prevaccine lineage. Cluster I appeared to be a major clone with a worldwide distribution. Typing of genes encoding acellular vaccine (ACV) antigens, ptxA, prn, fhaB, fim2, and fim3 revealed the emergence and increasing incidence of non-ACV alleles occurring in clusters I and IV, which may have been driven by ACV immune selection. Our findings suggest that B. pertussis, despite its high population homogeneity, is evolving in response to vaccination pressure with recent expansion of clones carrying variants of genes encoding ACV antigens.

Breaching the Barrier: Genome-Wide Investigation into the Role of a Primary Amine in Promoting E. coli Outer-Membrane Passage and Growth Inhibition by Ampicillin.

Gram-negative bacteria are problematic for antibiotic development due to the low permeability of their cell envelopes. To rationally design new antibiotics capable of breaching this barrier, more information is required about the specific components of the cell envelope that prevent the passage of compounds with different physiochemical properties. Ampicillin and benzylpenicillin are ß-lactam antibiotics with identical chemical structures except for a clever synthetic addition of a primary amine group in ampicillin, which promotes its accumulation in Gram-negatives. Previous work showed that ampicillin is better able to pass through the outer membrane porin OmpF in Escherichia coli compared to benzylpenicillin. It is not known, however, how the primary amine may affect interaction with other cell envelope components. This study applied TraDIS to identify genes that affect E. coli fitness in the presence of equivalent subinhibitory concentrations of ampicillin and benzylpenicillin, with a focus on the cell envelope. Insertions that compromised the outer membrane, particularly the lipopolysaccharide layer, were found to decrease fitness under benzylpenicillin exposure, but had less effect on fitness under ampicillin treatment. These results align with expectations if benzylpenicillin is poorly able to pass through porins. Disruption of genes encoding the AcrAB-TolC efflux system were detrimental to survival under both antibiotics, but particularly ampicillin. Indeed, insertions in these genes and regulators of acrAB-tolC expression were differentially selected under ampicillin treatment to a greater extent than insertions in ompF. These results suggest that maintaining ampicillin efflux may be more significant to E. coli survival than full inhibition of OmpF-mediated uptake. IMPORTANCE Due to the growing antibiotic resistance crisis, there is a critical need to develop new antibiotics, particularly compounds capable of targeting high-priority antibiotic-resistant Gram-negative pathogens. In order to develop new compounds capable of overcoming resistance a greater understanding of how Gram-negative bacteria are able to prevent the uptake and accumulation of many antibiotics is required. This study used a novel genome wide approach to investigate the significance of a primary amine group as a chemical feature that promotes the uptake and accumulation of compounds in the Gram-negative model organism Escherichia coli. The results support previous biochemical observations that the primary amine promotes passage through the outer membrane porin OmpF, but also highlight active efflux as a major resistance factor.

Genomic and phenotypic analyses of diverse non-clinical Acinetobacter baumannii strains reveals strain-specific virulence and resistance capacity.

Acinetobacter baumannii is a critically important pathogen known for its widespread antibiotic resistance and ability to persist in hospital-associated environments. Whilst the majority of A. baumannii infections are hospital-acquired, infections from outside the hospital have been reported with high mortality. Despite this, little is known about the natural environmental reservoir(s) of A. baumannii and the virulence potential underlying non-clinical strains. Here, we report the complete genome sequences of six diverse strains isolated from environments such as river, soil, and industrial sites around the world. Phylogenetic analyses showed that four of these strains were unrelated to representative nosocomial strains and do not share a monophyletic origin, whereas two had sequence types belonging to the global clone lineages GC1 and GC2. Further, the majority of these strains harboured genes linked to virulence and stress protection in nosocomial strains. These genotypic properties correlated well with in vitro virulence phenotypic assays testing resistance to abiotic stresses, serum survival, and capsule formation. Virulence potential was confirmed in vivo, with most environmental strains able to effectively kill Galleria mellonella greater wax moth larvae. Using phenomic arrays and antibiotic resistance profiling, environmental and nosocomial strains were shown to have similar substrate utilisation patterns although environmental strains were distinctly more sensitive to antibiotics. Taken together, these features of environmental A. baumannii strains suggest the existence of a strain-specific distinct gene pools for niche specific adaptation. Furthermore, environmental strains appear to be equally virulent as contemporary nosocomial strains but remain largely antibiotic sensitive.

Insertion sequence-driven evolution of Escherichia coli in chemostats.

Insertion sequence (IS) elements are present in almost all bacterial genomes and are mobile enough to provide genomic tools to differentiate closely related isolates. They can be used to estimate genetic diversity and identify fitness-enhancing mutations during evolution experiments. Here, we determined the genomic distribution of eight IS elements in 120 genomes sampled from Escherichia coli populations that evolved in glucose- and phosphate-limited chemostats by comparison to the ancestral pattern. No significant differential transposition of the various IS types was detected across the environments. The phylogenies revealed substantial diversity amongst clones sampled from each chemostat, consistent with the phenotypic diversity within populations. Two IS-related changes were common to independent chemostats, suggesting parallel evolution. One of them corresponded to insertions of IS1 elements within rpoS encoding the master regulator of stress conditions. The other parallel event was an IS5-dependent deletion including mutY involved in DNA repair, thereby providing the molecular mechanism of generation of mutator clones in these evolving populations. These deletions occurred in different co-existing genotypes within single populations and were of various sizes. Moreover, differential locations of IS elements combined with their transpositional activity provided evolved clones with different phenotypic landscapes. Therefore, IS elements strongly influenced the evolutionary processes in continuous E. coli cultures by providing a way to modify both the global regulatory network and the mutation rates of evolving cells.

The uncertain consequences of transferring bacterial strains between laboratories - rpoS instability as an example.

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.

Genomic identification of a novel mutation in hfq that provides multiple benefits in evolving glucose-limited populations of Escherichia coli.

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.