Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen.

Enterohemorrhagic Eschericia coli (EHEC) 0157:H7 must be able to survive the acid stress of gastric passage. Previous studies show that adhesion of EHEC 0157:H7 to human epithelial cells is increased if EHEC was pre-exposed to acid. Whole genome array analysis of EHEC reveals that the putative fimbrial gene yadK is significantly upregulated after EHEC exposure to acid treatments. In this study, a YadK deficient mutant (△yadK) in EHEC 0157:H7 wild-type (WT) strain 85-170 was constructed and phenotyped. The results of this study demonstrate that △yadK exposed to acid stress showed no acid-induced increase in bacteria-host cell adhesion observed in similarly acid-induced wild type cells. This study concludes that YadK plays a role in acid-induced host adhesion of EHEC 0157:H7. It also indicates that acid stress, which is a part of the host’s natural assault to resist invasion, may regulate factors responsible for enhanced bacteria-host attachment, resulting in increased EHEC virulence.

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Engineered Cyanophycin Synthetase (CphA) from Nostoc ellipsosporum Confers Enhanced CphA Activity and Cyanophycin Accumulation to Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01132-06 ◽

2006 ◽

Vol 72 (12) ◽

pp. 7652-7660 ◽

Cited By ~ 17

Author(s):

Tran Hai ◽

Kay M. Frey ◽

Alexander Steinbüchel

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Dry Matter ◽

Wild Type ◽

Acinetobacter Baylyi ◽

E Coli ◽

Recombinant Cells ◽

Hydrophobic Amino Acids ◽

Type Gene ◽

Nostoc Ellipsosporum

ABSTRACT The cyanophycin (CGP) synthetase gene (cphA NE1) of the transposon-induced argL mutant NE1 of the cyanobacterium Nostoc ellipsosporum, which exhibits a CGP-leaky phenotype during diazotrophical growth, was cloned and expressed in Escherichia coli strain TOP10. Its amino acid sequence exhibited high similarities to CphAs of other cyanobacteria. Recombinant cells of E. coli, which harbored a fragment comprising the complete cphA NE1 gene plus 400 bp of its downstream region in colinear orientation to the lacZ promoter, accumulated CGP up to 17 and 8.5% (wt/wt) of cellular dry matter (CDM) if cultivated in complex medium in the presence or absence of isopropyl-β-d-thiogalactopyranoside, respectively. Two truncated CphAs, lacking 31 (CphANE1del96) or 59 (CphANE1del180) amino acids of the C-terminal region, were derived from cphA NE1 by deleting 96 or 180 bp from its 3′ region through the introduction of stop codons. In comparison to the wild-type gene, cphA NE1del96 conferred about 2.1- to 2.2-fold-higher enzyme activity (up to 5.75 U/mg protein) on E. coli. Furthermore, these cells accumulated about twofold more CGP (up to 34.5% [wt/wt] of CDM) than cells expressing the wild-type gene. An engineered CphA possessing significantly enhanced activity and conferring the highest CGP content on E. coli is demonstrated. In contrast, CphANE1del180 was inactive and did not confer CGP accumulation on E. coli. Interestingly, a short conserved stretch of 4 to 5 hydrophobic amino acids is located in the protein region present in CphANE1del96 but absent in CphANE1del180. In addition, CphANE1 and CphANE1del96 are, besides CphA from Acinetobacter baylyi, the only CphAs exhibiting rigid substrate specificities that do not enable the incorporation of lysine instead of arginine into CGP.

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Overexpression of the wild-type gene coding for Escherichia coli DNA adenine methylase (dam)

Biochemical Journal ◽

10.1042/bj2830745 ◽

1992 ◽

Vol 283 (3) ◽

pp. 745-750 ◽

Cited By ~ 5

Author(s):

V U Nwosu

Keyword(s):

Escherichia Coli ◽

Cellular Protein ◽

Wild Type ◽

Recognition Sequence ◽

Methyl Transfer ◽

Gene Coding ◽

Dna Adenine Methylase ◽

Dam Methylase ◽

Pcr Method ◽

Type Gene

The gene coding for Escherichia coli dam methylase was isolated from a dam+ K12 strain by the PCR method. The gene was subcloned into an overexpression vector under the control of the strong lambda PL promoter. The resultant construct produced the dam methylase at about 20% of total cellular protein. Purification of the protein was achieved with two chromatography columns and yielded 6 mg of pure methylase per gram cell paste. The methylase readily methylates the synthetic dodecamer GACTGATCAGTC containing its recognition sequence (underlined). It also methylates a synthetic dodecamer containing the EcoRV recognition sequence GATATC. However, methyl transfer is to the second adenine in the EcoRV sequence.

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Novel Temperature-Sensitive Mutants of Escherichia coli That Are Unable To Grow in the Absence of Wild-Type tRNA6Leu

Journal of Bacteriology ◽

10.1128/jb.180.11.2931-2935.1998 ◽

1998 ◽

Vol 180 (11) ◽

pp. 2931-2935 ◽

Cited By ~ 15

Author(s):

Toru Nakayashiki ◽

Hachiro Inokuchi

Keyword(s):

Escherichia Coli ◽

Single Copy ◽

Temperature Sensitive ◽

Wild Type ◽

Suppressor Activity ◽

Temperature Sensitive Mutants ◽

Wobble Position ◽

Group I ◽

Group Ii ◽

Type Gene

ABSTRACT Escherichia coli has only a single copy of a gene for tRNA6 Leu (Y. Komine et al., J. Mol. Biol. 212:579–598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O 2-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA4 Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O 2-methyluridine) as its anticodon, tRNA6 Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA6 Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su+6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA6 Leu. These tRNA6 Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaAgene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA4 Leu restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA4 Leu poorly recognizes the UUG codon at 42°C and that the wild-type tRNA6 Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA6 Leu remains to be investigated.

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Structural and Functional Lesions in Brush Border of Human Polarized Intestinal Caco-2/TC7 Cells Infected by Members of the Afa/Dr Diffusely Adhering Family of Escherichia coli

Infection and Immunity ◽

10.1128/iai.68.10.5979-5990.2000 ◽

2000 ◽

Vol 68 (10) ◽

pp. 5979-5990 ◽

Cited By ~ 40

Author(s):

Isabelle Peiffer ◽

Julie Guignot ◽

Alain Barbat ◽

Christophe Carnoy ◽

Steve L. Moseley ◽

...

Keyword(s):

Escherichia Coli ◽

Brush Border ◽

Glucose Transporter ◽

Binding Capacity ◽

Cell Interaction ◽

Cytoskeletal Proteins ◽

Wild Type ◽

Fimbrial Adhesin ◽

Human Intestinal Cells ◽

Dependent Mechanism

ABSTRACT Diffusely adhering Escherichia coli (DAEC) strains expressing F1845 fimbrial adhesin or Dr hemagglutinin belonging to the Afa/Dr family of adhesins infect cultured polarized human intestinal cells through recognition of the brush border-associated decay-accelerating factor (DAF; CD55) as a receptor. The wild-type Afa/Dr DAEC strain C1845 has been shown to induce brush border lesions by an adhesin-dependent mechanism triggering apical F-actin rearrangements. In the present study, we undertook to further characterize cell injuries following the interaction of wild-type Afa/Dr DAEC strains C1845 and IH11128 expressing fimbrial F1845 adhesin and Dr hemagglutinin, respectively, with polarized, fully differentiated Caco-2/TC7 cells. In both cases, bacterium-cell interaction was followed by rearrangement of the major brush border-associated cytoskeletal proteins F-actin, villin, and fimbrin, proteins which play a pivotal role in brush border assembly. In contrast, distribution of G-actin, actin-depolymerizing factor, and tubulin was not modified. Using draE mutants, we found that a mutant in which cysteine replaces aspartic acid at position 54 conserved binding capacity but failed to induce F-actin disassembly. Accompanying the cytoskeleton injuries, we found that the distribution of brush border-associated functional proteins sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV), glucose transporter SGLT1, and fructose transporter GLUT5 was dramatically altered. In parallel, SI and DPPIV enzyme activity decreased.

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Chaperone DnaJ Influences the Formation of Biofilm by Escherichia coli

Polish Journal of Microbiology ◽

10.5604/01.3001.0009.2123 ◽

2015 ◽

Vol 64 (3) ◽

pp. 279-283 ◽

Cited By ~ 9

Author(s):

Anna Grudniak ◽

Jolanta Włodkowska ◽

Krystyna Wolska

Keyword(s):

Escherichia Coli ◽

Heat Shock ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Cell Physiology ◽

Wild Type ◽

Type Gene ◽

Biofilm Development

DnaJ chaperone, a member of the so called DnaK-DnaJ-GrpE chaperone machine plays an important role in cell physiology. The ability of Escherichia coli ΔdnaJ mutant to form biofilm was studied. It was shown that this mutant is impaired in biofilm development when exposed to 42°C for 2 h. The impairment in biofilm development was observed when the heat shock was applied either at the onset of biofilm formation or 2 h later. The biofilm formed was thinner and its structure was changed as compared to wild-type strain. This defect could be complemented by the introduction of a wild-type gene on a low-copy plasmid.

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Role of a novel fimbrial adhesin in acid-induced host adhesion of escherichia coli 0157:H7

10.32920/ryerson.14655759.v1 ◽

2021 ◽

Author(s):

Frances B Chingcuanco

Keyword(s):

Escherichia Coli ◽

Cell Adhesion ◽

Acid Stress ◽

Whole Genome ◽

Deficient Mutant ◽

Wild Type ◽

Array Analysis ◽

Genome Array ◽

Fimbrial Adhesin

Enterohemorrhagic Eschericia coli (EHEC) 0157:H7 must be able to survive the acid stress of gastric passage. Previous studies show that adhesion of EHEC 0157:H7 to human epithelial cells is increased if EHEC was pre-exposed to acid. Whole genome array analysis of EHEC reveals that the putative fimbrial gene yadK is significantly upregulated after EHEC exposure to acid treatments. In this study, a YadK deficient mutant (△yadK) in EHEC 0157:H7 wild-type (WT) strain 85-170 was constructed and phenotyped. The results of this study demonstrate that △yadK exposed to acid stress showed no acid-induced increase in bacteria-host cell adhesion observed in similarly acid-induced wild type cells. This study concludes that YadK plays a role in acid-induced host adhesion of EHEC 0157:H7. It also indicates that acid stress, which is a part of the host’s natural assault to resist invasion, may regulate factors responsible for enhanced bacteria-host attachment, resulting in increased EHEC virulence.

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Membrane-Bound Division Proteins DivIB and DivIC ofBacillus subtilis Function Solely through Their External Domains in both Vegetative and Sporulation Division

Journal of Bacteriology ◽

10.1128/jb.181.9.2710-2718.1999 ◽

1999 ◽

Vol 181 (9) ◽

pp. 2710-2718 ◽

Cited By ~ 31

Author(s):

Vittorio L. Katis ◽

R. Gerry Wake

Keyword(s):

Escherichia Coli ◽

The Other ◽

Wild Type ◽

Transmembrane Segment ◽

Ofbacillus Subtilis ◽

Transmembrane Segments ◽

Terminal Domain ◽

Division Site ◽

Membrane Bound ◽

Type Gene

ABSTRACT The Bacillus subtilis membrane-bound division proteins, DivIB and DivIC, each contain a single transmembrane segment flanked by a short cytoplasmic N-terminal domain and a larger external C-terminal domain. Both proteins become localized at the division site prior to septation. Mutagenesis of both divIB and divICwas performed whereby the sequences encoding the cytoplasmic domains were replaced by the corresponding sequence of the other gene. Finally, the cytoplasmic-plus-transmembrane-encoding domain of each protein was replaced by a totally foreign sequence not involved in division, that encodes the N-terminal-plus-transmembrane domains of theEscherichia coli TolR protein. B. subtilisstrains expressing the divIB and divIC hybrids, in the absence of the wild-type gene, were viable when grown under conditions in which the wild-type genes were found previously to be essential. Furthermore, these strains were able to sporulate to near normal levels. Thus, the cytoplasmic and transmembrane segments of DivIB and DivIC do not appear to have any specific functions other than to anchor these proteins correctly in the membrane. The implications of these findings are discussed.

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