Use of tRNA-Supplemented Host Strains for Expression of Heterologous Genes in E. coli

2003 ◽  
pp. 225-234 ◽  
Author(s):  
Carsten-Peter Carstens
Keyword(s):  
E Coli ◽  
Host Strains

scholarly journals Different impacts of naturally occurring variants of the globally disseminated carbapenemase-encoding plasmid pKpQIL on the biology of host strains

2018 ◽  
Author(s):  
Howard T. H. Saw ◽  
Mark A. Webber ◽  
Neil Woodford ◽  
Laura J. V. Piddock
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Host Range ◽  
Generation Time ◽  
Enterobacter Cloacae ◽  
Bacterial Host ◽  
E Coli ◽  
Naturally Occurring ◽  
Filter Mating ◽  
Host Strains

SynopsisKlebsiella-associated plasmid pKpQIL and its variant have been isolated globally. Our study aimed to determine whether a naturally occurring variant has altered host range and impacts on the fitness of different bacterial host strains. The plasmids pKpQIL-UK and pKpQIL-D2 were transferred from the original clinical isolate host strains of Klebsiella pneumoniae into Escherichia coli, Salmonella Typhimurium, Enterobacter cloacae and Serratia marcescens strains by filter-mating and conjugation frequencies determined and compared. The fitness of the resulting transconjugants was assessed by determining growth kinetics, ability to form a biofilm and persistence of the plasmids in each host was also measured. Transfer of either plasmid into Salmonella and S. marcescens was similar. However, pKpQIL-UK transferred into E. coli at a higher rate than did pKpQIL-D2; the reverse was found for E. cloacae. Both plasmids were rapidly lost from the E. coli population. Plasmid pKpQIL-UK, but not -D2, was able to persist in Salmonella. Although pKpQIL-UK imposed a greater fitness cost (inferred from an increased generation time) than -D2 on E. cloacae, it was able to persist as well as pKpQIL-D2 in this host. The pKpQIL-D2 plasmid did not confer any fitness benefit on any of the hosts under the conditions tested. Variants of the globally important pKpQIL plasmid have arisen in patients due to recombination. The impacts of the pKpQIL-UK plasmid and the -D2 variant in various Enterobacteriaceae are host-dependent. Continuing evolution of pKpQIL may alter its host range in the future.

scholarly journals Evaluating the Five Escherichia coli Derivative Strains as Platform for Arginine Deiminase Overproduction

2020 ◽  
Author(s):  
Sara Abdollahi ◽  
Mohammad Hossein Morowvat ◽  
Amir Savardashtaki ◽  
Cambyz Irajie ◽  
Sohrab Najafipour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Microtiter Plate ◽  
Arginine Deiminase ◽  
Expression Vectors ◽  
Efficacy Evaluation ◽  
Microtiter Plate Assay ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Host Strains

Abstract Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modified expression strains. This study attempted to evaluate the five host strains including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHuffle in terms of arginine deiminase (ADI) production and enzyme activity. Arginine deiminase (ADI) was chosen a bacterial enzyme which degrades L-arginine. It is effective in treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC) which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through CaCl 2 method. It was then transformed with the construct of pET3a-ADI using heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. All the five strains exhibited a significant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the efficacy evaluation of the five studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.

Colicins G and H and their host strains

1991 ◽  
Vol 37 (10) ◽  
pp. 751-757 ◽  
Author(s):  
D. E. Bradley
Keyword(s):  
Escherichia Coli ◽  
Key Words ◽  
Side Chains ◽  
Human Origin ◽  
Wild Type ◽  
E Coli ◽  
Molecular Weights ◽  
Protein Receptors ◽  
Type Strains ◽  
Host Strains

Escherichia coli strains CA46(pColG) and CA58(pColH) each apparently synthesized two generally similar bactericidal colicin proteins whose molecular weights were approximately 5 500 and 100 000. These proteins were more resistant to trypsin than representative colicins A, D, E1, and V. The smooth wild-type strains harbouring plasmids pColG and pColH were serotyped O169:NM and O30:NM, respectively, being typically associated with nonpathogenic E. coli of human origin. Rough and semirough variants, which were selected using resistance to novobiocin, were intrinsically insensitive to almost as many colicins (10 tested) as their parents. For this reason the wild-type strains would not be useful for identifying colicins G and H on the basis of immunity. The O antigenic side chains of both wild-type strains shielded three of the six bacteriophage protein receptors tested. Key words: colicin, protein, plasmid, O antigen, bacteriophage.

scholarly journals Specialised functions of two common plasmid mediated toxin-antitoxin systems, ccdAB and pemIK, in Enterobacteriaceae

2020 ◽  
Author(s):  
Alma Y. Wu ◽  
Muhammad Kamruzzaman ◽  
Jonathan R. Iredell
Keyword(s):  
Klebsiella Pneumoniae ◽  
Species Differences ◽  
Plasmid Stability ◽  
Bacterial Species ◽  
Type Ii ◽  
Stability Function ◽  
E Coli ◽  
Stability Functions ◽  
Resistance Plasmids ◽  
Host Strains

AbstractToxin-antitoxin systems (TAS) are commonly found on bacterial plasmids and are involved in plasmid maintenance. Even though the same TAS are present in a variety of plasmid types and bacterial species, differences in their sequences, expression and functions are not well defined. Here, we aimed to identify commonly occurring plasmid TAS in Escherichia coli and Klebsiella pneumoniae and compare the sequence, expression and plasmid stability function of their variants. 27 putative type II TAS were identified from 1063 plasmids of Klebsiella pneumoniae in GenBank. Among these, ccdAB and pemIK were found to be most common, also occurring in plasmids of E. coli. Comparisons of ccdAB variants, taken from E. coli and K. pneumoniae, revealed sequence differences, while pemIK variants from IncF and IncL/M plasmids were almost identical. Similarly, the expression and plasmid stability functions of ccdAB variants varied according to the host strain and species, whereas the expression and functions of pemIK variants were consistent among host strains. The specialised functions of some TAS may determine the host specificity and epidemiology of major antibiotic resistance plasmids.

scholarly journals Isogenic Lysogens of Diverse Shiga Toxin 2-Encoding Bacteriophages Produce Markedly Different Amounts of Shiga Toxin

1999 ◽  
Vol 67 (12) ◽  
pp. 6710-6714 ◽  
Author(s):  
Patrick L. Wagner ◽  
David W. K. Acheson ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Fragment Length ◽  
Toxin Production ◽  
E Coli ◽  
Length Polymorphisms ◽  
Shiga Toxin 2 ◽  
K 12 ◽  
Host Strains ◽  
Restriction Fragment Length

ABSTRACT We produced isogenic Escherichia coli K-12 lysogens of seven different Shiga toxin 2 (Stx2)-encoding bacteriophages derived from clinical Shiga toxin-producing E. coli (STEC) isolates of serotypes O157:H7, O145, O111, and O83 to assess the variability among these phages and determine if there were phage-related differences in toxin production. Phage genomic restriction fragment length polymorphisms (RFLP) and superinfection resistance studies revealed significant differences among these phages and allowed the seven phages to be placed into five distinct groups. Experiments revealed striking differences in spontaneous phage and toxin production that were correlated with the groupings derived from the RFLP and resistance studies. These results suggest that the genotype of the Stx2 prophage can influence the level of phage release and toxin expression by host strains and thus may be relevant to STEC pathogenesis.

scholarly journals Insertion Site Occupancy by stx2 Bacteriophages Depends on the Locus Availability of the Host Strain Chromosome

2007 ◽  
Vol 189 (18) ◽  
pp. 6645-6654 ◽  
Author(s):  
Ruth Serra-Moreno ◽  
Juan Jofre ◽  
Maite Muniesa
Keyword(s):  
Shiga Toxin ◽  
Insertion Site ◽  
Site Occupancy ◽  
Host Strain ◽  
Host Chromosome ◽  
Shiga Toxins ◽  
E Coli ◽  
Emergent Pathogen ◽  
Insertion Sites ◽  
Host Strains

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) is an emergent pathogen characterized by the expression of Shiga toxins, which are encoded in the genomes of lambdoid phages. These phages are infectious for other members of the Enterobacteriaceae and establish lysogeny when they integrate into the host chromosome. Five insertion sites, used mainly by these prophages, have been described to date. In the present study, the insertion of stx 2 prophages in these sites was analyzed in 168 STEC strains isolated from cattle. Additionally, insertion sites were determined for stx 2 phages which (i) converted diverse laboratory host strains, (ii) coexisted with another stx 2 prophage, and (iii) infected a recombinant host strain lacking the most commonly used insertion site. Results show that depending on the host strain, phages preferentially use one insertion site. For the most part, yehV is occupied in STEC strains while wrbA is preferentially selected by the same stx phages in E. coli laboratory strains. If this primary insertion site is unavailable, then a secondary insertion site is selected. It can be concluded that insertion site occupancy by stx phages depends on the host strain and on the availability of the preferred locus in the host strain.

scholarly journals Evaluation of the Staphylococcus aureus Class C Nonspecific Acid Phosphatase (SapS) as a Reporter for Gene Expression and Protein Secretion in Gram-Negative and Gram-Positive Bacteria

2007 ◽  
Vol 73 (22) ◽  
pp. 7232-7239 ◽  
Author(s):  
Erika du Plessis ◽  
Jacques Theron ◽  
Eldie Berger ◽  
Maureen Louw
Keyword(s):  
Staphylococcus Aureus ◽  
Acid Phosphatase ◽  
Bacillus Halodurans ◽  
Regulatory Sequences ◽  
Reporter System ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Class C ◽  
Host Strains

ABSTRACTA phosphatase secreted byStaphylococcus aureusstrain 154 has previously been characterized and classified as a new member of the bacterial class C family of nonspecific acid phosphatases. As the acid phosphatase activity can be easily detected with a cost-effective plate screen assay, quantitatively measured by a simple enzyme assay, and detected by zymography, its potential use as a reporter system was investigated. TheS. aureusacid phosphatase (sapS) gene has been cloned and expressed from its own regulatory sequences inEscherichia coli,Bacillus subtilis, andBacillus halodurans. Transcriptional and translational fusions of thesapSgene with selected heterologous promoters and signal sequences were constructed and expressed in all three of the host strains. From the range of promoters evaluated, the strongest promoter for heterologous protein production in each of the host strains was identified, i.e., theE. coli lacZpromoter inE. coli, theB. haloduransalkaline protease promoter inB. subtilis, and theB. haloduransσDpromoter inB. halodurans. This is the first report on the development of a class C acid phosphatase gene as a reporter gene with the advantage of being able to function in both gram-positive and gram-negative host strains.

scholarly journals E. Coli CB390 as an Indicator of Total Coliphages for Microbiological Assessment of Lime and Drying Bed Treated Sludge

Water ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 1833
Author(s):  
Camilo Venegas ◽  
Andrea C. Sánchez-Alfonso ◽  
Crispín Celis Zambrano ◽  
Mauricio González Mendez ◽  
Fidson-Juarismy Vesga
Keyword(s):  
Microbiological Quality ◽  
Solid Samples ◽  
E Coli ◽  
Maximum Reduction ◽  
Treatment Conditions ◽  
Recovery Capacity ◽  
Single Host ◽  
Semi Solid ◽  
Host Strains

The use of a single host strain that allows for an evaluation of the levels of total coliphages in any type of environmental sample would facilitate the detection of and reduction in complexity and costs, favoring countries or areas with technical and economic limitations. The CB390 strain is a candidate for this type of simultaneous determinations, mainly in water samples. The objective of the study was to establish the recovery capacity of the CB390 strain in solid and semi-solid samples and to evaluate the microbiological quality of the sludge generated and stabilized by lime and drying beds in two WWTPs in Colombia. The results of both matrices indicated that CB390 recovered similar numbers of total coliphages (p > 0.05) against the two host strains when evaluated separately. Only the drying bed treatment was able to reduce between 2.0 and 2.9 Log10 units for some microorganisms, while the addition of lime achieved a maximum reduction of 1.3 Log10 units for E. coli. In conclusion, the CB390 strain can be used in solid and semi-solid samples, and the treatment in a drying bed provided a product of microbiological quality. However, the results are influenced by the infrastructure of the WWTP, the treatment conditions, and the monitoring of the stabilization processes.

scholarly journals Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications

2021 ◽  
Vol 9 ◽  
Author(s):  
Arshpreet Bhatwa ◽  
Weijun Wang ◽  
Yousef I. Hassan ◽  
Nadine Abraham ◽  
Xiu-Zhen Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Growth Conditions ◽  
Industrial Applications ◽  
Expression Vectors ◽  
Bacterial Host ◽  
New Developments ◽  
E Coli ◽  
Host Strains

Recombinant proteins are becoming increasingly important for industrial applications, where Escherichia coli is the most widely used bacterial host for their production. However, the formation of inclusion bodies is a frequently encountered challenge for producing soluble and functional recombinant proteins. To overcome this hurdle, different strategies have been developed through adjusting growth conditions, engineering host strains of E. coli, altering expression vectors, and modifying the proteins of interest. These approaches will be comprehensively highlighted with some of the new developments in this review. Additionally, the unique features of protein inclusion bodies, the mechanism and influencing factors of their formation, and their potential advantages will also be discussed.

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