The Order Bacillales Hosts Functional Homologs of the WorrisomecfrAntibiotic Resistance Gene

ABSTRACTThecfrgene encodes the Cfr methyltransferase that methylates a single adenine in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to several classes of antibiotics that include drugs of clinical and veterinary importance. This paper describes a first step toward elucidating natural residences of the worrisomecfrgene and functionally similar genes. Threecfr-like genes from the orderBacillaleswere identified from BLAST searches and cloned into plasmids under the control of an inducible promoter. Expression of the genes was induced inEscherichia coli, and MICs for selected antibiotics indicate that thecfr-like genes confer resistance to PhLOPSa (phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A) antibiotics in the same way as thecfrgene. In addition, modification at A2503 on 23S rRNA was confirmed by primer extension. Finally, expression of the Cfr-like proteins was verified by SDS gel electrophoresis of whole-cell extracts. The work shows thatcfr-like genes exist in the environment and thatBacillalesare natural residences ofcfr-like genes.

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Resistance Gene Transfer during Treatments for Experimental Avian Colibacillosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05617-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 189-196 ◽

Cited By ~ 19

Author(s):

Alexandra Dheilly ◽

Laëtitia Le Devendec ◽

Gwenaëlle Mourand ◽

Axelle Bouder ◽

Eric Jouy ◽

...

Keyword(s):

Escherichia Coli ◽

Gene Transfer ◽

Resistance Gene ◽

Gel Electrophoresis ◽

Intestinal Tract ◽

Pulsed Field Gel Electrophoresis ◽

Content Type ◽

E Coli ◽

Plasmid Genes ◽

Antimicrobial Treatments

ABSTRACTAn experiment was conducted in animal facilities to compare the impacts of four avian colibacillosis treatments—oxytetracycline (OTC), trimethoprim-sulfadimethoxine (SXT), amoxicillin (AMX), or enrofloxacin (ENR)—on the susceptibility ofEscherichia coliin broiler intestinal tracts. Birds were first orally inoculated with rifampin-resistantE. colistrains bearing plasmid genes conferring resistance to fluoroquinolones (qnr), cephalosporins (blaCTX-MorblaFOX), trimethoprim-sulfonamides, aminoglycosides, or tetracyclines. Feces samples were collected before, during, and after antimicrobial treatments. The susceptibilities ofE. colistrains were studied, and resistance gene transfer was analyzed. An increase in the tetracycline-resistantE. colipopulation was observed only in OTC-treated birds, whereas multiresistantE. coliwas detected in the dominantE. colipopulations of SXT-, AMX-, or ENR-treated birds. Most multiresistantE. colistrains were susceptible to rifampin and exhibited various pulsed-field gel electrophoresis profiles, suggesting the transfer of one of the multiresistance plasmids from the inoculated strains to otherE. colistrains in the intestinal tract. In conclusion, this study clearly illustrates how, inE. coli, “old” antimicrobials may coselect antimicrobial resistance to recent and critical molecules.

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Acfr-Like Gene from Clostridium difficile Confers Multiple Antibiotic Resistance by the Same Mechanism as thecfrGene

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01274-15 ◽

2015 ◽

Vol 59 (9) ◽

pp. 5841-5843 ◽

Cited By ~ 33

Author(s):

Lykke H. Hansen ◽

Birte Vester

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Clostridium Difficile ◽

Primer Extension ◽

23S Rrna ◽

Multiple Antibiotic Resistance ◽

Gene Sequences ◽

Peptidyl Transferase ◽

Content Type

ABSTRACTThe Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA. Manycfr-like gene sequences in the databases code for unknown functions. This study confirms that a Cfr-like protein from aPeptoclostridium difficile(formerlyClostridium difficile) strain does function as a Cfr protein. The enzyme is expressed inEscherichia coliand shows elevated MICs for five classes of antibiotics. A primer extension stop indicates a modification at A2503 in 23S rRNA.

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Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00781-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5238-5246 ◽

Cited By ~ 9

Author(s):

Dongfei Han ◽

Ji-Young Ryu ◽

Robert A. Kanaly ◽

Hor-Gil Hur

Keyword(s):

Escherichia Coli ◽

Pseudomonas Putida ◽

Hypothetical Protein ◽

Aldehyde Group ◽

Agrobacterium Vitis ◽

T7 Promoter ◽

Content Type ◽

E Coli ◽

Cell Extracts ◽

Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

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Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00731-20 ◽

2020 ◽

Vol 86 (14) ◽

Author(s):

Dongchang Sun ◽

Xudan Mao ◽

Mingyue Fei ◽

Ziyan Chen ◽

Tingheng Zhu ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Natural Transformation ◽

Inducible Promoter ◽

Regulation Mechanism ◽

Type I ◽

Content Type ◽

E Coli ◽

Double Stranded Dna ◽

Dna Binding Site

ABSTRACT Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli. However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli. In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

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Prevalence of Antimicrobial Resistance and Transfer of Tetracycline Resistance Genes in Escherichia coli Isolates from Beef Cattle

Applied and Environmental Microbiology ◽

10.1128/aem.01511-15 ◽

2015 ◽

Vol 81 (16) ◽

pp. 5560-5566 ◽

Cited By ~ 29

Author(s):

Seung Won Shin ◽

Min Kyoung Shin ◽

Myunghwan Jung ◽

Kuastros Mekonnen Belaynehe ◽

Han Sang Yoo

Keyword(s):

Escherichia Coli ◽

South Korea ◽

Beef Cattle ◽

Resistance Gene ◽

Resistance Genes ◽

Tetracycline Resistance ◽

Tetracycline Resistance Gene ◽

Content Type ◽

E Coli ◽

Tetracycline Resistance Genes

ABSTRACTThe aim of this study was to investigate the prevalence and transferability of resistance in tetracycline-resistantEscherichia coliisolates recovered from beef cattle in South Korea. A total of 155E. coliisolates were collected from feces in South Korea, and 146 were confirmed to be resistant to tetracycline. The tetracycline resistance genetet(A) (46.5%) was the most prevalent, followed bytet(B) (45.1%) andtet(C) (5.8%). Strains carryingtet(A) plustet(B) andtet(B) plustet(C) were detected in two isolates each. In terms of phylogenetic grouping, 101 (65.2%) isolates were classified as phylogenetic group B1, followed in decreasing order by D (17.4%), A (14.2%), and B2 (3.2%). Ninety-one (62.3%) isolates were determined to be multidrug resistant by the disk diffusion method. MIC testing using the principal tetracyclines, namely, tetracycline, chlortetracycline, oxytetracycline, doxycycline, and minocycline, revealed that isolates carryingtet(B) had higher MIC values than isolates carryingtet(A). Conjugation assays showed that 121 (82.9%) isolates could transfer a tetracycline resistance gene to a recipient via the IncFIB replicon (65.1%). This study suggests that the high prevalence of tetracycline-resistantE. coliisolates in beef cattle is due to the transferability of tetracycline resistance genes betweenE. colipopulations which have survived the selective pressure caused by the use of antimicrobial agents.

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P212A Mutant of Dihydrodaidzein Reductase Enhances (S)-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

Applied and Environmental Microbiology ◽

10.1128/aem.03584-15 ◽

2016 ◽

Vol 82 (7) ◽

pp. 1992-2002 ◽

Cited By ~ 13

Author(s):

Pyung-Gang Lee ◽

Joonwon Kim ◽

Eun-Jung Kim ◽

EunOk Jung ◽

Bishnu Prasad Pandey ◽

...

Keyword(s):

Escherichia Coli ◽

Reaction System ◽

Gut Bacteria ◽

Site Directed Mutagenesis ◽

Anaerobic Growth ◽

Efficient Production ◽

Cell Reaction ◽

Whole Cell ◽

Content Type ◽

Postmenopausal Symptoms

ABSTRACT(S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis fromSlackia isoflavoniconvertensintoEscherichia coliBL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 μM and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis oftrans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%.

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Emergence of Colistin Resistance Gene mcr-1 in Cronobacter sakazakii Producing NDM-9 and in Escherichia coli from the Same Animal

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01444-16 ◽

2016 ◽

Vol 61 (2) ◽

Cited By ~ 29

Author(s):

Bao-Tao Liu ◽

Feng-Jing Song ◽

Ming Zou ◽

Zhi-Hui Hao ◽

Hu Shan

Keyword(s):

Escherichia Coli ◽

Resistance Gene ◽

Resistance Genes ◽

Cronobacter Sakazakii ◽

Colistin Resistance ◽

Content Type ◽

E Coli ◽

Carbapenem Resistant

ABSTRACT We report the presence of mcr-1 in Escherichia coli and carbapenem-resistant Cronobacter sakazakii from the same diseased chicken. The mcr-1 gene linked with ISApl1 was located on two different IncI2 plasmids, including one multidrug plasmid in E. coli, whereas fosA3-bla NDM-9 was on an IncB/O plasmid in C. sakazakii. The development of the fosA3-bla NDM-9 resistance region was mediated by IS26. The colocation of mcr-1 or bla NDM-9 with other resistance genes will accelerate the dissemination of the two genes.

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Plasmid with Colistin Resistance Genemcr-1in Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strains Isolated from Pig Slurry in Estonia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00443-16 ◽

2016 ◽

Vol 60 (11) ◽

pp. 6933-6936 ◽

Cited By ~ 29

Author(s):

Age Brauer ◽

Kaidi Telling ◽

Mailis Laht ◽

Piret Kalmus ◽

Irja Lutsar ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Resistance Gene ◽

Antibiotic Resistance Gene ◽

Dna Transfer ◽

The Other ◽

Pig Slurry ◽

Colistin Resistance ◽

Content Type ◽

Slurry Sample

ABSTRACTA plasmid carrying the colistin resistance genemcr-1was isolated from a pig slurry sample in Estonia. The gene was present on a 33,311-bp plasmid of the IncX4 group.mcr-1is the only antibiotic resistance gene on the plasmid, with the other genes mainly coding for proteins involved in conjugative DNA transfer (taxA,taxB,taxC,trbM, and thepilXoperon). The plasmid pESTMCR was present in three phylogenetically very differentEscherichia colistrains, suggesting that it has high potential for horizontal transfer.

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Suppression of Nonsense Mutations Induced by Expression of an RNA Complementary to a Conserved Segment of 23S rRNA

Journal of Bacteriology ◽

10.1128/jb.181.17.5257-5262.1999 ◽

1999 ◽

Vol 181 (17) ◽

pp. 5257-5262 ◽

Cited By ~ 5

Author(s):

Natalya S. Chernyaeva ◽

Emanuel J. Murgola ◽

Alexander S. Mankin

Keyword(s):

Escherichia Coli ◽

Reporter Genes ◽

Chain Termination ◽

23S Rrna ◽

Specific Interactions ◽

Peptidyl Transferase ◽

Nonsense Mutations ◽

Peptidyl Transferase Center ◽

Codon Positions

ABSTRACT We identified a short RNA fragment, complementary to theEscherichia coli 23S rRNA segment comprising nucleotides 735 to 766 (in domain II), which when expressed in vivo results in the suppression of UGA nonsense mutations in two reporter genes. Neither UAA nor UAG mutations, examined at the same codon positions, were suppressed by the expression of this antisense rRNA fragment. Our results suggest that a stable phylogenetically conserved hairpin at nucleotides 736 to 760 in 23S rRNA, which is situated close to the peptidyl transferase center, may participate in one or more specific interactions during peptide chain termination.

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Silica Gel for Enhanced Activity and Hypochlorite Protection of Cyanuric Acid Hydrolase in Recombinant Escherichia coli

mBio ◽

10.1128/mbio.01477-15 ◽

2015 ◽

Vol 6 (6) ◽

Cited By ~ 5

Author(s):

Adi Radian ◽

Kelly G. Aukema ◽

Alptekin Aksan ◽

Lawrence P. Wackett

Keyword(s):

Escherichia Coli ◽

Cyanuric Acid ◽

Uv Spectroscopy ◽

Water Disinfection ◽

Enzyme Inactivation ◽

Amine Group ◽

Acid Hydrolase ◽

Whole Cell ◽

Content Type ◽

Degradation Rates

ABSTRACTChlorinated isocyanuric acids are widely used water disinfectants that generate hypochlorite, but with repeated application, they build up cyanuric acid (CYA) that must be removed to maintain disinfection. 3-Aminopropyltriethoxysilane (APTES)-treatedEscherichia colicells expressing cyanuric acid hydrolase (CAH) fromMoorella thermoaceticaexhibited significantly high CYA degradation rates and provided protection against enzyme inactivation by hypochlorite (chlorine). APTES coating or encapsulation of cells had two benefits: (i) overcoming diffusion limitations imposed by the cell wall and (ii) protecting against hypochlorite inactivation of CAH activity. Cells encapsulated in APTES gels degraded CYA three times faster than nonfunctionalized tetraethoxysilane (TEOS) gels, and cells coated with APTES degraded CYA at a rate of 29 µmol/min per mg of CAH protein, similar to the rate with purified enzyme. UV spectroscopy, fluorescence spectroscopy, and scanning electron microscopy showed that the higher rates were due to APTES increasing membrane permeability and enhancing cyanuric acid diffusion into the cytoplasm to reach the CAH enzyme. Purified CAH enzyme was shown to be rapidly inactivated by hypochlorite. APTES aggregates surrounding cells protected via the amine groups reacting with hypochlorite as shown by pH changes, zeta potential measurements, and infrared spectroscopy. APTES-encapsulatedE. colicells expressing CAH degraded cyanuric acid at high rates in the presence of 1 to 10 ppm hypochlorite, showing effectiveness under swimming pool conditions. In contrast, CAH activity in TEOS gels or free cells was completely inactivated by hypochlorite. These studies show that commercially available silica materials can selectively enhance, protect, and immobilize whole-cell biocatalysts for specialized applications.IMPORTANCEHypochlorite is used in vast quantities for water disinfection, killing bacteria on surfaces, and washing and whitening. In pools, spas, and other waters, hypochlorite is frequently delivered as chlorinated isocyanuric acids that release hypochlorite and cyanuric acid. Over time, cyanuric acid accumulates and impairs disinfection and must be removed. The microbial enzyme cyanuric acid hydrolase can potentially remove cyanuric acid to restore disinfection and protect swimmers. Whole bacterial cells expressing cyanuric acid hydrolase were encapsulated in an inert silica matrix containing an amine group. The amine group serves to permeabilize the cell membrane and accelerate cyanuric acid degradation, and it also reacts with hypochlorite to protect against inactivation of cyanuric acid hydrolase. Methods for promoting whole-cell biocatalysis are important in biotechnology, and the present work illustrates approaches to enhance rates and protect against an inhibitory substance.

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