scholarly journals Fitness effects of CTX-M-15-encoding IncF plasmids on their native Escherichia coli ST131 H30Rx hosts

2021 ◽  
Author(s):  
Jana Palkovicova ◽  
Iva Kutilova ◽  
Javier DelaFuente ◽  
Matej Medvecky ◽  
Ivana Jamborova ◽  
...  
Keyword(s):  
Point Mutations ◽  
Plasmid Vector ◽  
Relative Fitness ◽  
Wild Type ◽  
E Coli ◽  
Fitness Effects ◽  
Significant Difference ◽  
Diverse Origin ◽  
Chromosomal Mutations ◽  
Free Strains

Objectives: The objective of this study was to investigate effects of large CTX-M-15-encoding IncF plasmids on the fitness of their native E. coli ST131 H30Rx hosts in order to understand possible plasmid-host coevolution. Methods: We selected five E. coli ST131 H30Rx strains of diverse origin, each carrying a multireplicon IncF plasmid encoding the gene blaCTX-M-15. The plasmid was eliminated from each isolate by displacement using an incompatible plasmid vector pMDP5_cureEC958. Whole-genome sequencing (WGS) was performed to obtain complete chromosome and plasmid sequences of wild-type isolates and to detect chromosomal mutations in plasmid-free strains. Competition assays were conducted to determine the relative fitness of plasmid-free clones compared to the corresponding wild-type isolates. Results: We were able to successfully eliminate the IncF plasmids from all of the wild-type strains using the curing vector pMDP5_cureEC958. The chromosomes of plasmid-free clones contained zero to six point mutations. Plasmid-free strains of three isolates showed no significant difference in relative fitness compared to the corresponding plasmid-free strains. In the two remaining isolates, the plasmids produced a small but significant fitness cost. Conclusion: We conclude that IncF plasmids produce moderate fitness effects in their E. coli ST131 H30Rx hosts. This fitness compatibility is likely to promote the maintenance of antibiotic resistance in this worrisome E. coli lineage.

scholarly journals Role of CheB and CheR in the Complex Chemotactic and Aerotactic Pathway of Azospirillum brasilense

2006 ◽  
Vol 188 (13) ◽  
pp. 4759-4768 ◽  
Author(s):  
Bonnie B. Stephens ◽  
Star N. Loar ◽  
Gladys Alexandre
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Azospirillum Brasilense ◽  
Model Organism ◽  
Spatial Gradient ◽  
Wild Type ◽  
Temporal Gradient ◽  
E Coli ◽  
Significant Difference

ABSTRACT It has previously been reported that the alpha-proteobacterium Azospirillum brasilense undergoes methylation-independent chemotaxis; however, a recent study revealed cheB and cheR genes in this organism. We have constructed cheB, cheR, and cheBR mutants of A. brasilense and determined that the CheB and CheR proteins under study significantly influence chemotaxis and aerotaxis but are not essential for these behaviors to occur. First, we found that although cells lacking CheB, CheR, or both were no longer capable of responding to the addition of most chemoattractants in a temporal gradient assay, they did show a chemotactic response (albeit reduced) in a spatial gradient assay. Second, in comparison to the wild type, cheB and cheR mutants under steady-state conditions exhibited an altered swimming bias, whereas the cheBR mutant and the che operon mutant did not. Third, cheB and cheR mutants were null for aerotaxis, whereas the cheBR mutant showed reduced aerotaxis. In contrast to the swimming bias for the model organism Escherichia coli, the swimming bias in A. brasilense cells was dependent on the carbon source present and cells released methanol upon addition of some attractants and upon removal of other attractants. In comparison to the wild type, the cheB, cheR, and cheBR mutants showed various altered patterns of methanol release upon exposure to attractants. This study reveals a significant difference between the chemotaxis adaptation system of A. brasilense and that of the model organism E. coli and suggests that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense.

scholarly journals Use of Inducible Feedback-ResistantN-Acetylglutamate Synthetase (argA) Genes for Enhanced Arginine Biosynthesis by Genetically EngineeredEscherichia coli K-12 Strains

1998 ◽  
Vol 64 (5) ◽  
pp. 1805-1811 ◽  
Author(s):  
B. S. Rajagopal ◽  
Joseph DePonte ◽  
Mendel Tuchman ◽  
Michael H. Malamy
Keyword(s):  
Feedback Inhibition ◽  
Expression System ◽  
Point Mutations ◽  
Normal Growth ◽  
Growth Conditions ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
E Coli ◽  
Genes Encoding ◽  
K 12

ABSTRACT The goal of this work was to construct Escherichia colistrains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. colistrains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33–38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain “arg boxes,” which titrate the ArgR repressor protein, with or without the E. coli carABgenes encoding carbamyl phosphate synthetase and the argIgene for ornithine transcarbamylase. The free arginine production rates of “arg-derepressed” E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt)argA. The expression system with fbr argAproduced 7- to 35-fold more arginine than a system overexpressingcarAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5α strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carABand argI genes. Plasmids containing wt or fbrargA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.

scholarly journals Reengineering of a Corynebacterium glutamicuml-Arginine and l-Citrulline Producer

2009 ◽  
Vol 75 (6) ◽  
pp. 1635-1641 ◽  
Author(s):  
Masato Ikeda ◽  
Satoshi Mitsuhashi ◽  
Kenji Tanaka ◽  
Mikiro Hayashi
Keyword(s):  
Point Mutations ◽  
Amino Acid Biosynthesis ◽  
The Other ◽  
No Production ◽  
Wild Type ◽  
Acid Biosynthesis ◽  
E Coli ◽  
Lysine Producer ◽  
Efficient Producer ◽  
Type Strains

ABSTRACT Toward the creation of a robust and efficient producer of l-arginine and l-citrulline (arginine/citrulline), we have performed reengineering of a Corynebacterium glutamicum strain by using genetic information of three classical producers. Sequence analysis of their arg operons identified three point mutations (argR123, argG92 up, and argG45) in one producer and one point mutation (argB26 or argB31) in each of the other two producers. Reconstitution of the former three mutations or of each argB mutation on a wild-type genome led to no production. Combined introduction of argB26 or argB31 with argR123 into a wild type gave rise to arginine/citrulline production. When argR123 was replaced by an argR-deleted mutation (ΔargR), the production was further increased. The best mutation set, ΔargR and argB26, was used to screen for the highest productivity in the backgrounds of different wild-type strains of C. glutamicum. This yielded a robust producer, RB, but the production was still one-third of that of the best classical producer. Transcriptome analysis revealed that the arg operon of the classical producer was much more highly upregulated than that of strain RB. Introduction of leuC456, a mutation derived from a classical l-lysine producer and provoking global induction of the amino acid biosynthesis genes, including the arg operon, into strain RB led to increased production but incurred retarded fermentation. On the other hand, replacement of the chromosomal argB by heterologous Escherichia coli argB, natively insensitive to arginine, caused a threefold-increased production without retardation, revealing that the limitation in strain RB was the activity of the argB product. To overcome this, in addition to argB26, the argB31 mutation was introduced into strain RB, which caused higher deregulation of the enzyme and resulted in dramatically increased production, like the strain with E. coli argB. This reconstructed strain displayed an enhanced performance, thus allowing significantly higher productivity of arginine/citrulline even at the suboptimal 38°C.

scholarly journals Contribution of Long Polar Fimbriae to the Virulence of Rabbit-Specific Enteropathogenic Escherichia coli

2004 ◽  
Vol 72 (3) ◽  
pp. 1230-1239 ◽  
Author(s):  
Hayley J. Newton ◽  
Joan Sloan ◽  
Vicki Bennett-Wood ◽  
Louise M. Adams ◽  
Roy M. Robins-Browne ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Open Reading Frames ◽  
Wild Type ◽  
E Coli ◽  
Severe Diarrhea ◽  
Early Stages ◽  
Shared Identity ◽  
Significant Difference ◽  
Specific Pathogen

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a major of cause of diarrhea among children in developing countries. Although EPEC is a human specific pathogen, some related strains are natural pathogens of animals, including laboratory-bred rabbits. We have identified two chromosomal loci in rabbit-specific EPEC (REPEC) O15:H− strain 83/39, which are predicted to encode long polar fimbriae (LPF). lpfR154 was identical to a fimbrial gene cluster, lpfO113 , identified previously in enterohemorrhagic E. coli (EHEC) O113:H21. The second locus, lpfR141 , comprised a novel sequence with five predicted open reading frames, lpfA to lpfE, that encoded long fine fimbriae in nonfimbriated E. coli ORN103. The predicted products of lpfR141 shared identity with components of the lpfABCC′DE gene cluster from EHEC O157:H7, and the fimbriae were similar in morphology and length to LPF from EHEC O157:H7. Interruption of lpfR141 resulted in significant attenuation of REPEC 83/39 for rabbits with respect to the early stages of colonization and severity of diarrhea. However, there was no significant difference in the number of bacteria shed at later time points or in overall body weight and mortality rate of rabbits infected with lpfR141 mutant strains or wild-type REPEC 83/39. Although rabbits infected with the lpfR141 mutants did not develop severe diarrhea, there was evidence of attaching and effacing histopathology, which was indistinguishable in morphology, location, and extent compared to rabbits infected with wild-type REPEC 83/39. The results suggested that lpfR141 contributes to the early stages of REPEC-mediated disease and that this is important for the development of severe diarrhea in susceptible animals.

scholarly journals LT(K63/R72), a New Mutant of Escherichia coli Heat-labile Enterotoxin, Exhibits Characteristics More Similar to LT(K63) than LT(R72)

2005 ◽  
Vol 37 (2) ◽  
pp. 126-132 ◽  
Author(s):  
Qiang Feng ◽  
Jun Yang ◽  
Ping Luo ◽  
Wei-Jun Zhang ◽  
Quan-Ming Zou
Keyword(s):  
Escherichia Coli ◽  
Production Rate ◽  
Wild Type ◽  
Th2 Response ◽  
E Coli ◽  
Heat Labile ◽  
Significant Difference ◽  
Low Toxic ◽  
The Stability ◽  
Type Response

Abstract LT(K63), a non-toxic mutant and LT(R72), a low toxic mutant of E. coli heat-labile enterotoxin are frequently used mucosal adjuvants. In many cases, the adjuvanticity of LT(K63) is lower than that of LT (R72), but LT(K63), which induces a mixed Th1/Th2 response, exhibits a higher level of protection than LT (R72) which induces a polarized Th2-type response. To utilize the advantages of both adjuvants, a doublemutation LT(K63/R72) was generated and purified. The characterization results showed that there was no significant difference in production rate and immunogenicity between wild type LT and LT mutants. The results also showed that the toxicity and the trypsin sensitivity of LT(K63/R72) are between that of LT(K63) and LT(R72). Using HPLC, when samples in an OHpak SB-800 column were eluted by denatural buffer (TEAN containing 10 mg/ml SDS), we found the stability of LT(K63/R72) was higher than that of LT(R72) and lower than that of LT(K63). Through further analyzes, we found that LT(K63/R72) exhibits characteristics more closely related to LT(K63) than LT(R72).

Random mutagenesis suggests that sequence errors are not a major cause of variation in the activity of individual molecules of β-galactosidase

2012 ◽  
Vol 90 (4) ◽  
pp. 540-547 ◽  
Author(s):  
Douglas B. Craig ◽  
Thomas Schwab ◽  
Reinhard Sterner
Keyword(s):  
Single Molecule ◽  
Random Sequence ◽  
Random Mutagenesis ◽  
Error Rates ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
E Coli ◽  
Significant Difference ◽  
Gene Libraries ◽  
Sequence Errors

Wild-type Escherichia coli lacZ was subjected to error-prone PCR to generate two plasmid-encoded gene libraries containing approximately 2.6 (SD 1.9) nucleotide exchanges resulting in 1.8 (SD 1.4) amino-acid substitutions. The libraries were used, along with a plasmid containing wild-type lacZ, to transform E. coli lacking genomic lacZ. Cells expressing functional β-galactosidase were identified by blue/white screening. Cell lysates containing the populations of heterogeneously mutagenized β-galactosidase were subjected to single molecule assays using a capillary electrophoresis laser-induced fluorescence-based protocol. There was no significant difference in the average catalytic rate between the random mutagenized and wild-type enzyme populations. Furthermore, there was no clear pattern between error rates and the variances in the population catalytic rates. This suggests that random sequence errors are not a substantial source of the catalytic heterogeneity of this enzyme.

scholarly journals gyrA Mutations in Ciprofloxacin-Resistant Bartonella bacilliformis Strains Obtained In Vitro

2003 ◽  
Vol 47 (1) ◽  
pp. 383-386 ◽  
Author(s):  
Michael F. Minnick ◽  
Zachary R. Wilson ◽  
Laura S. Smitherman ◽  
D. Scott Samuels
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Dna Gyrase ◽  
Wild Type ◽  
Bartonella Bacilliformis ◽  
E Coli ◽  
A Subunit ◽  
Resistant Strains ◽  
Single Point Mutations

ABSTRACT We isolated and characterized mutants of Bartonella bacilliformis that are resistant to the fluoroquinolone antibiotic ciprofloxacin, which targets the A subunit of DNA gyrase. Mutants had single point mutations in the gyrA gene that changed either Asp-90 to Gly or Asp-95 to Asn and had 3- or 16-fold higher resistance, respectively, to ciprofloxacin than did wild-type B. bacilliformis. Asp-95 is homologous to Asp-87 of Escherichia coli GyrA and is a common residue mutated in fluoroquinolone-resistant strains of other bacteria. This is the first report of a mutation at an Asp-90 homologue, which corresponds to Asp-82 in E. coli GyrA.

Construction and Characterization of Escherichia coli O157:H7 Strains Expressing Firefly Luciferase and Green Fluorescent Protein and Their Use in Survival Studies‡

1997 ◽  
Vol 60 (10) ◽  
pp. 1167-1173 ◽  
Author(s):  
PINA M. FRATAMICO ◽  
MING Y. DENG ◽  
TERENCE P. STROBAUGH ◽  
SAMUEL A. PALUMBO
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Orange Juice ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Escherichia Coli O157 ◽  
Apple Cider ◽  
E Coli ◽  
Significant Difference ◽  
Green Fluorescent

The firefly (Photinus pyralis) luciferase (luc) gene on plasmid vector pBESTluc and the Aequorea victoria green fluorescent protein (gfp) gene on plasmid vector pGFP were introduced into strains of Escherichia coli O157:H7. The recombinant E. coli strains were indistinguishable from their parent strains in biochemical and immunological assays and in a multiplex PCR reaction. There was no significant difference in the growth kinetics of the luc-bearing recombinants and the parent strains. At 37°C all of the recombinant strains maintained the vectors and expressed luciferase and the green fluorescent protein when grown both with and without antibiotic selection. Individual colonies of luc-bearing E. coli strains were readily luminescent in the dark after being sprayed with a solution of 1 mM beetle luciferin. The recombinants containing pGFP emitted bright green fluorescence when excited with UV light and the addition of any other proteins, substrates, or cofactors was not required. The green fluorescent protein-expressing E. coli O157:H7 strains were used in studies examining the survival of the organism in apple cider and in orange juice. In apple cider the organism declined to undetectable levels in 24 days at refrigeration temperature while in orange juice the strains survived with only small decreases in number during the 24-day sampling period. These recombinant E. coli O157:H7 strains, containing readily identifiable and stable markers, could be useful as positive controls in microbial assays as well as in studies monitoring bacterial survival and the behavior of E. coli O157:H7 in foods and in a food processing environment.

scholarly journals Thrombomodulin Gene Mutations Associated with Thromboembolic Diseases

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3497-3497
Author(s):  
Qingyun Wang ◽  
Liang Tang ◽  
Bei Hu ◽  
Yu Hu ◽  
Han Liu
Keyword(s):  
Venous Thrombosis ◽  
Cultured Cells ◽  
Conflicts Of Interest ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Gene Mutations ◽  
Wild Type ◽  
Transmembrane Glycoprotein ◽  
Significant Difference ◽  
Artery Disease

Abstract Background Thrombomodulin (TM), encoded by the THBD gene, is an endothelial transmembrane glycoprotein that acts as a cofactor for thrombin in the activation of protein C (PC). It has been clearly demonstrated that the anticoagulant and profibrinolytic functions of the thrombin-TM-PC system play critical roles in the prevention of thromboembolic diseases. Methods and Results Using direct sequencing, six novel mutations were identified in THBD gene of eight patients with either arterial or venous thrombosis: c.376G>T (p.Asp126Tyr), c.569C>G (p.Ser190Trp), c.659T>G (p.Leu220X), c.1208G>A (p.Arg403Lys), c.1288G>A (p.Gly430Ser), c.*6_*23del and c.*23_*40del. Of the mutations, c.376G>T and c.*6_*23del were detected in a 10-year old child with venous thrombosis (VTE), who died one year later. The c.376G>T point mutation predicting p.Asp126Tyr in thrombomodulin protein was also detected in a 38-year old VTE patient. The nonsense mutation c.659T>G recurred in a 55-year old patient with coronary artery disease (CAD) and a 46-year old VTE patient, who died from repeated venous thrombosis two years later. The wild type and the mutant thrombomodulin were expressed in HEK-293t cells. Two mutations were found to have reduced TM protein expression compared to wild-type (100%), Ser190Trp (69.1% ±5%, P < .05), p.Leu220X (19.1% ± 7.6%, P < .001). Comparing to wild type, four point mutations diminished the cofactor activity of TM according to the reduced level of activation of PC in cultured cells. As for the two 18-bp deletions, a psiCHECKTM-2 vector containing wild type and mutant 3'UTR of TM was constructed. Compared with the wild-type, the deletions significantly reduced the reporter gene-expression level. The antigen level and activity in cultured cells with c.376G>T have no significant difference with the wild type, indicating that the mutant may cause a thrombophilic state through other unknown pathological mechanisms. Conclusion The results suggest that gene mutation of thrombomodulin may be important in the pathogenesis of thrombotic diseases. Further study on genetics of thrombosis should focus on resequencing of THBD gene in different populations. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effect of the Bacillus atrophaeus subsp. globigii Spo0F H101R Mutation on Strain Fitness

2012 ◽  
Vol 78 (24) ◽  
pp. 8601-8610 ◽  
Author(s):  
Doncho V. Zhelev ◽  
Mia Hunt ◽  
Anna Le ◽  
Christopher Dupuis ◽  
Suelynn Ren ◽  
...  
Keyword(s):  
Developmental Process ◽  
Point Mutations ◽  
Relative Fitness ◽  
Evolutionary Constraint ◽  
Wild Type ◽  
Low Oxygen ◽  
Bacillus Atrophaeus ◽  
Experimental Conditions ◽  
Content Type ◽  
Fitness Advantage

ABSTRACTSporulation is a critical developmental process inBacillusspp. that, once initiated, removes the possibility of further growth until germination. Therefore, the threshold conditions triggering sporulation are likely to be subject to evolutionary constraint. Our previous studies revealed two spontaneous hypersporulating mutants ofBacillus atrophaeussubsp.globigii, both containing point mutations in thespo0Fgene. One of these strains (Detrick-2; contains thespo0F101allele with a C:T [His101Arg] substitution) had been deliberately selected in the early 1940s as an anthrax surrogate. To determine whether the experimental conditions used during the selection of the “military” strains could have supported the emergence of hypersporulating variants, the relative fitness of strain Detrick-2 was measured in several experimental settings modeled on experimental conditions employed during its development in the 1940s as a simulant. The congenic strain Detrick-1 contained a wild-typespo0Fgene and sporulated like the wild-type strain. The relative fitness of Detrick-1 and Detrick-2 was evaluated in competition experiments using quantitative single nucleotide polymorphism (SNP)-specific real-time PCR assays directed at the C:T substitution. The ancestral strain Detrick-1 had a fitness advantage under all conditions tested except when competing cultures were subjected to frequent heat shocks. The hypersporulating strain gained the maximum fitness advantage when cultures were grown at low oxygen tension and when heat shock was applied soon after the formation of the first heat-resistant spores. This is interpreted as gain of fitness by the hypersporulating strain in fast-changing fluctuating environments as a result of the increased rate of switching to the sporulating phenotype.

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