Decoupling growth and production by removing the origin of replication from a bacterial chromosome

AbstractEfficient production of biochemicals and proteins in cell factories frequently benefits from a two-stage bioprocess in which growth and production phases are decoupled. Here we describe a novel growth switch based on the permanent removal of the origin of replication (oriC) from the Escherichia coli chromosome. Without oriC, cells cannot initiate a new round of replication and they stop growing while their metabolism remains active. Our system relies on a serine recombinase from bacteriophage phiC31 whose expression is controlled by the temperature-sensitive cI857 repressor from phage lambda. Reporter protein expression in switched cells continues after cessation of growth, leading to protein levels up to five times higher compared to non-switching cells. Switching induces a unique physiological state that is different from both normal exponential and stationary phases. Switched cells remain in this state even when not growing, retain their protein synthesis capacity, and do not induce proteins associated with the stationary phase. Our switcher technology is potentially useful for a range of products and applicable in many bacterial species for decoupling growth and production.

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AnEscherichia colinitrogen starvation response is important for mutualistic coexistence withRhodopseudomonas palustris

10.1101/262139 ◽

2018 ◽

Author(s):

Alexandra L. McCully ◽

Megan G. Behringer ◽

Jennifer R. Gliessman ◽

Evgeny V. Pilipenko ◽

Jeffrey L. Mazny ◽

...

Keyword(s):

Escherichia Coli ◽

Nitrogen Starvation ◽

Physiological State ◽

Rhodopseudomonas Palustris ◽

Genetically Engineered ◽

Individual Species ◽

Starvation Response ◽

E Coli ◽

Protein Levels ◽

Stable Coexistence

AbstractMicrobial mutualistic cross-feeding interactions are ubiquitous and can drive important community functions. Engaging in cross-feeding undoubtedly affects the physiology and metabolism of individual species involved. However, the nature in which an individual’s physiology is influenced by cross-feeding and the importance of those physiological changes for the mutualism have received little attention. We previously developed a genetically tractable coculture to study bacterial mutualisms. The coculture consists of fermentativeEscherichia coliand phototrophicRhodopseudomonas palustris. In this coculture, E. coli anaerobically ferments sugars into excreted organic acids as a carbon source for R. palustris. In return, a genetically-engineered R. palustris constitutively converts N2into NH4+, providingE. coliwith essential nitrogen. Using RNA-seq and proteomics, we identified transcript and protein levels that differ in each partner when grown in coculture versus monoculture. When in coculture withR. palustris, E. coligene-expression changes resembled a nitrogen starvation response under the control of the transcriptional regulator NtrC. By genetically disruptingE. coliNtrC, we determined that a nitrogen starvation response is important for a stable coexistence, especially at lowR. palustrisNH4+excretion levels. Destabilization of the nitrogen starvation regulatory network resulted in variable growth trends and in some cases, extinction. Our results highlight that alternative physiological states can be important for survival within cooperative cross-feeding relationships.ImportanceMutualistic cross-feeding between microbes within multispecies communities is widespread. Studying how mutualistic interactions influence the physiology of each species involved is important for understanding how mutualisms function and persist in both natural and applied settings. Using a bacterial mutualism consisting ofRhodopseudomonas palustrisandEscherichia coligrowing cooperatively through bidirectional nutrient exchange, we determined that anE. colinitrogen starvation response is important for maintaining a stable coexistence. The lack of anE. colinitrogen starvation response ultimately destabilized the mutualism and, in some cases, led to community collapse after serial transfers. Our findings thus inform on the potential necessity of an alternative physiological state for mutualistic coexistence with another species compared to the physiology of species grown in isolation.

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Induction of Escherichia coli and Salmonella typhimurium into the viable but nonculturable state following chlorination of wastewater

Journal of Water and Health ◽

10.2166/wh.2005.040 ◽

2005 ◽

Vol 3 (3) ◽

pp. 249-257 ◽

Cited By ~ 81

Author(s):

James D. Oliver ◽

Maya Dagher ◽

Karl Linden

Keyword(s):

Escherichia Coli ◽

Salmonella Typhimurium ◽

Stationary Phases ◽

Health Hazard ◽

Physiological State ◽

Treated Wastewater ◽

Vbnc State ◽

Viable But Nonculturable ◽

Nonculturable Cells ◽

Nonculturable State

We examined the effects of chlorine disinfection on Escherichia coli and Salmonella typhimurium in secondary-treated wastewater to determine whether such treatment might induce these bacteria into the viable but nonculturable (VBNC) state. In this state, cells lose culturability but retain viability and the potential to revert to the metabolically active and infectious state. To examine the effects of chlorination on cells in different physiological states, cells from the logarithmic and stationary phases, or nutrient starved, or grown in natural wastewater, were studied. Isogenic cells with and without plasmids were also examined. Whereas a mixture of free and combined chlorine, as occurs under typical wastewater disinfection, was found to be rapidly lethal to most cells, regardless of their physiological state or plasmid content, c. 104 of the original 106 cells ml−1 did survive in the VBNC state. While we were not successful in resuscitating these cells to the culturable state, the presence of such nonculturable cells in treated wastewater offers a potential public health hazard.

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Non-Caloric Artificial Sweeteners Modulate the Expression of Key Metabolic Genes in the Omnipresent Gut Microbe Escherichia coli

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000504511 ◽

2019 ◽

Vol 29 (1-6) ◽

pp. 43-56 ◽

Cited By ~ 1

Author(s):

Rizwan Mahmud ◽

Saadlee Shehreen ◽

Shayan Shahriar ◽

Md Siddiqur Rahman ◽

Sharif Akhteruzzaman ◽

...

Keyword(s):

Escherichia Coli ◽

Metabolic Pathways ◽

Bacterial Species ◽

Physiological State ◽

Well Being ◽

Artificial Sweeteners ◽

Diabetic Patients ◽

Dietary Intakes ◽

Metabolic Genes ◽

Gut Microbe

The human gut is inhabited by several hundred different bacterial species. These bacteria are closely associated with our health and well-being. The composition of these diverse commensals is influenced by our dietary intakes. Non-caloric artificial sweeteners (NAS) have gained global popularity, particularly among diabetic patients, due to their perceived health benefits, such as reduction of body weight and maintenance of blood glucose level compared to caloric sugars. Recent studies have reported that these artificial sweeteners can alter the composition of gut microbiota and, thus, affect our normal physiological state. Here, we investigated the effect of aspartame and acesulfame potassium (ace-K), two popular NAS, in a commercial formulation on the growth and metabolic pathways of omnipresent gut commensal Escherichia coliby analyzing the relative expression levels of the key genes, which control over twenty important metabolic pathways. Treatment with NAS preparation (aspartame and ace-K) modulates the growth of E. colias well as inducing the expression of important metabolic genes associated with glucose (pfkA, sucA, aceE, pfkB, lpdA), nucleotide (tmk, adk, tdk, thyA), and fatty acid (fabI) metabolisms, among others. Several of the affected geneswere previously reported to be important for the colonization of the microbes in the gut. These findings may shed light on the mechanism of alteration of gut microbes and their metabolism by NAS.

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The symmetrical pattern of base-pair substitutions rates across the chromosome in Escherichia coli has multiple causes

10.1101/523696 ◽

2019 ◽

Cited By ~ 1

Author(s):

Brittany A. Niccum ◽

Heewook Lee ◽

Wazim MohammedIsmail ◽

Haixu Tang ◽

Patricia L. Foster

Keyword(s):

Escherichia Coli ◽

Base Pair ◽

Bacterial Species ◽

Replication Timing ◽

Mutation Rates ◽

Whole Genome ◽

Origin Of Replication ◽

Symmetrical Pattern ◽

Major Factors ◽

Eukaryotic Genomes

AbstractMutation accumulation experiments followed by whole-genome sequencing have revealed that for several bacterial species the rate of base-pair substitutions is not constant across the chromosome but varies in a wave-like pattern symmetrical about the origin of replication. The experiments reported here demonstrate that in Escherichia coli several interacting factors determine the wave. Perturbing replication timing, progression, or the structure of the terminus disrupts the pattern. Biases in error-correction by proofreading and mismatch repair are major factors. The activities of the nucleoid binding proteins, HU and Fis, are important, suggesting that mutation rates increase when highly structured DNA is replicated. These factors should apply to most bacterial, and possibly eukaryotic, genomes, and imply that different areas of the genome evolve at different rates.

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Assessment of Production Conditions for Efficient Use of Escherichia coli in High-Yield Heterologous Recombinant Selenoprotein Synthesis

Applied and Environmental Microbiology ◽

10.1128/aem.70.9.5159-5167.2004 ◽

2004 ◽

Vol 70 (9) ◽

pp. 5159-5167 ◽

Cited By ~ 62

Author(s):

Olle Rengby ◽

Linda Johansson ◽

Lars A. Carlson ◽

Elena Serini ◽

Alexios Vlamis-Gardikas ◽

...

Keyword(s):

Escherichia Coli ◽

Elongation Factor ◽

Thioredoxin Reductase ◽

Exponential Phase ◽

Vector System ◽

Efficient Production ◽

Late Exponential Phase ◽

E Coli ◽

Cell Factories ◽

Production Conditions

ABSTRACT The production of heterologous selenoproteins in Escherichia coli necessitates the design of a secondary structure in the mRNA forming a selenocysteine insertion sequence (SECIS) element compatible with SelB, the elongation factor for selenocysteine insertion at a predefined UGA codon. SelB competes with release factor 2 (RF2) catalyzing translational termination at UGA. Stoichiometry between mRNA, the SelB elongation factor, and RF2 is thereby important, whereas other expression conditions affecting the yield of recombinant selenoproteins have been poorly assessed. Here we expressed the rat selenoprotein thioredoxin reductase, with titrated levels of the selenoprotein mRNA under diverse growth conditions, with or without cotransformation of the accessory bacterial selA, selB, and selC genes. Titration of the selenoprotein mRNA with a pBAD promoter was performed in both TOP10 and BW27783 cells, which unexpectedly could not improve yield or specific activity compared to that achieved in our prior studies. Guided by principal component analysis, we instead discovered that the most efficient bacterial selenoprotein production conditions were obtained with the high-transcription T7lac-driven pET vector system in presence of the selA, selB, and selC genes, with induction of production at late exponential phase. About 40 mg of rat thioredoxin reductase with 50% selenocysteine content could thereby be produced per liter bacterial culture. These findings clearly illustrate the ability of E. coli to upregulate the selenocysteine incorporation machinery on demand and that this is furthermore strongly augmented in late exponential phase. This study also demonstrates that E. coli can indeed be utilized as cell factories for highly efficient production of heterologous selenoproteins such as rat thioredoxin reductase.

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