Molecular Characterization and Identification of Calnexin 1 As a Radiation Biomarker from Tradescantia BNL4430

Calnexin (CNX) is an integral membrane protein that functions as a chaperone in the endoplasmic reticulum for the correct folding of proteins under stress conditions, rendering organisms tolerant under adverse conditions. Studies have investigated the cytogenetic effects of gamma irradiation (Ɣ-IR) on plants, but information on the molecular response under Ɣ-IR remains limited. Previously, we constructed a cDNA library of an irradiation-sensitive bioindicator plant, Tradescantia BNL4430 (T-4430) under Ɣ-IR, in which the Calnexin-1 gene was highly upregulated at 50 mGy treatment. TrCNX1 encodes a 61.4 kDa protein with conserved signature motifs similar to already reported CNX1s. TrCNX1 expression was evaluated by semiquantitative reverse transcriptase PCR and quantitative real-time PCR and was ubiquitously expressed in various tissues and highly upregulated in flower petals under 50 mGy Ɣ-IR stress. The protective function of TrCNX1 was investigated by overexpression of TrCNX1 in an Escherichia coli BL21(DE3) heterologous system. Using plate assay, we showed that TrCNX1 increased the viability of E. coli transformants under both UV-B and Ɣ-IR compared with the control, demonstrating that TrCNX1 functions under irradiation stress. TrCNX1 may enhance irradiation stress tolerance in crops and act as a radio marker gene to monitor the effects of radiation.

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Polyester Cloth–Based Hybridization Array System for Identification of Enterohemorrhagic Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-116 ◽

2012 ◽

Vol 75 (9) ◽

pp. 1691-1697 ◽

Cited By ~ 15

Author(s):

BURTON W. BLAIS ◽

MARTINE GAUTHIER ◽

MYLÈNE DESCHÊNES ◽

GEORGE HUSZCZYNSKI

Keyword(s):

Escherichia Coli ◽

Marker Gene ◽

Enterohemorrhagic Escherichia Coli ◽

Oligonucleotide Probes ◽

E Coli ◽

Polyester Cloth ◽

Gene Profiles ◽

Pcr Products ◽

Different Strains ◽

Immunoenzymatic Assay

A cloth-based hybridization array system (CHAS) was developed for the identification of foodborne colony isolates of seven priority enterohemorrhagic Escherichia coli (EHEC-7) serogroups targeted by U.S. food inspection programs. Gene sequences associated with intimin; Shiga-like toxins 1 and 2; and the antigenic markers O26, O45, O103, O111, O121, O145, and O157 were amplified in a multiplex PCR incorporating a digoxigenin label, and detected by hybridization of the PCR products with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. The EHEC-7 CHAS exhibited 100% inclusivity and 100% exclusivity characteristics with respect to detection of the various markers among 89 different E. coli strains, with various marker gene profiles and 15 different strains of non–E. coli bacteria.

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Incidence and Characterization of Integrons, Genetic Elements Mediating Multiple-Drug Resistance, in AvianEscherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.12.2925 ◽

1999 ◽

Vol 43 (12) ◽

pp. 2925-2929 ◽

Cited By ~ 165

Author(s):

Lydia Bass ◽

Cynthia A. Liebert ◽

Margie D. Lee ◽

Anne O. Summers ◽

David G. White ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Gene ◽

Multiple Drug Resistance ◽

Marker Gene ◽

Clinical Isolates ◽

Multiple Antibiotic Resistance ◽

Class 1 Integron ◽

E Coli ◽

Class 1

ABSTRACT Antibiotic resistance among avian bacterial isolates is common and is of great concern to the poultry industry. Approximately 36% (n = 100) of avian, pathogenic Escherichia coli isolates obtained from diseased poultry exhibited multiple-antibiotic resistance to tetracycline, oxytetracycline, streptomycin, sulfonamides, and gentamicin. Clinical avian E. coli isolates were further screened for the presence of markers for class 1 integrons, the integron recombinase intI1 and the quaternary ammonium resistance gene qacEΔ1, in order to determine the contribution of integrons to the observed multiple-antibiotic resistance phenotypes. Sixty-three percent of the clinical isolates were positive for the class 1 integron markersintI1 and qacEΔ1. PCR analysis with the conserved class 1 integron primers yielded amplicons of approximately 1 kb from E. coli isolates positive for intI1 andqacEΔ1. These PCR amplicons contained the spectinomycin-streptomycin resistance gene aadA1. Further characterization of the identified integrons revealed that many were part of the transposon Tn21, a genetic element that encodes both antibiotic resistance and heavy-metal resistance to mercuric compounds. Fifty percent of the clinical isolates positive for the integron marker gene intI1 as well as for theqacEΔ1 and aadA1 cassettes also contained the mercury reductase gene merA. The correlation between the presence of the merA gene with that of the integrase and antibiotic resistance genes suggests that these integrons are located in Tn21. The presence of these elements among avianE. coli isolates of diverse genetic makeup as well as inSalmonella suggests the mobility of Tn21 among pathogens in humans as well as poultry.

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Competing Substrates for the Bifunctional Diaminopimelic Acid Epimerase/Glutamate Racemase Modulate Peptidoglycan Synthesis in Chlamydia trachomatis

Infection and Immunity ◽

10.1128/iai.00401-20 ◽

2020 ◽

Vol 89 (1) ◽

pp. e00401-20

Author(s):

Raghuveer Singh ◽

Jessica A. Slade ◽

Mary Brockett ◽

Daniel Mendez ◽

George W. Liechti ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Diaminopimelic Acid ◽

Competition Strategy ◽

Content Type ◽

E Coli ◽

Glutamate Racemase ◽

Heterologous System ◽

Substrate Competition

ABSTRACTThe Chlamydia trachomatis genome encodes multiple bifunctional enzymes, such as DapF, which is capable of both diaminopimelic acid (DAP) epimerase and glutamate racemase activity. Our previous work demonstrated the bifunctional activity of chlamydial DapF in vitro and in a heterologous system (Escherichia coli). In the present study, we employed a substrate competition strategy to demonstrate DapFCt function in vivo in C. trachomatis. We reasoned that, because DapFCt utilizes a shared substrate-binding site for both racemase and epimerase activities, only one activity can occur at a time. Therefore, an excess of one substrate relative to another must determine which activity is favored. We show that the addition of excess l-glutamate or meso-DAP (mDAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess l-glutamate reduced in vivo synthesis of mDAP by C. trachomatis to undetectable levels, thus confirming that excess racemase substrate led to inhibition of DapFCt DAP epimerase activity. We previously showed that expression of dapFCt in a murI (racemase) ΔdapF (epimerase) double mutant of E. coli rescues the d-glutamate auxotrophic defect. Addition of excess mDAP inhibited growth of this strain, but overexpression of dapFCt allowed the mutant to overcome growth inhibition. These results confirm that DapFCt is the primary target of these mDAP and l-glutamate treatments. Our findings demonstrate that suppression of either the glutamate racemase or epimerase activity of DapF compromises the growth of C. trachomatis. Thus, a substrate competition strategy can be a useful tool for in vivo validation of an essential bifunctional enzyme.

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Intestinal Metabolites Influence Macrophage Phagocytosis and Clearance of Bacterial Infection

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.622491 ◽

2021 ◽

Vol 11 ◽

Author(s):

Amy A. O’Callaghan ◽

Elaine Dempsey ◽

Namrata Iyer ◽

Sarah Stiegeler ◽

Kevin Mercurio ◽

...

Keyword(s):

Bacterial Infection ◽

Metabolic Flux ◽

Marker Gene ◽

Intestinal Lumen ◽

Microbial Fermentation ◽

Macrophage Phenotype ◽

E Coli ◽

Macrophage Phagocytosis ◽

Infection Treatment ◽

By Products

The metabolite-rich environment that is the intestinal lumen contains metabolic by-products deriving from microbial fermentation and host cell metabolism, with resident macrophages being constantly exposed to this metabolic flux. Succinate, lactate and itaconate are three metabolites secreted by primed macrophages due to a fragmented tri-carboxylic acid (TCA) cycle. Additionally, succinate and lactate are known by-products of microbial fermentation. How these metabolites impact biological functioning of resident macrophages particularly in response to bacterial infection remains poorly understood. We have investigated the potential influence of these metabolites on macrophage phagocytosis and clearance of Escherichia coli (E. coli) infection. Treatment of murine bone-marrow-derived macrophages (BMDMs) with succinate reduced numbers of intracellular E. coli early during infection, while lactate-treated BMDMs displayed no difference throughout the course of infection. Treatment of BMDMs with itaconate lead to higher levels of intracellular E. coli early in the infection with bacterial burden subsequently reduced at later time-points compared to untreated macrophages, indicative of enhanced engulfment and killing capabilities of macrophages in response to itaconate. Expression of engulfment mediators MARCKS, RhoB, and CDC42 were reduced or unchanged following succinate or lactate treatment and increased in itaconate-treated macrophages following E. coli infection. Nitric oxide (NO) levels varied while pro- and anti-inflammatory cytokines differed in secretory levels in all metabolite-treated macrophages post-infection with E. coli or in response to lipopolysaccharide (LPS) stimulation. Finally, the basal phenotypic profile of metabolite-treated macrophages was altered according to marker gene expression, describing how fluid macrophage phenotype can be in response to the microenvironment. Collectively, our data suggests that microbe- and host-derived metabolites can drive distinct macrophage functional phenotypes in response to infection, whereby succinate and itaconate regulate phagocytosis and bactericidal mechanisms, limiting the intracellular bacterial niche and impeding the pathogenesis of infection.

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Fitness of Enterohemorrhagic Escherichia coli (EHEC)/Enteroaggregative E. coli O104:H4 in Comparison to That of EHEC O157: Survival Studies in Food andIn Vitro

Applied and Environmental Microbiology ◽

10.1128/aem.01796-16 ◽

2016 ◽

Vol 82 (21) ◽

pp. 6326-6334 ◽

Cited By ~ 5

Author(s):

Christina Böhnlein ◽

Jan Kabisch ◽

Diana Meske ◽

Charles M. A. P. Franz ◽

Rohtraud Pichner

Keyword(s):

Escherichia Coli ◽

Meat Product ◽

Enterohemorrhagic Escherichia Coli ◽

Content Type ◽

E Coli ◽

Adverse Conditions ◽

Log Reduction ◽

Fermented Sausages ◽

Survival Studies

ABSTRACTIn 2011, one of the world's largest outbreaks of hemolytic-uremic syndrome (HUS) occurred, caused by a rareEscherichia coliserotype, O104:H4, that shared the virulence profiles of Shiga toxin-producingE. coli(STEC)/enterohemorrhagicE. coli(EHEC) and enteroaggregativeE. coli(EAEC). The persistence and fitness factors of the highly virulent EHEC/EAEC O104:H4 strain, grown either in food orin vitro, were compared with those ofE. coliO157 outbreak-associated strains. The log reduction rates of the different EHEC strains during the maturation of fermented sausages were not significantly different. Both the O157:NM and O104:H4 serotypes could be shown by qualitative enrichment to be present after 60 days of sausage storage. Moreover, the EHEC/EAEC O104:H4 strain appeared to be more viable thanE. coliO157:H7 under conditions of decreased pH and in the presence of sodium nitrite. Analysis of specific EHEC strains in experiments with an EHEC inoculation cocktail showed a dominance of EHEC/EAEC O104:H4, which could be isolated from fermented sausages for 60 days. Inhibitory activities of EHEC/EAEC O104:H4 toward severalE. colistrains, including serotype O157 strains, could be determined. Our study suggests that EHEC/EAEC O104:H4 is well adapted to the multiple adverse conditions occurring in fermented raw sausages. Therefore, it is strongly recommended that STEC strain cocktails composed of several serotypes, instead ofE. coliO157:H7 alone, be used in food risk assessments. The enhanced persistence of EHEC/EAEC O104:H4 as a result of its robustness, as well as the production of bacteriocins, may account for its extraordinary virulence potential.IMPORTANCEIn 2011, a severe outbreak caused by an EHEC/EAEC serovar O104:H4 strain led to many HUS sequelae. In this study, the persistence of the O104:H4 strain was compared with those of other outbreak-relevant STEC strains under conditions of fermented raw sausage production. Both O157:NM and O104:H4 strains could survive longer during the production of fermented sausages thanE. coliO157:H7 strains.E. coliO104:H4 was also shown to be well adapted to the multiple adverse conditions encountered in fermented sausages, and the secretion of a bacteriocin may explain the competitive advantage of this strain in an EHEC strain cocktail. Consequently, this study strongly suggests that enhanced survival and persistence, and the presumptive production of a bacteriocin, may explain the increased virulence of the O104:H4 outbreak strain. Furthermore, this strain appears to be capable of surviving in a meat product, suggesting that meat should not be excluded as a source of potentialE. coliO104:H4 infection.

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Adhesion of Campylobacter jejuni Is Increased in Association with Foodborne Bacteria

Microorganisms ◽

10.3390/microorganisms8020201 ◽

2020 ◽

Vol 8 (2) ◽

pp. 201

Author(s):

Anja Klančnik ◽

Ivana Gobin ◽

Barbara Jeršek ◽

Sonja Smole Možina ◽

Darinka Vučković ◽

...

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Campylobacter Jejuni ◽

Biofilm Formation ◽

Risk Evaluation ◽

Acanthamoeba Castellanii ◽

Host Cells ◽

E Coli ◽

Adverse Conditions ◽

Ultrathin Sections

The aim of this study was to evaluate Campylobacter jejuni NTCT 11168 adhesion to abiotic and biotic surfaces when grown in co-culture with Escherichia coli ATCC 11229 and/or Listeria monocytogenes 4b. Adhesion of C. jejuni to polystyrene and to Caco-2 cells and Acanthamoeba castellanii was lower for at least 3 log CFU/mL compared to E. coli and L. monocytogenes. Electron micrographs of ultrathin sections revealed interactions of C. jejuni with host cells. In co-culture with E. coli and L. monocytogenes, adhesion of C. jejuni to all tested surfaces was significantly increased for more than 1 log CFU/mL. There was 10% higher aggregation for C. jejuni than for other pathogens, and high co-aggregation of co-cultures of C. jejuni with E. coli and L. monocytogenes. These data show that C. jejuni in co-cultures with E. coli and L. monocytogenes present significantly higher risk than C. jejuni as mono-cultures, which need to be taken into account in risk evaluation. C. jejuni adhesion is a prerequisite for their colonization, biofilm formation, and further contamination of the environment. C. jejuni survival under adverse conditions as a factor in their pathogenicity and depends on their adhesion to different surfaces, not only as individual strains, but also in co-cultures with other bacteria like E. coli and L. monocytogenes.

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Ecological and Technical Mechanisms for Cross-Reaction of Human Fecal Indicators with Animal Hosts

Applied and Environmental Microbiology ◽

10.1128/aem.02319-19 ◽

2019 ◽

Vol 86 (5) ◽

Cited By ~ 5

Author(s):

Shuchen Feng ◽

Warish Ahmed ◽

Sandra L. McLellan

Keyword(s):

False Positive ◽

Marker Gene ◽

Fecal Pollution ◽

Marker Genes ◽

Indicator Organisms ◽

Sequencing Data ◽

Content Type ◽

E Coli ◽

Cross Reactions ◽

Nonspecific Amplification

ABSTRACT Quantitative PCR (qPCR) assays for human/sewage marker genes have demonstrated sporadic positive results in animal feces despite their high specificities to sewage and human feces. It is unclear whether these positive reactions are caused by true occurrences of microorganisms containing the marker gene (i.e., indicator organisms) or nonspecific amplification (false positive). The distribution patterns of human/sewage indicator organisms in animals have not been explored in depth, which is crucial for evaluating a marker gene’s true- or false-positive reactions. Here, we analyzed V6 region 16S rRNA gene sequences from 257 animal fecal samples and tested a subset of 184 using qPCR for human/sewage marker genes. Overall, specificities of human/sewage marker genes within sequencing data were 99.6% (BacV6-21), 96.9% (Lachno3), and 96.1% (HF183, indexed by its inferred V6 sequence). Occurrence of some true cross-reactions was associated with atypical compositions of organisms within the genera Blautia or Bacteroides. For human/sewage marker qPCR assays, specificities were 96.7% (HF183/Bac287R), 96.2% (BacV6-21), 95.6% (human Bacteroides [HB]), and 94.0% (Lachno3). Select assays duplexed with either Escherichia coli or Enterococcus spp. were also validated. Most of the positive qPCR results in animals were low level and, on average, 2 orders of magnitude lower than the copy numbers of E. coli and Enterococcus spp. The lower specificity in qPCR assays compared to sequencing data was mainly caused by amplification of sequences highly similar to the marker gene and not the occurrence of the exact marker sequence in animal fecal samples. IMPORTANCE Identifying human sources of fecal pollution is critical to remediate sanitation concerns. Large financial investments are required to address these concerns; therefore, a high level of confidence in testing results is needed. Human fecal marker genes validated in this study showed high specificity in both sequencing data and qPCR results. Human marker sequences were rarely found in individual animals, and in most cases, the animals had atypical microbial communities. Sequencing also revealed the presence of closely related organisms that could account for nonspecific amplification in certain assays. Both the true cross-reactions and the nonspecific amplification had low signals well below E. coli or Enterococcus levels and likely would not impact the assay’s ability to reliably detect human fecal pollution. No animal source had multiple human/sewage marker genes present; therefore, using a combination of marker genes would increase the confidence of human fecal pollution detection.

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Stress-Induced MazF-Mediated Proteins in Escherichia coli

mBio ◽

10.1128/mbio.00340-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 8

Author(s):

Akanksha Nigam ◽

Tamar Ziv ◽

Adi Oron-Gottesman ◽

Hanna Engelberg-Kulka

Keyword(s):

Escherichia Coli ◽

Proteomic Analysis ◽

Normal Growth ◽

Growth Conditions ◽

Translation Machinery ◽

Content Type ◽

E Coli ◽

Adverse Conditions ◽

Almost All ◽

Induced Proteins

ABSTRACT Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. By that means, under stress, the induced MazF generates a stress-induced translation machinery (STM) composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated through the chromosomally borne mazF gene. We show that the mRNAs of almost all of them are characterized by the presence of an ACA site up to 100 nucleotides upstream of the AUG initiator. Therefore, under stressful conditions, induced MazF processes mRNAs that are translated by STM. Furthermore, the presence of the ACA sites far upstream (up to 100 nucleotides) of the AUG initiator may still permit translation by the canonical translation machinery. Thus, such dual-translation mechanisms enable the bacterium under stress also to prepare proteins for immediate functions while coming back to normal growth conditions. IMPORTANCE The stress response, the strategy that bacteria have developed in order to cope up with all kinds of adverse conditions, is so far understood at the level of transcription. Our previous findings of a uniquely modified stress-induced translation machinery (STM) generated in E. coli under stress by the endoribonucleolytic activity of the toxin MazF opens a new chapter in understanding microbial physiology under stress at the translational level. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated by chromosomally borne MazF through STM.

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Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery

Journal of Bacteriology ◽

10.1128/jb.00331-08 ◽

2008 ◽

Vol 191 (1) ◽

pp. 333-346 ◽

Cited By ~ 44

Author(s):

Gouzel Karimova ◽

Carine Robichon ◽

Daniel Ladant

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Membrane Protein ◽

Integral Membrane Protein ◽

Dependent Manner ◽

E Coli ◽

Division Site ◽

Inner Membrane Protein ◽

Two Hybrid

ABSTRACT Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of E. coli cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many E. coli cell division proteins and to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the ftsQ1(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the E. coli cell division machinery.

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Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.

The Journal of Cell Biology ◽

10.1083/jcb.127.6.1617 ◽

1994 ◽

Vol 127 (6) ◽

pp. 1617-1626 ◽

Cited By ~ 635

Author(s):

M Furuse ◽

M Itoh ◽

T Hirase ◽

A Nagafuchi ◽

S Yonemura ◽

...

Keyword(s):

Amino Acids ◽

Fusion Protein ◽

Tight Junctions ◽

Integral Membrane Protein ◽

Deletion Mutants ◽

Glutathione S Transferase ◽

E Coli ◽

Vitro Binding ◽

Peripheral Proteins

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.

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