Underground isoleucine biosynthesis pathways in E. coli

The promiscuous activities of enzymes provide fertile ground for the evolution of new metabolic pathways. Here, we systematically explore the ability of E. coli to harness underground metabolism to compensate for the deletion of an essential biosynthetic pathway. By deleting all threonine deaminases, we generated a strain in which isoleucine biosynthesis was interrupted at the level of 2-ketobutyrate. Incubation of this strain under aerobic conditions resulted in the emergence of a novel 2-ketobutyrate biosynthesis pathway based upon the promiscuous cleavage of O-succinyl-L-homoserine by cystathionine γ-synthase (MetB). Under anaerobic conditions, pyruvate formate-lyase enabled 2-ketobutyrate biosynthesis from propionyl-CoA and formate. Surprisingly, we found this anaerobic route to provide a substantial fraction of isoleucine in a wild-type strain when propionate is available in the medium. This study demonstrates the selective advantage underground metabolism offers, providing metabolic redundancy and flexibility which allow for the best use of environmental carbon sources.

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Adherence to abiotic surface induces SOS response in Escherichia coli K-12 strains under aerobic and anaerobic conditions

Microbiology ◽

10.1099/mic.0.075317-0 ◽

2014 ◽

Vol 160 (9) ◽

pp. 1964-1973 ◽

Cited By ~ 10

Author(s):

Suelen B. Costa ◽

Ana Carolina C. Campos ◽

Ana Claudia M. Pereira ◽

Ana Luiza de Mattos-Guaraldi ◽

Raphael Hirata Júnior ◽

...

Keyword(s):

Biofilm Formation ◽

Wild Type Strain ◽

Sos Response ◽

Wild Type ◽

Abiotic Surface ◽

Anaerobic Conditions ◽

E Coli ◽

Endonuclease V ◽

Sos Induction ◽

K 12

During the colonization of surfaces, Escherichia coli bacteria often encounter DNA-damaging agents and these agents can induce several defence mechanisms. Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species (ROS) generated by chemical and physical agents or by metabolism. In this work, we have evaluated whether the interaction with an abiotic surface by mutants derived from E. coli K-12 deficient in some enzymes that are part of BER causes DNA damage and associated filamentation. Moreover, we studied the role of endonuclease V (nfi gene; 1506 mutant strain) in biofilm formation. Endonuclease V is an enzyme that is involved in DNA repair of nitrosative lesions. We verified that endonuclease V is involved in biofilm formation. Our results showed more filamentation in the xthA mutant (BW9091) and triple xthA nfo nth mutant (BW535) than in the wild-type strain (AB1157). By contrast, the mutant nfi did not present filamentation in biofilm, although its wild-type strain (1466) showed rare filaments in biofilm. The filamentation of bacterial cells attaching to a surface was a consequence of SOS induction measured by the SOS chromotest. However, biofilm formation depended on the ability of the bacteria to induce the SOS response since the mutant lexA Ind− did not induce the SOS response and did not form any biofilm. Oxygen tension was an important factor for the interaction of the BER mutants, since these mutants exhibited decreased quantitative adherence under anaerobic conditions. However, our results showed that the presence or absence of oxygen did not affect the viability of BW9091 and BW535 strains. The nfi mutant and its wild-type did not exhibit decreased biofilm formation under anaerobic conditions. Scanning electron microscopy was also performed on the E. coli K-12 strains that had adhered to the glass, and we observed the presence of a structure similar to an extracellular matrix that depended on the oxygen tension. In conclusion, it was proven that bacterial interaction with abiotic surfaces can lead to SOS induction and associated filamentation. Moreover, we verified that endonuclease V is involved in biofilm formation.

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CexE Is A Virulence Factor ofCitrobacter rodentiumAnd Present In Enteric Pathogens Of Humans

10.1101/375527 ◽

2018 ◽

Author(s):

Zachary Rivas ◽

Ryan M. McCormack ◽

Becky Adkins ◽

George P. Munson

Keyword(s):

Virulence Factor ◽

Wild Type Strain ◽

Selective Advantage ◽

Enteric Pathogens ◽

Wild Type ◽

E Coli ◽

Adult Mice ◽

Providencia Alcalifaciens

AbstractCexE is a 12 kDa protein that was originally reported to be present in just three strains of enterotoxigenicEscherichia coli(ETEC); a frequent causes of diarrheal illnesses worldwide. However, an examination of recently sequenced genomes has revealed that CexE is actually present in a majority of ETEC strains. Homologs of CexE are also present in enteroaggregativeE. coli(EAEC) and other enteric pathogens includingYersinia enterocolitica, Providencia alcalifaciens, andCitrobacter rodentium. CexE and its homologs are expressed within virulence regulons of ETEC, EAEC, andC. rodentium. This, along with its distribution across several species of enteric pathogens, suggest that CexE confers a selective advantage to these pathogens. However, this hypothesis has yet to be testedin vivo. Here we demonstrate that CexE is conditionally secreted to the external leaf of the outer membrane of ETEC. Although CexE does not appear to play a role in adherencein vitro, it does facilitate colonization of murine intestinal tissues byC. rodentium in vivo. In adult mice wild-type bacteria reached significantly higher loads and were shed in higher numbers than acexE::kanmutant. A similar trend was observed in neonatal mice. In addition, all of the neonates infected with the wild-type strain succumbed to infection within 16 days of inoculation. In contrast, 45% of the neonates infected with thecexE::kanstrain survived for the 30 day duration of the experiment. These finding indicate that CexE is a conditionally secreted virulence factor that increases the colonization of hosts by enteric pathogens.

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Resource reallocation in engineered Escherichia coli strains with reduced genomes

10.1101/2020.10.19.346155 ◽

2020 ◽

Author(s):

Ernesto S. Nakayasu ◽

Adam M. Chazin-Gray ◽

Ryan M. Francis ◽

Arielle M. Eaton ◽

Deanna L. Auberry ◽

...

Keyword(s):

Amino Acid ◽

Biosynthetic Pathway ◽

Wild Type Strain ◽

Acid Metabolism ◽

Evolutionary Stability ◽

Proteomic Profiling ◽

Wild Type ◽

Resource Reallocation ◽

E Coli ◽

Optimal Balance

AbstractA major challenge in synthetic biology is properly balancing evolved and engineered functions without compromising microbial fitness. Many microbial proteins are not required for growth in regular laboratory conditions, but it is unclear what fraction of the proteome can be eliminated to increase bioproduction and maintain fitness. Here, we investigated the effects of massive genome reduction in E. coli on the expression level and evolutionary stability of a model biosynthetic pathway to produce the pigment protodeoxyviolacein (PDV). We identified an amino acid metabolism imbalance and compromised growth that were correlated with elimination of genes associated with significant proteome fraction. Proteomic profiling suggested that increased amino acid pools are responsible for an alleviation of fitness defects associated with PDV expression. In addition, all strains with genome reductions that significantly affected the proteome exhibited decreased stability of PDV production compared to the wild-type strain under persistent PDV expression conditions despite the alleviation of fitness defects. These findings exhibit the importance of balancing evolved functions with engineered ones to achieve an optimal balance of fitness and bioproduction.

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Adaptive Laboratory Evolution of Cupriavidus necator H16 for Carbon Co-Utilization with Glycerol

International Journal of Molecular Sciences ◽

10.3390/ijms20225737 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5737 ◽

Cited By ~ 2

Author(s):

Miriam González-Villanueva ◽

Hemanshi Galaiya ◽

Paul Staniland ◽

Jessica Staniland ◽

Ian Savill ◽

...

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Carbon Sources ◽

Strain Engineering ◽

Cupriavidus Necator ◽

Superior Performance ◽

Wild Type ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution ◽

Cupriavidus Necator H16

Cupriavidus necator H16 is a non-pathogenic Gram-negative betaproteobacterium that can utilize a broad range of renewable heterotrophic resources to produce chemicals ranging from polyhydroxybutyrate (biopolymer) to alcohols, alkanes, and alkenes. However, C. necator H16 utilizes carbon sources to different efficiency, for example its growth in glycerol is 11.4 times slower than a favorable substrate like gluconate. This work used adaptive laboratory evolution to enhance the glycerol assimilation in C. necator H16 and identified a variant (v6C6) that can co-utilize gluconate and glycerol. The v6C6 variant has a specific growth rate in glycerol 9.5 times faster than the wild-type strain and grows faster in mixed gluconate–glycerol carbon sources compared to gluconate alone. It also accumulated more PHB when cultivated in glycerol medium compared to gluconate medium while the inverse is true for the wild-type strain. Through genome sequencing and expression studies, glycerol kinase was identified as the key enzyme for its improved glycerol utilization. The superior performance of v6C6 in assimilating pure glycerol was extended to crude glycerol (sweetwater) from an industrial fat splitting process. These results highlight the robustness of adaptive laboratory evolution for strain engineering and the versatility and potential of C. necator H16 for industrial waste glycerol valorization.

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Development of mutants of Gliocladium virens tolerant to benomyl

Canadian Journal of Microbiology ◽

10.1139/m90-084 ◽

1990 ◽

Vol 36 (7) ◽

pp. 484-489 ◽

Cited By ~ 12

Author(s):

G. C. Papavizas ◽

D. P. Roberts ◽

K. K. Kim

Keyword(s):

Carbon Source ◽

Type Strain ◽

Wild Type Strain ◽

Carbon Sources ◽

Sclerotium Rolfsii ◽

Wild Type ◽

Gliocladium Virens ◽

Rhizosphere Competence ◽

Filter Paper Cellulase ◽

Type Strains

Aqueous suspensions of conidia of Gliocladium virens strains Gl-3 and Gl-21 were exposed to both ultraviolet radiation and ethyl methanesulfonate. Two mutants of Gl-3 and three of Gl-21 were selected for tolerance to benomyl at 10 μg∙mL−1, as indicated by growth and conidial germination on benomyl-amended potato dextrose agar. The mutants differed considerably from their respective wild-type strains in appearance, growth habit, sporulation, carbon-source utilization, and enzyme activity profiles. Of 10 carbon sources tested, cellobiose, xylose, and xylan were the best for growth, galactose and glucose were intermediate, and arabinose, ribose, and rhamnose were poor sources of carbon. The wild-type strains and the mutants did not utilize cellulose as the sole carbon source for growth. Two benomyl-tolerant mutants of Gl-3 produced less cellulase (β-1,4-glucosidase, carboxymethylcellulase, filter-paper cellulase) than Gl-3. In contrast, mutants of Gl-21 produced more cellulase than the wild-type strain. Only Gl-3 provided control of blight on snapbean caused by Sclerotium rolfsii. Wild-type strain Gl-21 and all mutants from both strains were ineffective biocontrol agents. Key words: Gliocladium, benomyl tolerance, Sclerotium, rhizosphere competence.

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Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

Journal of Bacteriology ◽

10.1128/jb.183.17.5187-5197.2001 ◽

2001 ◽

Vol 183 (17) ◽

pp. 5187-5197 ◽

Cited By ~ 302

Author(s):

Vanessa Sperandio ◽

Alfredo G. Torres ◽

Jorge A. Girón ◽

James B. Kaper

Keyword(s):

Escherichia Coli ◽

Quorum Sensing ◽

Type Strain ◽

Wild Type Strain ◽

Regulatory Mechanism ◽

Sos Response ◽

Enterohemorrhagic Escherichia Coli ◽

Trna Genes ◽

Wild Type ◽

E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

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Mutations Conferring Aminoglycoside and Spectinomycin Resistance in Borrelia burgdorferi

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.2.445-452.2006 ◽

2006 ◽

Vol 50 (2) ◽

pp. 445-452 ◽

Cited By ~ 39

Author(s):

Daniel Criswell ◽

Virginia L. Tobiason ◽

J. Stephen Lodmell ◽

D. Scott Samuels

Keyword(s):

16S Rrna ◽

Borrelia Burgdorferi ◽

Type Strain ◽

Wild Type Strain ◽

Small Subunit ◽

Wild Type ◽

Spectinomycin Resistance ◽

E Coli ◽

Resistant Mutants

ABSTRACT We have isolated and characterized in vitro mutants of the Lyme disease agent Borrelia burgdorferi that are resistant to spectinomycin, kanamycin, gentamicin, or streptomycin, antibiotics that target the small subunit of the ribosome. 16S rRNA mutations A1185G and C1186U, homologous to Escherichia coli nucleotides A1191 and C1192, conferred >2,200-fold and 1,300-fold resistance to spectinomycin, respectively. A 16S rRNA A1402G mutation, homologous to E. coli A1408, conferred >90-fold resistance to kanamycin and >240-fold resistance to gentamicin. Two mutations were identified in the gene for ribosomal protein S12, at a site homologous to E. coli residue Lys-87, in mutants selected in streptomycin. Substitutions at codon 88, K88R and K88E, conferred 7-fold resistance and 10-fold resistance, respectively, to streptomycin on B. burgdorferi. The 16S rRNA A1185G and C1186U mutations, associated with spectinomycin resistance, appeared in a population of B. burgdorferi parental strain B31 at a high frequency of 6 × 10−6. These spectinomycin-resistant mutants successfully competed with the wild-type strain during 100 generations of coculture in vitro. The aminoglycoside-resistant mutants appeared at a frequency of 3 × 10−9 to 1 ×10−7 in a population and were unable to compete with wild-type strain B31 after 100 generations. This is the first description of mutations in the B. burgdorferi ribosome that confer resistance to antibiotics. These results have implications for the evolution of antibiotic resistance, because the 16S rRNA mutations conferring spectinomycin resistance have no significant fitness cost in vitro, and for the development of new selectable markers.

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The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.184.10.2850-2853.2002 ◽

2002 ◽

Vol 184 (10) ◽

pp. 2850-2853 ◽

Cited By ~ 39

Author(s):

Annie Conter ◽

Rachel Sturny ◽

Claude Gutierrez ◽

Kaymeuang Cam

Keyword(s):

Escherichia Coli ◽

Type Strain ◽

Wild Type Strain ◽

Cell Envelope ◽

Wild Type ◽

E Coli ◽

Induced Stress ◽

Mutant Strains ◽

Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

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Expression of Different-Size Transcripts from theclpP-clpX Operon of Escherichia coli during Carbon Deprivation

Journal of Bacteriology ◽

10.1128/jb.182.23.6630-6637.2000 ◽

2000 ◽

Vol 182 (23) ◽

pp. 6630-6637 ◽

Cited By ~ 13

Author(s):

Chin Li ◽

Yi Ping Tao ◽

Lee D. Simon

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Type Strain ◽

Wild Type Strain ◽

Transcription Termination ◽

Carbon Starvation ◽

Lower Percentage ◽

Wild Type ◽

Lower Efficiency ◽

E Coli

ABSTRACT Transcription of the clpP-clpX operon ofEscherichia coli leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entireclpP-clpX operon, whereas the shorter transcript contains the entire clpP gene but none of the clpXcoding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the ς32-dependent promoter of the clpP-clpXoperon within 15 min after the start of carbon starvation. Expression of the clpP-clpX operon from the promoters upstream of theclpP gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation. E. coli rpoBmutations that affect transcription termination efficiency alter the ratio of the shorter clpP-clpX transcript to the longer transcript. The E. coli rpoB3595 mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the rpoB8 mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter clpP-clpXtranscript when E. coli is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.

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Biosynthesis of lysine in Saccharomyces cerevisiae: properties and spectrophotometric determination of homocitrate synthase activity

Canadian Journal of Microbiology ◽

10.1139/m76-246 ◽

1976 ◽

Vol 22 (11) ◽

pp. 1664-1667 ◽

Cited By ~ 11

Author(s):

Gary S. Gray ◽

J. K. Bhattacharjee

Keyword(s):

Saccharomyces Cerevisiae ◽

Biosynthetic Pathway ◽

Wild Type Strain ◽

Wild Type ◽

Temperature Optimum ◽

Acetyl Coa ◽

Metal Chelating ◽

Binding Agents ◽

Homocitrate Synthase

A rapid assay is described for homocitrate synthase (EC 4.1.3.21) of the lysine biosynthetic pathway of Saccharomyces cerevisiae. The α-ketoglutarate-dependent cleavage of acetyl-coA was measured spectrophotometrically as decrease in absorbance at 600 nm in the presence of 2, 6-dichlorophenol-indophenol and enzyme from the wild-type strain X2180. This activity was also present in a citrate synthaseless glutamate auxotroph glu3, and the activity was inhibited by 5 mML-lysine. Radioactive homocitric acid was obtained from a reaction mixture containing [1-14C]acetyl-coA. Homocitrate synthase activity was dependent upon time, both substrates, and enzyme. The activity exhibited a pH and temperature optimum of 7.5–8.0 and 32 °C, respectively, and was inhibited by metal-chelating and sulfhydryl-binding agents.

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