scholarly journals GENETIC ANALYSIS OF AN ESCHERICHIA COLI MUTANT WITH A LESION IN STABLE RNA TURNOVER

Genetics ◽  
1974 ◽  
Vol 76 (2) ◽  
pp. 185-194
Author(s):  
Yoshinari Ohnishi

ABSTRACT A mutant that rapidly degrades more than 80% of its rRNA and tRNA under defined conditions was genetically analyzed. Two genes, srnA and srnB, are separately located, and the mutated alleles of both are required for degradation of stable RNA in cultures treated with rifampicin at 42°. srnA is closely linked to tsx by matings and transduction tests; by P1 transduction, the gene order is lac (9 min) proC (9.55 min) tsx (9.8 min) srnA (about 10 min) purE (12 min) rnsA (14.4 min). srnB is not yet completely mapped, but is outside the lac-rnsA region, probably in the region between 75 and 90 min.—The product of the rnsA gene, RNase I, is a potent endonuclease of E. coli, and the only one known that can attack ribosomes and tRNA. However, not only are the srn lesions genetically separate from rnsA, but also, derivatives of an srn strain were prepared lacking RNase I, and they retain the Srn- phenotype. Thus, no correlation of rapid RNA turnover and RNase I activity has been found.

1994 ◽  
Vol 180 (6) ◽  
pp. 2147-2153 ◽  
Author(s):  
M Pizza ◽  
M R Fontana ◽  
M M Giuliani ◽  
M Domenighini ◽  
C Magagnoli ◽  
...  

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.


mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Sagi Huja ◽  
Yaara Oren ◽  
Eva Trost ◽  
Elzbieta Brzuszkiewicz ◽  
Dvora Biran ◽  
...  

ABSTRACTHere we present an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78 that represent the major cause of avian colisepticemia, an invasive infection caused by avian pathogenicEscherichia coli(APEC) strains. It is associated with high mortality and morbidity, resulting in significant economic consequences for the poultry industry. To understand the genetic basis of the virulence of avian septicemic E. coli, we sequenced the entire genome of a clinical isolate of serotype O78—O78:H19 ST88 isolate 789 (O78-9)—and compared it with three publicly available APEC O78 sequences and one complete genome of APEC serotype O1 strain. Although there was a large variability in genome content between the APEC strains, several genes were conserved, which are potentially critical for colisepticemia. Some of these genes are present in multiple copies per genome or code for gene products with overlapping function, signifying their importance. A systematic deletion of each of these virulence-related genes identified three systems that are conserved in all septicemic strains examined and are critical for serum survival, a prerequisite for septicemia. These are the plasmid-encoded protein, the defective ETT2 (E. colitype 3 secretion system 2) type 3 secretion system ETT2sepsis, and iron uptake systems. Strain O78-9 is the only APEC O78 strain that also carried the regulon coding for yersiniabactin, the iron binding system of theYersiniahigh-pathogenicity island. Interestingly, this system is the only one that cannot be complemented by other iron uptake systems under iron limitation and in serum.IMPORTANCEAvian colisepticemia is a severe systemic disease of birds causing high morbidity and mortality and resulting in severe economic losses. The bacteria associated with avian colisepticemia are highly antibiotic resistant, making antibiotic treatment ineffective, and there is no effective vaccine due to the multitude of serotypes involved. To understand the disease and work out strategies to combat it, we performed an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78, the major cause of the disease. We identified several potential virulence factors, conserved in all the colisepticemic strains examined, and determined their contribution to growth in serum, an absolute requirement for septicemia. These findings raise the possibility that specific vaccines or drugs can be developed against these critical virulence factors to help combat this economically important disease.


2007 ◽  
Vol 189 (14) ◽  
pp. 4975-4983 ◽  
Author(s):  
Blaine A. Legaree ◽  
Calvin B. Adams ◽  
Anthony J. Clarke

ABSTRACT Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.


2004 ◽  
Vol 186 (8) ◽  
pp. 2418-2429 ◽  
Author(s):  
Jay E. Johnson ◽  
Laura L. Lackner ◽  
Cynthia A. Hale ◽  
Piet A. J. de Boer

ABSTRACT The MinC division inhibitor is required for accurate placement of the septal ring at the middle of the Escherichia coli cell. The N-terminal domain of MinC (ZMinC) interferes with FtsZ assembly, while the C-terminal domain (DMinC) mediates both dimerization and complex formation with either MinD or DicB. Binding to either of these activators greatly enhances the division-inhibitory activity of MinC in the cell. The MinD ATPase plays a crucial role in the rapid pole-to-pole oscillation of MinC that is proposed to force FtsZ ring formation to midcell. DicB is encoded by one of the cryptic prophages on the E. coli chromosome (Qin) and is normally not synthesized. Binding of MinD or DicB to DMinC produces complexes that have high affinities for one or more septal ring-associated targets. Here we show that the FtsZ-binding protein ZipA is required for both recruitment of the DMinC/DicB complex to FtsZ rings and the DicB-inducible division block normally seen in MinC+ cells. In contrast, none of the known FtsZ-associated factors, including ZipA, FtsA, and ZapA, appear to be specifically required for targeting of the DMinC/MinD complex to rings, implying that the two MinC/activator complexes must recognize distinct features of FtsZ assemblies. MinD-dependent targeting of MinC may occur in two steps of increasing topological specificity: (i) recruitment of MinC from the cytoplasm to the membrane, and (ii) specific targeting of the MinC/MinD complex to nascent septal ring assemblies on the membrane. Using membrane-tethered derivatives of MinC, we obtained evidence that both of these steps contribute to the efficiency of MinC/MinD-mediated division inhibition.


2020 ◽  
Vol 4 (1) ◽  
pp. 49
Author(s):  
Gracielle Oliveira Sabbag Cunha ◽  
Ana Paula Terezan ◽  
Andreia Pereira Matos ◽  
Marcela Carmen De Melo Burger ◽  
Paulo Cezar Vieira ◽  
...  

This study evaluated the antimicrobial activity of isolated compounds and semisynthetic derivatives from Miconia ferruginata (Melastomataceae) against five microorganisms: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 6623), Pseudomonas aeruginosa (ATCC 15442), and Candida albicans (ATCC 10231). The isomeric mixture of ursolic and oleanolic acids was active against S. aureus (MIC = 250 μg mL-1) and against E. coli, B. subtilis, and P. aeruginosa (MIC = 500 μg mL-1). The flavone 5,6,7-trihydroxy-4’-methoxyflavone and the methyl esters, semisynthetic derivatives of a mixture of ursolic and oleanolic acids, showed no activity against the tested microorganisms. These results suggest that the carboxyl group present in the triterpenes may contribute to antimicrobial activity.


Author(s):  
Alex B. Benedict ◽  
Joshua D. Chamberlain ◽  
Diana G. Calvopina ◽  
Joel S. Griffitts

Abstract Background The bacteriophage T7 gene 10 ribosome binding site (g10RBS) has long been used for robust expression of recombinant proteins in Escherichia coli. This RBS consists of a Shine–Dalgarno (SD) sequence augmented by an upstream translational “enhancer” (Enh) element, supporting protein production at many times the level seen with simple synthetic SD-containing sequences. The objective of this study was to dissect the g10RBS to identify simpler derivatives that exhibit much of the original translation efficiency. Methods and results Twenty derivatives of g10RBS were tested using multiple promoter/reporter gene contexts. We have identified one derivative (which we call “CON_G”) that maintains 100% activity in E. coli and is 33% shorter. Further minimization of CON_G results in variants that lose only modest amounts of activity. Certain nucleotide substitutions in the spacer region between the SD sequence and initiation codon show strong decreases in translation. When testing these 20 derivatives in the alphaproteobacterium Agrobacterium fabrum, most supported strong reporter protein expression that was not dependent on the Enh. Conclusions The g10RBS derivatives tested in this study display a range of observed activity, including a minimized version (CON_G) that retains 100% activity in E. coli while being 33% shorter. This high activity is evident in two different promoter/reporter sequence contexts. The array of RBS sequences presented here may be useful to researchers in need of fine-tuned expression of recombinant proteins of interest.


2018 ◽  
Vol 6 (21) ◽  
Author(s):  
Yasufumi Matsumura ◽  
Gisele Peirano ◽  
Johann D. D. Pitout

ABSTRACT We report here the complete genome sequence of Escherichia coli J53, which is used as a recipient in conjugation experiments and is a laboratory strain derived from E. coli K-12. This genome sequence will help in the development of a comprehensive genetic analysis of conjugative elements.


2018 ◽  
Vol 6 (25) ◽  
Author(s):  
Yasufumi Matsumura ◽  
Masaki Yamamoto ◽  
Satoshi Nakano ◽  
Miki Nagao

ABSTRACT We report here the complete genome sequence of Escherichia coli ME8067, an azide-resistant laboratory strain used for conjugation experiments. The ME8067 genome was closely related to E. coli strain K-12 substrain W3110. This genome sequence will support further genetic analysis of conjugative elements.


2015 ◽  
Vol 197 (8) ◽  
pp. 1507-1514 ◽  
Author(s):  
Ziad W. El-Hajj ◽  
Elaine B. Newman

ABSTRACTAlthoughEscherichia coliis a very small (1- to 2-μm) rod-shaped cell, here we describe anE. colimutant that forms enormously long cells in rich media such as Luria broth, as long indeed as 750 μm. Theseextremelyelongated (eel) cells are as long as the longest bacteria known and have no internal subdivisions. They are metabolically competent, elongate rapidly, synthesize DNA, and distribute cell contents along this length. They lack only the ability to divide. The concentration of the essential cell division protein FtsZ is reduced in these eel cells, and increasing this concentration restores division.IMPORTANCEEscherichia coliis usually a very small bacterium, 1 to 2 μm long. We have isolated a mutant that forms enormously long cells, 700 times longer than the usualE. colicell.E. colifilaments that form under other conditions usually die within a few hours, whereas our mutant is fully viable even when it reaches such lengths. This mutant provides a useful tool for the study of aspects ofE. coliphysiology that are difficult to investigate with small cells.


Genetics ◽  
1982 ◽  
Vol 100 (1) ◽  
pp. 7-18
Author(s):  
E C Cox ◽  
D L Horner

ABSTRACT In this paper we report on the isolation and genetic analysis of a series of strong mutators mapping at five minutes on the E. coli chromosome. These mutations are dominant and show no evidence of interaction in merodiploids. Cultures grown in broth medium exhibit mutant frequencies five to six orders of magnitude higher than mut+ strains. Cultures propagated in minimal salts media mutate at rates one to three orders higher than wild-type. Three-factor crosses have been used to order these mutators relative to metD, proA, and a Tn10 insertion near five minutes.


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