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.

Bacteria of the Burkholderia cepacia complex are cyanogenic under biofilm and colonial growth conditions.

BACKGROUND: The Burkholderia cepacia complex (Bcc) is a collection of nine genotypically distinct but phenotypically similar species. They show wide ecological diversity and include species that are used for promoting plant growth and bio-control as well species that are opportunistic pathogens of vulnerable patients. Over recent years the Bcc have emerged as problematic pathogens of the CF lung. Pseudomonas aeruginosa is another important CF pathogen. It is able to synthesise hydrogen cyanide (HCN), a potent inhibitor of cellular respiration. We have recently shown that HCN production by P. aeruginosa may have a role in CF pathogenesis. This paper describes an investigation of the ability of bacteria of the Bcc to make HCN. RESULTS: The genome of Burkholderia cenocepacia has 3 putative HCN synthase encoding (hcnABC) gene clusters. B. cenocepacia and all 9 species of the Bcc complex tested were able to make cyanide at comparable levels to P. aeruginosa, but only when grown surface attached as colonies or during biofilm growth on glass beads. In contrast to P. aeruginosa and other cyanogenic bacteria, cyanide was not detected during planktonic growth of Bcc strains. CONCLUSION: All species in the Bcc are cyanogenic when grown as surface attached colonies or as biofilms.

Oxygen, cyanide and energy generation in the cystic fibrosis pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram-negative, rod-shaped bacterium that belongs to the gamma-proteobacteria. This clinically challenging, opportunistic pathogen occupies a wide range of niches from an almost ubiquitous environmental presence to causing infections in a wide range of animals and plants. P. aeruginosa is the single most important pathogen of the cystic fibrosis (CF) lung. It causes serious chronic infections following its colonisation of the dehydrated mucus of the CF lung, leading to it being the most important cause of morbidity and mortality in CF sufferers. The recent finding that steep O2 gradients exist across the mucus of the CF-lung indicates that P. aeruginosa will have to show metabolic adaptability to modify its energy metabolism as it moves from a high O2 to low O2 and on to anaerobic environments within the CF lung. Therefore, the starting point of this review is that an understanding of the diverse modes of energy metabolism available to P. aeruginosa and their regulation is important to understanding both its fundamental physiology and the factors significant in its pathogenicity. The main aim of this review is to appraise the current state of knowledge of the energy generating pathways of P. aeruginosa. We first look at the organisation of the aerobic respiratory chains of P. aeruginosa, focusing on the multiple primary dehydrogenases and terminal oxidases that make up the highly branched pathways. Next, we will discuss the denitrification pathways used during anaerobic respiration as well as considering the ability of P. aeruginosa to carry out aerobic denitrification. Attention is then directed to the limited fermentative capacity of P. aeruginosa with discussion of the arginine deiminase pathway and the role of pyruvate fermentation. In the final part of the review, we consider other aspects of the biology of P. aeruginosa that are linked to energy metabolism or affected by oxygen availability. These include cyanide synthesis, which is oxygen-regulated and can affect the operation of aerobic respiratory pathways, and alginate production leading to a mucoid phenotype, which is regulated by oxygen and energy availability, as well as having a role in the protection of P. aeruginosa against reactive oxygen species. Finally, we consider a possible link between cyanide synthesis and the mucoid switch that operates in P. aeruginosa during chronic CF lung infection.

Cyanide measurements in bacterial culture and sputum.

Cyanide is produced by a few bacterial species, including Pseudomonas aeruginosa, and it has a role in the opportunistic infections of this bacterium including in cystic fibrosis lung infections. We describe two methods for determining cyanide in culture and patient sputum samples. One uses an ion-selective electrode to provide a convenient, rapid method of cyanide quantitation in culture or sputum, and the second is a semiquantitative method using Feigl-Anger paper that is useful for screening large numbers of bacterial strains for cyanide production.

A metabolic trade-off between phosphate and glucose utilization in Escherichia coli.

Getting the most out of available nutrients is a key challenge that all organisms face. Little is known about how they optimize and balance the simultaneous utilization of multiple elemental resources. We investigated the effects of long-term phosphate limitation on carbon metabolism of the model organism Escherichia coli using chemostat cultures. We profiled metabolic changes in the growth medium over time and found evidence for an increase in fermentative metabolism despite the aerobic conditions. Using full-genome sequencing and competition experiments, we found that fitness under phosphate-limiting conditions was reproducibly increased by a mutation preventing flux through succinate in the tricarboxylic acid cycle. In contrast, these mutations reduced competitive ability under carbon limitation, and thus reveal a conflicting metabolic benefit in the role of the TCA cycle in environments limited by inorganic phosphate and glucose.

The mucoid switch in Pseudomonas aeruginosa represses quorum sensing systems and leads to complex changes to stationary phase virulence factor regulation.

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the airways of Cystic Fibrosis (CF) patients during which it adapts and undergoes clonal expansion within the lung. It commonly acquires inactivating mutations of the anti-sigma factor MucA leading to a mucoid phenotype, caused by excessive production of the extracellular polysaccharide alginate that is associated with a decline in lung function. Alginate production is believed to be the key benefit of mucA mutations to the bacterium in the CF lung. A phenotypic and gene expression characterisation of the stationary phase physiology of mucA22 mutants demonstrated complex and subtle changes in virulence factor production, including cyanide and pyocyanin, that results in their down-regulation upon entry into stationary phase but, (and in contrast to wildtype strains) continued production in prolonged stationary phase. These findings may have consequences for chronic infection if mucoid P. aeruginosa were to continue to make virulence factors under non-growing conditions during infection. These changes resulted in part from a severe down-regulation of both AHL-and AQ (PQS)-dependent quorum sensing systems. In trans expression of the cAMP-dependent transcription factor Vfr restored both quorum sensing defects and virulence factor production in early stationary phase. Our findings have implications for understanding the evolution of P. aeruginosa during CF lung infection and it demonstrates that mucA22 mutation provides a second mechanism, in addition to the commonly occurring lasR mutations, of down-regulating quorum sensing during chronic infection this may provide a selection pressure for the mucoid switch in the CF lung.

Culture history and population heterogeneity as determinants of bacterial adaptation: the adaptomics of a single environmental transition.

Diversity in adaptive responses is common within species and populations, especially when the heterogeneity of the frequently large populations found in environments is considered. By focusing on events in a single clonal population undergoing a single transition, we discuss how environmental cues and changes in growth rate initiate a multiplicity of adaptive pathways. Adaptation is a comprehensive process, and stochastic, regulatory, epigenetic, and mutational changes can contribute to fitness and overlap in timing and frequency. We identify culture history as a major determinant of both regulatory adaptations and microevolutionary change. Population history before a transition determines heterogeneities due to errors in translation, stochastic differences in regulation, the presence of aged, damaged, cheating, or dormant cells, and variations in intracellular metabolite or regulator concentrations. It matters whether bacteria come from dense, slow-growing, stressed, or structured states. Genotypic adaptations are history dependent due to variations in mutation supply, contingency gene changes, phase variation, lateral gene transfer, and genome amplifications. Phenotypic adaptations underpin genotypic changes in situations such as stress-induced mutagenesis or prophage induction or in biofilms to give a continuum of adaptive possibilities. Evolutionary selection additionally provides diverse adaptive outcomes in a single transition and generally does not result in single fitter types. The totality of heterogeneities in an adapting population increases the chance that at least some individuals meet immediate or future challenges. However, heterogeneity complicates the adaptomics of single transitions, and we propose that subpopulations will need to be integrated into future population biology and systems biology predictions of bacterial behavior.

The identification of global patterns and unique signatures of proteins across 14 environments using outer membrane proteomics of bacteria.

We test the hypothesis that organisms sourced from different environments exhibit unique fingerprints in macromolecular composition. Experimentally, we followed proteomic changes with 14 different sub-lethal environmental stimuli in Escherichia coli at controlled growth rates. The focus was on the outer membrane sub-proteome, which is known to be extremely sensitive to environmental controls. The analyses surprisingly revealed that pairs of proteins belonging to very different regulons, such as Slp and OmpX or FadL and OmpF, have the closest patterns of change with the 14 conditions. Fe-limited and cold-cultured bacteria have the most distinct global patterns of spot changes, but the patterns with fast growth and oxygen limitation are the closest amongst the 14 environments. These unexpected but statistically robust results suggest that we have an incomplete picture of bacterial regulation across different stress responses; baseline choices and growth-rate influences are probably underestimated factors in such systems-level analysis. In terms of our aim of getting a unique profile for each of the 14 investigated environments, we find that it is unnecessary to compare all the proteins in a proteome and that a panel of five proteins is sufficient for identification of environmental fingerprints. This demonstrates the future feasibility of tracing the history of contaminating bacteria in hospitals, foods or industrial settings as well as for released organisms and biosecurity purposes.

Metabolic profiling of Pseudomonas aeruginosa demonstrates that the anti-sigma factor MucA modulates osmotic stress tolerance.

Metabolic footprinting has shown enormous potential as a phenotyping tool and we are interested in applying it to understand the physiology of the opportunistic pathogen Pseudomonas aeruginosa during its chronic infection of the lungs of cystic fibrosis patients. The selection pressures of surviving in the CF lung environment lead to genetic adaptations of the bacterium. A common adaptation is mutation of the mucA gene, resulting in a loss-of-function mutation to the anti-sigma factor MucA, which leads to a mucoid phenotype as a consequence of the overproduction of the extracellular polysaccharide alginate. However, apart from the mucoid phenotype little is known about the overall metabolic and physiological changes caused by mucA mutation. We investigated the pleiotropic metabolic effects of this mutation using time-resolved metabolic footprinting (extracellular metabolomics), and found changes in the levels of various metabolites associated with osmotic tolerance, including glycine-betaine, trehalose and glutamate. Physiological experiments confirmed that the isogenic mucA22 mutant is less resistant to osmotic stress than the parental PA01 wild-type strain, but only in the stationary phase of growth. Quantitative comparison of the endometabolome of the cells showed differences in the accumulation of osmoprotective metabolites by the wild-type and mucA22 mutant strains, suggesting a switch in osmo-protectant preference from glycine-betaine to trehalose.

Hypertonic Saline Therapy in Cystic Fibrosis: Do Population Shifts Caused by the Osmotic Sensitivity of Infecting Bacteria Explain the Effectiveness of this Treatment?

Cystic fibrosis (CF) is caused by a defect in the CF transmembrane regulator that leads to depletion and dehydration of the airway surface liquid (ASL) of the lung epithelium, providing an environment that can be infected by bacteria leading to increased morbidity and mortality. Pseudomonas aeruginosa chronically infects more than 80% of CF patients and one hallmark of infection is the emergence of a mucoid phenotype associated with a worsening prognosis and more rapid decline in lung function. Hypertonic saline (HS) is a clinically proven treatment that improves mucociliary clearance through partial rehydration of the ASL of the lung. Strikingly, while HS therapy does not alter the prevalence of P. aeruginosa in the CF lung it does decrease the frequency of episodes of acute, severe illness known as infective exacerbations among CF patients. In this article, we propose a hypothesis whereby the positive clinical effects of HS treatment are explained by the osmotic sensitivity of the mucoid sub-population of P. aeruginosa in the CF lung leading to selection against this group in favor of the osmotically resistant non-mucoid variants.