scholarly journals Transcriptional Analysis and Functional Characterization of a Gene Pair Encoding Iron-Regulated Xenocin and Immunity Proteins of Xenorhabdus nematophila

2008 ◽  
Vol 190 (11) ◽  
pp. 3877-3885 ◽  
Author(s):  
Jitendra Singh ◽  
Nirupama Banerjee
Keyword(s):  
Bacterial Species ◽  
Functional Characterization ◽  
Inducible Promoter ◽  
Transcriptional Analysis ◽  
Transcriptional Unit ◽  
Xenorhabdus Nematophila ◽  
Immunity Protein ◽  
E Coli ◽  
Larval Stages

ABSTRACT We describe a two-gene cluster encoding a bacteriocin, xenocin, and the cognate immunity protein in the insect-pathogenic bacterium Xenorhabdus nematophila, which infects and kills larval stages of the common crop pest Helicoverpa armigera. The two genes, xcinA and ximB, are present in the genome as a single transcriptional unit, which is regulated under SOS conditions. The stress-inducible promoter was activated by mitomycin C, glucose, and Fe3+ depletion and at an elevated temperature when it was tested in Escherichia coli cells. Expression of the xenocin protein alone in E. coli inhibited the growth of this organism. The growth inhibition was abolished when the immunity protein was also present. A recombinant xenocin-immunity protein complex inhibited the growth of E. coli indicator cells when it was added exogenously to a growing culture. Xenocin is an endoribonuclease with an enzymatically active C-terminal domain. Six resident bacterial species (i.e., Bacillus, Enterobacter, Enterococcus, Citrobacter, Serratia, and Stenotrophomonas species) from the H. armigera gut exhibited sensitivity to recombinant xenocin when the organisms were grown under iron-depleted conditions and at a high temperature. Xenocin also inhibited the growth of two Xenorhabdus isolates. This study demonstrates that Fe3+ depletion acts as a common cue for synthesis of xenocin by X. nematophila and sensitization of the target strains to the bacteriocin.

scholarly journals Expression, Purification, and Functional Characterization of Atypical Xenocin, Its Immunity Protein, and Their Domains from Xenorhabdus nematophila

2013 ◽  
Vol 2013 ◽  
pp. 1-9
Author(s):  
Jitendra Singh Rathore
Keyword(s):  
Protein Complexes ◽  
Functional Characterization ◽  
Single Step ◽  
Soil Nematode ◽  
Xenorhabdus Nematophila ◽  
Toxic Activity ◽  
Immunity Protein ◽  
The Family ◽  
Receptor Domain

Xenorhabdus nematophila, a gram-negative bacterium belonging to the family Enterobacteriaceae is a natural symbiont of a soil nematode from the family Steinernematidae. In this study cloning, expression, and purification of broad range iron regulated multidomain bacteriocin called xenocin from X. nematophila (66 kDa, encoded by xcinA gene) and its multidomain immunity protein (42 kDa, encoded by ximB gene) have been done. xcinA-ximB (N′ terminal 270 bp), translocation, and translocation-receptor domain of xcinA, ximB, and its hemolysin domain were cloned, expressed, and purified by single step Ni-NTA chromatography under native conditions. In the functional characterization, neutralization of xcinA toxicity by immunity domain of ximB gene was determined by endogenous assay. Exogenous toxic assays results showed that only the purified recombinant xenocin-immunity domain (10 kDa) protein complex had toxic activity. Atypical cognate immunity protein (42 kDa) of xenocin was fusion of immunity domain (10 kDa) and hemolysin domain (32 kDa). In silico analysis of immunity protein revealed its similarity with hemolysin and purine NTPase like proteins. Hemolytic activity was not observed in immunity protein or in its various domains; however, full-length immunity protein lacking Walker motif showed ATPase activity. Finally, using circular dichroism performed secondary structural analyses of all the recombinant proteins/protein complexes.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the Adenylyl Cyclase Genesgs-1by Analysis of a Mutational Spectrum inCaenorhabditis elegans

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 133-142 ◽  
Author(s):  
Celine Moorman ◽  
Ronald H A Plasterk
Keyword(s):  
Life Span ◽  
Adenylyl Cyclase ◽  
Neuronal Degeneration ◽  
Functional Characterization ◽  
Adenylyl Cyclases ◽  
Loss Of Function ◽  
C Elegans ◽  
Larval Stages ◽  
Pharyngeal Pumping

AbstractThe sgs-1 (suppressor of activated Gαs) gene encodes one of the four adenylyl cyclases in the nematode C. elegans and is most similar to mammalian adenylyl cyclase type IX. We isolated a complete loss-of-function mutation in sgs-1 and found it to result in animals with retarded development that arrest in variable larval stages. sgs-1 mutant animals exhibit lethargic movement and pharyngeal pumping and (while not reaching adulthood) have a mean life span that is >50% extended compared to wild type. An extensive set of reduction-of-function mutations in sgs-1 was isolated in a screen for suppressors of a neuronal degeneration phenotype induced by the expression of a constitutively active version of the heterotrimeric Gαs subunit of C. elegans. Although most of these mutations change conserved residues within the catalytic domains of sgs-1, mutations in the less-conserved transmembrane domains are also found. The sgs-1 reduction-of-function mutants are viable and have reduced locomotion rates, but do not show defects in pharyngeal pumping or life span.

scholarly journals Construction and Characterization of a proU-gfp Transcriptional Fusion That Measures Water Availability in a Microbial Habitat

2002 ◽  
Vol 68 (9) ◽  
pp. 4604-4612 ◽  
Author(s):  
Catherine A. Axtell ◽  
Gwyn A. Beattie
Keyword(s):  
Pseudomonas Syringae ◽  
Water Availability ◽  
Water Deprivation ◽  
Bacterial Species ◽  
Transcriptional Fusion ◽  
Bacterial Biosensor ◽  
E Coli ◽  
Quantitative Manner ◽  
Microbial Habitat

ABSTRACT We constructed and characterized a transcriptional fusion that measures the availability of water to a bacterial cell. This fusion between the proU promoter from Escherichia coli and the reporter gene gfp was introduced into strains of E. coli, Pantoea agglomerans, and Pseudomonas syringae. The proU-gfp fusion in these bacterial biosensor strains responded in a quantitative manner to water deprivation caused by the presence of NaCl, Na2SO4, KCl, or polyethylene glycol (molecular weight, 8000). The fusion was induced to a detectable level by NaCl concentrations of as low as 10 mM in all three bacterial species. Water deprivation induced proU-gfp expression in both planktonic and surface-associated cells; however, it induced a higher level of expression in the surface-associated cells. Following the introduction of P. agglomerans biosensor cells onto bean leaves, the cells detected a significant decrease in water availability within only 5 min. After 30 min, the populations were exposed, on average, to a water potential equivalent to that imposed by approximately 55 mM NaCl. These results demonstrate the effectiveness of a proU-gfp-based biosensor for evaluating water availability on leaves. Furthermore, the inducibility of proU-gfp in multiple bacterial species illustrates the potential for tailoring proU-gfp-based biosensors to specific habitats.

scholarly journals Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli

2021 ◽  
Author(s):  
I. B. Trindade ◽  
G. Hernandez ◽  
E. Lebègue ◽  
F. Barrière ◽  
T. Cordeiro ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Paramagnetic Nmr ◽  
E Coli ◽  
The Past ◽  
Internal Cavities ◽  
Structural Differences ◽  
Bond Strengths ◽  
Redox Bohr Effect ◽  
Micromolar Range

AbstractIron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the “palm” domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe–2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins. Graphic abstract

Sa1806 Molecular and Functional Characterization of Fimh in Crohn's Disease Associated E. Coli

2013 ◽  
Vol 144 (5) ◽  
pp. S-310
Author(s):  
Brendan Chandler ◽  
Belgin Dogan ◽  
Ellen J. Scherl ◽  
Kenneth W. Simpson
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Functional Characterization ◽  
E Coli

scholarly journals First Report of a Foodborne Salmonella enterica Serovar Gloucester (4:i:l,w) ST34 Strain Harboring blaCTX–M–55 and qnrS Genes Located in IS26-Mediated Composite Transposon

2021 ◽  
Vol 12 ◽  
Author(s):  
Lili Li ◽  
Rikke Heidemann Olsen ◽  
Anhua Song ◽  
Jian Xiao ◽  
Chong Wang ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Structural Unit ◽  
Bacterial Species ◽  
Meat Products ◽  
Sequence Type ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Long Read ◽  
Phenotypic And Genotypic Characterization

Extended-spectrum β-lactamases (ESBLs) production and (fluoro)quinolone (FQ) resistance among Salmonella pose a public health threat. The objective of this study was the phenotypic and genotypic characterization of an ESBL-producing and nalidixic acid-resistant Salmonella enterica serovar Gloucester isolate (serotype 4:i:l,w) of sequence type 34 (ST34) from ready-to-eat (RTE) meat products in China. Whole-genome short and long read sequencing (HiSeq and MinION) results showed that it contained blaCTX–M–55, qnrS1, and tetB genes, with blaCTX–M–55 and qnrS1 located in chromosomal IS26-mediated composite transposon (IS26–qnrS1–IS3–Tn3–orf–blaCTX–M–55–ISEcp1–IS26). The same genetic structure was found in the chromosome of S. enterica subsp. enterica serovar Typhimurium strain and in several plasmids of Escherichia coli, indicating that the IS26-mediated composite transposon in the chromosome of S. Gloucester may originate from plasmids of E. coli and possess the ability to disseminate to Salmonella and other bacterial species. Besides, the structural unit qnrS1–IS3–Tn3–orf–blaCTX–M–55 was also observed to be linked with ISKpn19 in both the chromosomes and plasmids of various bacteria species, highlighting the contribution of the insertion sequences (IS26 and ISKpn19) to the co-dissemination of blaCTX–M–55 and qnrS1. To our knowledge, this is the first description of chromosomal blaCTX–M–55 and qnrS in S. Gloucester from RTE meat products. Our work expands the host range and provides additional evidence of the co-transfer of blaCTX–M–55 and qnrS1 among different species of Salmonella through the food chain.

scholarly journals Expression, secretion and functional characterization of three laccases in E. coli

2022 ◽  
Vol 7 (1) ◽  
pp. 474-480
Author(s):  
Yating Mo ◽  
Hou Ip Lao ◽  
Sau Wa Au ◽  
Ieng Chon Li ◽  
Jeremy Hu ◽  
...  
Keyword(s):  
Functional Characterization ◽  
E Coli

scholarly journals Isolation and characterization of bacteria residing in the oral, gut, and fecal samples of different pheasant species

2023 ◽  
Vol 83 ◽  
Author(s):  
M. Mushtaq ◽  
S. M. Bukhari ◽  
S. Ahmad ◽  
A. Khattak ◽  
M. B. Chattha ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Gut Contents ◽  
Phasianus Colchicus ◽  
Gut Content ◽  
E Coli ◽  
Colony Forming Unit ◽  
Isolation And Characterization ◽  
Staphylococcus Spp ◽  
Campylobacter Spp

Abstract There is a paucity of research conducted on microbial prevalence in pheasants. The microbiota of captive birds has zoonotic significance and must be characterize. Present study is therefore planned to assess the microbiota from oral, fecal and gut content of captive avian species. It will be helpful in characterization of harmful microbes. Different samples taken from oral, gut and feces of ring-necked pheasants (Phasianus colchicus), green pheasants (Phasianus versicolor), golden pheasant (Chrysolophus pictus) and silver pheasant (Lophura nycthemera). Samples were collected, diluted, and inoculated onto different agar plates (MacConkey, SS agar, MSA and nutrient agar) for cultivation of bacterial species. Colonies of E.coli, Staphylococcus spp. Brachyspira spp. and Campylobacter spp were observed based on colony morphology. Colony forming unit showed E. coli as frequently found bacteria in fecal, oral and gut contents of all the above pheasants. The overall significance difference was found among bacterial species of golden pheasants, green pheasant, ring-necked pheasant, and silver pheasants. It was concluded that E.coli is predominant isolated from heathy pheasants followed by Campylobacter, Staphylococcus and Brachyspira.

Isolation and Characterization of Levoglucosan Metabolizing Bacteria

2021 ◽  
Author(s):  
Ajay S. Arya ◽  
Minh T. H. Hang ◽  
Mark A. Eiteman
Keyword(s):  
Recombinant Expression ◽  
Bacterial Species ◽  
Soluble Carbohydrate ◽  
Rrna Gene ◽  
Gene Products ◽  
16S Rrna Gene Sequences ◽  
E Coli ◽  
Isolation And Characterization ◽  
Charred Wood

Bacteria were isolated from wastewater and soil containing charred wood remnants based on their ability to use levoglucosan as a sole carbon source and on their levoglucosan dehydrogenase (LGDH) activity. On the basis of their 16S rRNA gene sequences, these bacteria represented diverse genera of Microbacterium, Paenibacillus , Shinella , and Klebsiella . Genomic sequencing of the isolates verified that two isolates represented novel species, Paenibacillus athensensis MEC069 T and Shinella sumterensis MEC087 T , while the remaining isolates were closely related to either Microbacterium lacusdiani or Klebsiella pneumoniae . The genetic sequence of LGDH, lgdA , was found in the genomes of these four isolates as well as Pseudarthrobacter phenanthrenivorans Sphe3. The identity of the P. phenanthrenivorans LGDH was experimentally verified following recombinant expression in E. coli . Comparison of the putative genes surrounding lgdA in the isolate genomes indicated that several other gene products facilitate the bacterial catabolism of levoglucosan, including a putative sugar isomerase and several transport proteins. Importance Levoglucosan is the most prevalent soluble carbohydrate remaining after high temperature pyrolysis of lignocellulosic biomass, but it is not fermented by typical production microbes such as Escherichia coli and Saccharomyces cerevisiae . A few fungi metabolize levoglucosan via the enzyme levoglucosan kinase, while several bacteria metabolize levoglucosan via levoglucosan dehydrogenase. This study describes the isolation and characterization of four bacterial species which degrade levoglucosan. Each isolate is shown to contain several genes within an operon involved in levoglucosan degradation, furthering our understanding of bacteria which metabolize levoglucosan.

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