Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis

The Escherichia coli CysG protein (sirohaem synthase) catalyses four separate reactions that are required for the transformation of uroporphyrinogen III into sirohaem, initially two S-adenosyl-L-methionine-dependent transmethylations at positions 2 and 7, mediated through the C-terminal, or CysGA, catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal, or CysGB, catalytic domain of the enzyme. This report describes how the deletion of the NAD+-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysGB but does not affect the catalytic activity of CysGA, whereas the mutation of a number of phylogenetically conserved residues within CysGA is detrimental to the transmethylation reaction but does not affect the activity of CysGB. Further studies have shown that CysGB is not essential for cobalamin biosynthesis because the presence of the Salmonella typhimurium CobI operon with either cysGA or the Pseudomonas denitrificans cobA are sufficient for the synthesis of cobyric acid in an E. coli cysG deletion strain. Evidence is also presented to suggest that a gene within the S. typhimurium CobI operon might act as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.

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The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Catalysts ◽

10.3390/catal11080926 ◽

2021 ◽

Vol 11 (8) ◽

pp. 926

Author(s):

Maria C. Martins ◽

Susana F. Fernandes ◽

Bruno A. Salgueiro ◽

Jéssica C. Soares ◽

Célia V. Romão ◽

...

Keyword(s):

Amino Acids ◽

Catalytic Domain ◽

Reductase Activity ◽

Conserved Residues ◽

Mutant Proteins ◽

E Coli ◽

C Terminus ◽

Bona Fide ◽

Oxygen Reductase ◽

Soluble Enzymes

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

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Escherichia coli Contamination of Vegetables Grown in Soils Fertilized with Noncomposted Bovine Manure: Garden-Scale Studies

Applied and Environmental Microbiology ◽

10.1128/aem.70.11.6420-6427.2004 ◽

2004 ◽

Vol 70 (11) ◽

pp. 6420-6427 ◽

Cited By ~ 57

Author(s):

Steven C. Ingham ◽

Jill A. Losinski ◽

Matthew P. Andrews ◽

Jane E. Breuer ◽

Jeffry R. Breuer ◽

...

Keyword(s):

Escherichia Coli ◽

Tap Water ◽

Silty Clay ◽

Silt Loam ◽

Silty Clay Loam ◽

Manure Application ◽

E Coli ◽

Fertilized Soil ◽

Low Levels ◽

National Organic Program

ABSTRACT In this study we tested the validity of the National Organic Program (NOP) requirement for a ≥120-day interval between application of noncomposted manure and harvesting of vegetables grown in manure-fertilized soil. Noncomposted bovine manure was applied to 9.3-m2 plots at three Wisconsin sites (loamy sand, silt loam, and silty clay loam) prior to spring and summer planting of carrots, radishes, and lettuce. Soil and washed (30 s under running tap water) vegetables were analyzed for indigenous Escherichia coli. Within 90 days, the level of E. coli in manure-fertilized soil generally decreased by about 3 log CFU/g from initial levels of 4.2 to 4.4 log CFU/g. Low levels of E. coli generally persisted in manure-fertilized soil for more than 100 days and were detected in enriched soil from all three sites 132 to 168 days after manure application. For carrots and lettuce, at least one enrichment-negative sample was obtained ≤100 days after manure application for 63 and 88% of the treatments, respectively. The current ≥120-day limit provided an even greater likelihood of not detecting E. coli on carrots (≥1 enrichment-negative result for 100% of the treatments). The rapid maturation of radishes prevented conclusive evaluation of a 100- or 120-day application-to-harvest interval. The absolute absence of E. coli from vegetables harvested from manure-fertilized Wisconsin soils may not be ensured solely by adherence to the NOP ≥120-day limit. Unless pathogens are far better at colonizing vegetables than indigenous E. coli strains are, it appears that the risk of contamination for vegetables grown in Wisconsin soils would be elevated only slightly by reducing the NOP requirement to ≥100 days.

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Mechanically Ventilated Broiler Sheds: a Possible Source of Aerosolized Salmonella, Campylobacter, and Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01380-09 ◽

2009 ◽

Vol 75 (23) ◽

pp. 7417-7425 ◽

Cited By ~ 43

Author(s):

H. N. Chinivasagam ◽

T. Tran ◽

L. Maddock ◽

A. Gale ◽

P. J. Blackall

Keyword(s):

Escherichia Coli ◽

Most Probable Number ◽

Production Cycle ◽

Airborne Bacteria ◽

Probable Number ◽

Mechanically Ventilated ◽

The Public ◽

E Coli ◽

Poultry Farms ◽

Low Levels

ABSTRACT This study assessed the levels of two key pathogens, Salmonella and Campylobacter, along with the indicator organism Escherichia coli in aerosols within and outside poultry sheds. The study ranged over a 3-year period on four poultry farms and consisted of six trials across the boiler production cycle of around 55 days. Weekly testing of litter and aerosols was carried out through the cycle. A key point that emerged is that the levels of airborne bacteria are linked to the levels of these bacteria in litter. This hypothesis was demonstrated by E. coli. The typical levels of E. coli in litter were ∼108 CFU g−1 and, as a consequence, were in the range of 102 to 104 CFU m−3 in aerosols, both inside and outside the shed. The external levels were always lower than the internal levels. Salmonella was only present intermittently in litter and at lower levels (103 to 105 most probable number [MPN] g−1) and consequently present only intermittently and at low levels in air inside (range of 0.65 to 4.4 MPN m−3) and once outside (2.3 MPN m−3). The Salmonella serovars isolated in litter were generally also isolated from aerosols and dust, with the Salmonella serovars Chester and Sofia being the dominant serovars across these interfaces. Campylobacter was detected late in the production cycle, in litter at levels of around 107 MPN g−1. Campylobacter was detected only once inside the shed and then at low levels of 2.2 MPN m−3. Thus, the public health risk from these organisms in poultry environments via the aerosol pathway is minimal.

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Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

Journal of Bacteriology ◽

10.1128/jb.188.6.2163-2172.2006 ◽

2006 ◽

Vol 188 (6) ◽

pp. 2163-2172 ◽

Cited By ~ 212

Author(s):

Paul W. King ◽

Matthew C. Posewitz ◽

Maria L. Ghirardi ◽

Michael Seibert

Keyword(s):

Escherichia Coli ◽

Catalytic Domain ◽

Expression System ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

E Coli ◽

Functional Studies ◽

Hydrogenase Maturation ◽

Iron Sulfur ◽

And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

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O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.152.1.534-537.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 534-537

Author(s):

S Mitra ◽

B C Pal ◽

R S Foote

Keyword(s):

Escherichia Coli ◽

Dna Methyltransferase ◽

Wild Type ◽

E Coli ◽

Methylguanine Dna Methyltransferase ◽

Synthetic Dna ◽

In The Wild ◽

Low Levels ◽

Enzyme Molecules ◽

Dna Substrate

O(6)-Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3H-labeled O(6)-methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase.

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Eliminating false positives in a qPCR assay for the detection of the uidA gene in Escherichia coli

Journal of Water and Health ◽

10.2166/wh.2013.201 ◽

2013 ◽

Vol 11 (3) ◽

pp. 382-386 ◽

Cited By ~ 1

Author(s):

Richard Kibbee ◽

Natalie Linklater ◽

Banu Örmeci

Keyword(s):

Escherichia Coli ◽

Quantitative Polymerase Chain Reaction ◽

Qpcr Assay ◽

Uida Gene ◽

Chain Reaction ◽

Taq Polymerase ◽

E Coli ◽

Gene Copies ◽

Polymerase Chain ◽

Low Levels

Due to contaminant Escherichia coli DNA present in recombinant Taq polymerase reagents, it is not possible to reliably detect low levels of E. coli in samples using the quantitative polymerase chain reaction (qPCR) assay. Native Taq polymerase was successfully used in this study to detect five uidA gene copies (5 fg of genomic DNA) of the uidA gene.

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Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease

Biochemical Journal ◽

10.1042/bj20120649 ◽

2012 ◽

Vol 446 (2) ◽

pp. 311-320 ◽

Cited By ~ 14

Author(s):

Anders Tryggvesson ◽

Frida M. Ståhlberg ◽

Axel Mogk ◽

Kornelius Zeth ◽

Adrian K. Clarke

Keyword(s):

Escherichia Coli ◽

Active Sites ◽

Terminal Sequence ◽

Core Complex ◽

Clp Protease ◽

E Coli ◽

Loop Region ◽

N Terminus ◽

Specific Association ◽

The Stability

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC–ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

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Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

Journal of Bacteriology ◽

10.1128/jb.183.2.570-579.2001 ◽

2001 ◽

Vol 183 (2) ◽

pp. 570-579 ◽

Cited By ~ 66

Author(s):

Michal Gropp ◽

Yael Strausz ◽

Miriam Gross ◽

Gad Glaser

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Catalytic Domain ◽

Mutational Analysis ◽

Amino Acid Starvation ◽

E Coli ◽

Terminal Domain ◽

Negative Effect ◽

And Control ◽

Two Hybrid

ABSTRACT The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD inrelA + cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

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Horizontal Transmission of Escherichia coli O157:H7 during Cattle Housing

Journal of Food Protection ◽

10.4315/0362-028x-67.12.2651 ◽

2004 ◽

Vol 67 (12) ◽

pp. 2651-2656 ◽

Cited By ~ 41

Author(s):

P. McGEE ◽

L. SCOTT ◽

J. J. SHERIDAN ◽

B. EARLEY ◽

N. LEONARD

Keyword(s):

Escherichia Coli ◽

Drinking Water ◽

Animal Behavior ◽

Horizontal Transmission ◽

Escherichia Coli O157 ◽

E Coli ◽

Holstein Friesian ◽

Water Feed ◽

Low Levels ◽

The Right

Ruminant livestock, particularly cattle, is considered the primary reservoir of Escherichia coli O157:H7. This study examines the transmission of E. coli O157:H7 within groups of cattle during winter housing. Holstein Friesian steers were grouped in six pens of five animals. An animal inoculated with and proven to be shedding a marked strain of E. coli O157: H7 was introduced into each pen. Fecal (rectal swabs) and hide samples (900 cm2 from the right rump) were taken from the 36 animals throughout the study. Water, feed, and gate or partition samples from each pen were also examined. Within 24 h of introducing the inoculated animals into the pens, samples collected from the drinking water, pen barriers, and animal hides were positive for the pathogen. Within 48 h, the hides of 20 (66%) of 30 cohort animals from the six pens were contaminated with E. coli O157:H7. The first positive fecal samples from the noninoculated cohort animals were detected 3 days after the introduction of the inoculated steers. During the 23 days of the study, 15 of 30 cohort animals shed the marked E. coli O157: H7 strain in their feces on at least one occasion. Animal behavior in the pens was monitored during a 12-h period using closed circuit television cameras. The camera footage showed an average of 13 instances of animal grooming in each pen per hour. The study suggests that transmission of E. coli O157:H7 between animals may occur following ingestion of the pathogen at low levels and that animal hide may be an important source of transmission.

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Evaluation of Immunocompetent Urinary Tract Infected Balb/C Mouse Model For the Study of Antibiotic Resistance Development Using Escherichia Coli CFT073 Infection

Antibiotics ◽

10.3390/antibiotics8040170 ◽

2019 ◽

Vol 8 (4) ◽

pp. 170 ◽

Cited By ~ 1

Author(s):

Ashok Chockalingam ◽

Sharron Stewart ◽

Lin Xu ◽

Adarsh Gandhi ◽

Murali K. Matta ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Urinary Tract ◽

Bladder Tissue ◽

Resistance Development ◽

Once Daily ◽

E Coli ◽

Low Levels ◽

Tract Infections

Urinary tract infections (UTI) are common worldwide and are becoming increasingly difficult to treat because of the development of antibiotic resistance. Immunocompetent murine models of human UTI have been used to study pathogenesis and treatment but not for investigating resistance development after treatment with antibiotics. In this study, intravesical inoculation of uropathogenic Escherichia coli CFT073 in immunocompetent Balb/c mice was used as a model of human UTI. The value of the model in investigating antibiotic exposure on in vivo emergence of antibiotic resistance was examined. Experimentally infected mice were treated with 20 or 200 mg/kg ampicillin, 5 or 50 mg/kg ciprofloxacin, or 100 or 1000 mg/kg of fosfomycin. Ampicillin and ciprofloxacin were given twice daily at 8 h intervals, and fosfomycin was given once daily. Antibiotic treatment began 24 h after bacterial inoculation and ended after 72 h following the initial treatment. Although minimum inhibitory concentrations (MIC) for the experimental strain of E. coli were exceeded at peak concentrations in tissues and consistently in urine, low levels of bacteria persisted in tissues in all experiments. E. coli from bladder tissue, kidney, and urine grew on plates containing 1× MIC of antibiotic, but none grew at 3× MIC. This model is not suitable for studying emergent resistance but might serve to examine bacterial persistence.

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