scholarly journals Conservation and Variation between Rhodobacter capsulatus and Escherichia coli Tat Systems

2006 ◽  
Vol 188 (22) ◽  
pp. 7807-7814 ◽  
Author(s):  
Ute Lindenstrauß ◽  
Thomas Brüser
Keyword(s):  
Escherichia Coli ◽  
Dimethyl Sulfoxide ◽  
Formate Dehydrogenase ◽  
Rhodobacter Capsulatus ◽  
Translational Coupling ◽  
E Coli ◽  
Biochemical Assays ◽  
Tat System ◽  
Respiration Deficiency ◽  
Physiological And Biochemical

ABSTRACT The Tat system allows the translocation of folded and often cofactor-containing proteins across biological membranes. Here, we show by an interspecies transfer of a complete Tat translocon that Tat systems are largely, but not fully, interchangeable even between different classes of proteobacteria. The Tat apparatus from the α-proteobacterium Rhodobacter capsulatus was transferred to a Tat-deficient Escherichia coli strain, which is a γ-proteobacterium. Similar to that of E. coli, the R. capsulatus Tat system consists of three components, rc-TatA, rc-TatB, and rc-TatC. A fourth gene (rc-tatF) is present in the rc-tatABCF operon which has no apparent relevance for translocation. The translational starts of rc-tatC and rc-tatF overlap in four nucleotides (ATGA) with the preceding tat genes, pointing to efficient translational coupling of rc-tatB, rc-tatC, and rc-tatF. We show by a variety of physiological and biochemical assays that the R. capsulatus Tat system functionally targets the E. coli Tat substrates TorA, AmiA, AmiC, and formate dehydrogenase. Even a Tat substrate from a third organism is accepted, demonstrating that usually Tat systems and Tat substrates from different proteobacteria are compatible with each other. Only one exceptional Tat substrate of E. coli, a membrane-anchored dimethyl sulfoxide (DMSO) reductase, was not targeted by the R. capsulatus Tat system, resulting in a DMSO respiration deficiency. Although the general features of Tat substrates and translocons are similar between species, the data indicate that details in the targeting pathways can vary considerably.

scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1421-1431 ◽  
Author(s):  
Patrice Bruscella ◽  
Laure Cassagnaud ◽  
Jeanine Ratouchniak ◽  
Gaël Brasseur ◽  
Elisabeth Lojou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acidithiobacillus Ferrooxidans ◽  
Biochemical Properties ◽  
Gene Encoding ◽  
Iron Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
Tat System ◽  
Micro Organisms

The gene encoding a putative high-potential iron–sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron–sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a ΔtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

scholarly journals Antibacterial effect of Dimethyl sulfoxide extract of Aloe vera (Aloe barbadensis) leaf gel against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae

Mediscope ◽  
2020 ◽  
Vol 7 (2) ◽  
pp. 67-74 ◽  
Author(s):  
Syed Didarul Haque ◽  
Abu Md Mayeenuddin Al Amin ◽  
Baishakhi Islam ◽  
Nazia Nazneen ◽  
Syeda Noorjahan Karim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Klebsiella Pneumoniae ◽  
Dimethyl Sulfoxide ◽  
Antibacterial Effect ◽  
Aloe Vera ◽  
Biologically Active ◽  
Diffusion Method ◽  
E Coli

An exploratory study based on laboratory experiment was carried out to determine the antibacterial effect of Dimethyl sulfoxide (DMSO) extract of Aloe vera leaf gel (DAE) against standard strains of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae in the Department of Pharmacology & Therapeutics in collaboration with the Department of Microbiology, Mymensingh Medical College, Mymensingh, Bangladesh. DMSO extract was used in five different concentrations (100, 200, 300, 400 and 500 μg/ml). Dose dependent inhibitory effect was seen against the test organisms using disc diffusion method. Zone of inhibition (ZOI) were 8 mm, 13 mm, 15 mm, 16 mm and 21 mm against S. aureus; 0 mm, 8 mm, 13 mm, 15 mm and 18 mm against P. aeruginosa; 8 mm, 11 mm, 13 mm, 16 mm and 20 mm against E. coli; 0 mm, 9 mm, 12 mm, 14 mm and 18 mm against K. pneumoniae at 100, 200, 300, 400 and 500 μg/ml respectively. The minimum inhibitory concentration (MIC) was assessed by broth dilution technique. The MICs of DAE for S. aureus, P. aeruginosa, E. coli and K. pneumoniae were 300 μg/ml, 400 μg/ml, 400 μg/ml and 450 μg/ml respectively. From the study it was observed that DMSO extract of Aloe vera leaf gel possesses antibacterial effect against the test pathogens. The findings highlight the need for further extensive study to detect and isolate the biologically active ingredients present in the Aloe vera leaves which are responsible for antibacterial effect. Hopefully, that would lead to the discovery of new and more potent antimicrobial agents originated from Aloe vera. Mediscope Vol. 7, No. 2: July 2020, Page 67-74

scholarly journals Contribution of the Twin Arginine Translocation System to the Virulence of Enterohemorrhagic Escherichia coli O157:H7

2003 ◽  
Vol 71 (9) ◽  
pp. 4908-4916 ◽  
Author(s):  
Nathalie Pradel ◽  
Changyun Ye ◽  
Valérie Livrelli ◽  
Jianguo Xu ◽  
Bernard Joly ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Immunoblot Analysis ◽  
Vero Cells ◽  
Soft Agar ◽  
Enterohemorrhagic Escherichia Coli ◽  
E Coli ◽  
Twin Arginine Translocation ◽  
Tat System ◽  
K 12

ABSTRACT Shiga toxin-producing Escherichia coli O157:H7 is a major food-borne infectious pathogen. In order to analyze the contribution of the twin arginine translocation (TAT) system to the virulence of E. coli O157:H7, we deleted the tatABC genes of the O157:H7 EDL933 reference strain. The mutant displayed attenuated toxicity on Vero cells and completely lost motility on soft agar plates. Further analyses revealed that the ΔtatABC mutation impaired the secretion of the Shiga toxin 1 (Stx1) and abolished the synthesis of H7 flagellin, which are two major known virulence factors of enterohemorrhagic E. coli O157:H7. Expression of the EDL933 stxAB 1 genes in E. coli K-12 conferred verotoxicity on this nonpathogenic strain. Remarkably, cytotoxicity assay and immunoblot analysis showed, for the first time, an accumulation of the holotoxin complex in the periplasm of the wild-type strain and that a much smaller amount of StxA1 and reduced verotoxicity were detected in the ΔtatC mutant cells. Together, these results establish that the TAT system of E. coli O157:H7 is an important virulence determinant of this enterohemorrhagic pathogen.

In vivo associations of Escherichia coli NarJ with a peptide of the first 50 residues of nitrate reductase catalytic subunit NarG

2009 ◽  
Vol 55 (2) ◽  
pp. 179-188 ◽  
Author(s):  
Haiming Li ◽  
Raymond J. Turner
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Catalytic Subunit ◽  
Bimolecular Fluorescence Complementation ◽  
E Coli ◽  
N Terminus ◽  
Tat System

The catalytic subunit of many Escherichia coli redox enzymes bares a twin-arginine translocation (Tat)-dependent signal peptide in its precursor, which directs the redox enzyme complex to this Sec-independent pathway. NarG of the E. coli nitrate reductase NarGHI complex possesses a vestige twin-arginine motif at its N terminus. During the cofactor insertion, and assembly and folding of the NarG–NarH complex, a chaperone protein, NarJ, is thought to interact with the N terminus and an unknown second site of NarG. Our previous in vitro study provided evidence that NarJ’s role shows some Tat system dependence. In this work, we investigated the associations of NarJ with a peptide of the first 50 residues of NarG (NarG50) in living cells. Two approaches were used: the Förster resonance energy transfer (FRET) based on yellow fluorescent protein – cyan fluorescent protein (YFP–CFP) and the bimolecular fluorescence complementation (BiFC). Compared with the wild-type (WT) E. coli cotransformants expressing both NarJ–YFP and NarG50–CFP, tat gene mutants gave an apparent FRET efficiency (Eapp) that was on the order of 25%–40% lower. These experiments implied a Tat system dependency of the in vivo associations between NarJ and the NarG50 peptide. In the BiFC assay, a 4-fold lower specific fluorescence intensity was observed for the E. coli WT cotransformants expressing both NarJ–Yc and NarG50–Yn than for its tat mutants, again suggesting a Tat dependence of the interactions. Fluorescence microscopy showed a “dot”/unipolar distribution of the reassembled YFP–NarJ:NarG50 both in WT and tat mutants, demonstrating a distinct localization of the interaction. Thus, although the degree of the interaction shows Tat dependence, the cell localization is less so. Taken together, these data further support that NarJ’s activity on NarG may be assisted by the Tat system.

scholarly journals Facile synthesis of bioactive Allitol from D-psicose by coexpression of ribitol dehydrogenase and formate dehydrogenase in Escherichia Coli

2018 ◽  
Vol 4 ◽  
Author(s):  
Hinawi A.M. Hassanin ◽  
Mohammed A.A. Eassa ◽  
Bo Jiang
Keyword(s):  
Escherichia Coli ◽  
Substrate Concentration ◽  
Formate Dehydrogenase ◽  
Maximum Yield ◽  
Optimum Conditions ◽  
Facile Synthesis ◽  
Nadh Regeneration ◽  
E Coli ◽  
Ribitol Dehydrogenase

Coexpression of formate dehydrogenase (FDH) and ribitol dehydrogenase (RDH) in Escherichia coli was used for the synthesis of Allitol from D-psicose. FDH was coexpressed with RDH for continuous NADH regeneration. The results revealed that the optimum conditions for allitol production occurred at pH 7.0 and 30 °C. Allitol reached the maximum yield of 19.2 mg at 2.0% substrate concentration after 48 hours. Using D-psicose as a substrate, allitol was successfully produced using an engineered E. coli coexpressed with RDH and FDH.

Functional Complementation in Escherichia coli of Different Phytoene Desaturase Genes and Analysis of Accumulated Carotenes

1991 ◽  
Vol 46 (11-12) ◽  
pp. 1045-1051 ◽  
Author(s):  
Hartmut Linden ◽  
Norihiko Misawa ◽  
Daniel Chamovitz ◽  
Iris Pecker ◽  
Joseph Hirschberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Phytoene Desaturase ◽  
Reaction Products ◽  
Double Bonds ◽  
E Coli ◽  
Synechococcus Pcc 7942 ◽  
Lycopene Cyclase ◽  
Pcc 7942 ◽  
Cis Isomer

Three different phytoene desaturase genes, from Rhodobacter capsulatus, Erwinia uredovora, and Synechococcus PCC 7942, have been functionally complemented with a gene construct from E. uredovora which encodes all enzymes responsible for formation of 15-cis phytoene in Escherichia coli. As indicated by the contrasting reaction products detected in the pigmented E. coli cells after co-transformation, a wide functional diversity of these three different types of phytoene desaturases can be concluded. The carotenes formed by the phytoene desaturase from R. capsulatus were trans-neurosporene with three additional double bonds and two cis isomers. Furthermore, small amounts of three ζ-carotene isomers (2 double bonds more than phytoene) and phytofluene (15-cis and all-trans with + 1 double bond) were detected as inter- mediates. When the subsequent genes from E. uredovora which encode for lycopene cyclase and β-carotene hydroxylase were present, neurosporene, the phytoene desaturase product of R. capsulatus, was subsequently converted to the monocyclic β-zeacarotene and its mono- hydroxylation product. The most abundant carotene resulting from phytoene desaturation by the E. uredovora enzyme was trans-lycopene together with a cis isomer. In addition, bisdehy-drolycopene was also formed. The reaction products of Synechococcus phytoene desaturase were two cis isomers of ζ-carotene and only small amounts of trans-ζ-carotene including 15-cis. The I50 values for flurtamone and diphenylamine to inhibit phytoene desaturation were determined and differential inhibition was observed for diphenylamine.

scholarly journals Asymmetric reduction of racemic sulfoxides by dimethyl sulfoxide reductases from Rhodobacter capsulatus, Escherichia coli and Proteus species

Microbiology ◽  
1998 ◽  
Vol 144 (8) ◽  
pp. 2247-2253 ◽  
Author(s):  
S. P. Hanlon ◽  
D. L. Graham ◽  
P. J. Hogan ◽  
R. A. Holt ◽  
C. D. Reeve ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dimethyl Sulfoxide ◽  
Rhodobacter Capsulatus ◽  
Asymmetric Reduction ◽  
Proteus Species

Amplification of the put genes and identification of the put gene products in Escherichia coli K12

1980 ◽  
Vol 58 (10) ◽  
pp. 787-796 ◽  
Author(s):  
Janet M. Wood ◽  
David Zadworny
Keyword(s):  
Escherichia Coli ◽  
Dehydrogenase Activity ◽  
Gene Products ◽  
Proline Dehydrogenase ◽  
Transport Activity ◽  
E Coli ◽  
Proline Transport ◽  
Escherichia Coli K12 ◽  
Biochemical Assays ◽  
Transport Defect

The utilization of L-proline as carbon or nitrogen source for the growth of Escherichia coli K12 requires the activities of an L-proline porter (PP-I) and a bifunctional L-proline dehydrogenase – Δ1-pyrroline carboxylate dehydrogenase. PP-I is inactivated by mutations at putP and the bifunctional dehydrogenase is encoded in the adjacent locus, putA, at 22 min on the chromosome map. Two additional loci, proP (at 92 min) and proT (at 82 min), have also been implicated in L-proline transport. We have studied four ColE1/E. coli K12 hybrid plasmids from the plasmid bank prepared by Clarke and Carbon. Each of these plasmids was shown previously to complement an L-proline transport defect in E. coli. Genetic complementation analysis and biochemical assays of L-proline transport and L-proline dehydrogenase activity show that three of these hybrid plasmids bear the putPA region of the E. coli chromosome (plasmids pLC4-45, pLC10-29, and pLC43-41). The fourth plasmid, pLC35-38, specifically enhances the L-proline transport activity of its host bacteria but not their L-proline dehydrogenase activity. It probably encodes putP. We have used these plasmids in an E. coli minicell system to identify the putA and putP gene products.

scholarly journals Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

2018 ◽  
Author(s):  
Huan Fang ◽  
Dong Li ◽  
Jie Kang ◽  
Pingtao Jiang ◽  
Jibin Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Biosynthetic Pathway ◽  
Rhodobacter Capsulatus ◽  
De Novo ◽  
Vitamin B ◽  
E Coli ◽  
De Novo Biosynthesis ◽  
Optimization Of Fermentation ◽  
Aerobic And Anaerobic

ABSTRACTThe only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea, and the only unknown step in its biosynthesis is the production of the intermediate adenosylcobinamide phosphate. Here, using genetic and metabolic engineering, we generated an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. Excitingly, the BluE and CobC enzymes from Rhodobacter capsulatus transform L-threonine into (R)-1-Amino-2-propanol O-2-Phosphate, which is then condensed with adenosylcobyric acid to yield adenosylcobinamide phosphate by either CobD from the aeroic R. capsulatus or CbiB from the anerobic Salmonella typhimurium. These findings suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increased the vitamin B12 yield of a recombinant E. coli strain by more than ∼250-fold to 307.00 µg/g DCW via metabolic engineering and optimization of fermentation conditions. Beyond our scientific insights about the aerobic and anaerobic pathways and our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.

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