scholarly journals Characterization of Two Unique Cold-Active Lipases Derived from a Novel Deep-Sea Cold Seep Bacterium

2021 ◽  
Vol 9 (4) ◽  
pp. 802
Author(s):  
Chenchen Guo ◽  
Rikuan Zheng ◽  
Ruining Cai ◽  
Chaomin Sun ◽  
Shimei Wu
Keyword(s):  
Deep Sea ◽  
Bacterial Species ◽  
Deep Ocean ◽  
Cold Seep ◽  
Bacterial Strains ◽  
E Coli ◽  
Cold Active ◽  
Genes Encoding ◽  
Potential Inhibitors

The deep ocean microbiota has unexplored potential to provide enzymes with unique characteristics. In order to obtain cold-active lipases, bacterial strains isolated from the sediment of the deep-sea cold seep were screened, and a novel strain gcc21 exhibited a high lipase catalytic activity, even at the low temperature of 4 °C. The strain gcc21 was identified and proposed to represent a new species of Pseudomonas according to its physiological, biochemical, and genomic characteristics; it was named Pseudomonas marinensis. Two novel encoding genes for cold-active lipases (Lipase 1 and Lipase 2) were identified in the genome of strain gcc21. Genes encoding Lipase 1 and Lipase 2 were respectively cloned and overexpressed in E. coli cells, and corresponding lipases were further purified and characterized. Both Lipase 1 and Lipase 2 showed an optimal catalytic temperature at 4 °C, which is much lower than those of most reported cold-active lipases, but the activity and stability of Lipase 2 were much higher than those of Lipase 1 under different tested pHs and temperatures. In addition, Lipase 2 was more stable than Lipase 1 when treated with different metal ions, detergents, potential inhibitors, and organic solvents. In a combination of mutation and activity assays, catalytic triads of Ser, Asp, and His in Lipase 1 and Lipase 2 were demonstrated to be essential for maintaining enzyme activity. Phylogenetic analysis showed that both Lipase 1 and Lipase 2 belonged to lipase family III. Overall, our results indicate that deep-sea cold seep is a rich source for novel bacterial species that produce potentially unique cold-active enzymes.

scholarly journals Functional Analysis of Genes in the rfbLocus of Leptospira borgpetersenii Serovar Hardjo Subtype Hardjobovis

2000 ◽  
Vol 68 (7) ◽  
pp. 3793-3798 ◽  
Author(s):  
Dieter M. Bulach ◽  
Thareerat Kalambaheti ◽  
Alejandro de la Peña-Moctezuma ◽  
Ben Adler
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Open Reading Frames ◽  
New Host ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Leptospira Borgpetersenii ◽  
Nucleotide Sugars

ABSTRACT Lipopolysaccharide (LPS) is a key antigen in immunity to leptospirosis. Its biosynthesis requires enzymes for the biosynthesis and polymerization of nucleotide sugars and the transport through and attachment to the bacterial membrane. The genes encoding these functions are commonly clustered into loci; for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis, this locus, named rfb, spans 36.7 kb and contains 31 open reading frames, of which 28 have been assigned putative functions on the basis of sequence similarity. Characterization of the function of these genes is hindered by the fact that it is not possible to construct isogenic mutant strains in Leptospira. We used two approaches to circumvent this problem. The first was to clone the entire locus into a heterologous host system and determine if a “recombinant” LPS or polysaccharide was synthesized in the new host. The second approach used putative functions to identify mutants in other bacterial species whose mutations might be complemented by genes on the leptospiralrfb locus. This approach was used to investigate the function of three genes in the leptospiral rfb locus and demonstrated function for orfH10, which complemented awbpM strain of Pseudomonas aeruginosa, andorfH13, which complemented an rfbW strain ofVibrio cholerae. However, despite the similarity of OrfH11 to WecC, a wecC strain of E. coli was not complemented by orfH11. The predicted protein encoded byorfH8 is similar to GalE from a number of organisms. ASalmonella enterica serovar Typhimurium strain producing no GalE was used as a background in which orfH8 produced detectable GalE enzyme activity.

scholarly journals Molecular characterization of clinical carbapenem-resistant Enterobacterales from Qatar

2021 ◽  
Author(s):  
Fatma Ben Abid ◽  
Clement K. M. Tsui ◽  
Yohei Doi ◽  
Anand Deshmukh ◽  
Christi L. McElheny ◽  
...  
Keyword(s):  
Common Species ◽  
Clinical Samples ◽  
Whole Genome ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Sequence Types ◽  
Common Sequence

AbstractOne hundred forty-nine carbapenem-resistant Enterobacterales from clinical samples obtained between April 2014 and November 2017 were subjected to whole genome sequencing and multi-locus sequence typing. Klebsiella pneumoniae (81, 54.4%) and Escherichia coli (38, 25.5%) were the most common species. Genes encoding metallo-β-lactamases were detected in 68 (45.8%) isolates, and OXA-48-like enzymes in 60 (40.3%). blaNDM-1 (45; 30.2%) and blaOXA-48 (29; 19.5%) were the most frequent. KPC-encoding genes were identified in 5 (3.6%) isolates. Most common sequence types were E. coli ST410 (8; 21.1%) and ST38 (7; 18.4%), and K. pneumoniae ST147 (13; 16%) and ST231 (7; 8.6%).

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 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 Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

2018 ◽  
Author(s):  
Krithika Rajagopalan ◽  
Jonathan Dworkin
Keyword(s):  
Biochemical Characterization ◽  
Regulatory Protein ◽  
Biochemical Properties ◽  
Bacterial Genomes ◽  
Phosphatase Inhibitors ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Genes Encoding ◽  
Number Of Bacteria

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli,which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not inE. coli.Here, we describe a previously unknown eSTP encoded by anE. coliORF,yegK,and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely relatedE. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.ImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacteriumE. colidemonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

scholarly journals The characterization of ESBL genes in Escherichia coli and Klebsiella pneumoniae causing nosocomial infections in Vietnam

2013 ◽  
Vol 7 (12) ◽  
pp. 922-928 ◽  
Author(s):  
Nguyen Hoang Thu Trang ◽  
Tran Vu Thieu Nga ◽  
James I Campbell ◽  
Nguyen Trong Hiep ◽  
Jeremy Farrar ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
Nosocomial Infections ◽  
Enteric Bacteria ◽  
E Coli ◽  
Plasmid Analysis ◽  
Resistance Plasmids ◽  
Genes Encoding ◽  
Third Generation Cephalosporins

Background: Extended-spectrum β-lactamases (ESBLs) are enzymes capable of hydrolyzing oxyimino-β-lactams and inducing resistance to third generation cephalosporins. The genes encoding ESBLs are widespread and generally located on highly transmissible resistance plasmids. We aimed to investigate the complement of ESBL genes in E. coli and Klebsiella pneumoniae causing nosocomial infections in hospitals in Ho Chi Minh City, Vietnam. Methodology: Thirty-two non-duplicate isolates of E. coli and Klebsiella pneumoniae causing nosocomial infections, isolated between March and June 2010, were subjected to antimicrobial susceptibility testing. All isolates were PCR-amplified to detect the blaSHV, blaTEM and blaCTX-M ESBL genes and subjected to plasmid analysis. Results: We found that co-resistance to multiple antimicrobials was highly prevalent, and we report the predominance of the blaCTX-M-15 and blaCTX-M-27 genes, located on highly transmissible plasmids ranging from 50 to 170 kb in size. Conclusions: Our study represents a snap shot of ESBL-producing enteric bacteria causing nosocomial infections in this setting. We suggest that antimicrobial resistance in nosocomial E. coli and Klebsiella pneumoniae is rampant in Vietnam and ESBL organisms are widespread. In view of these data and the dramatic levels of antimicrobial resistance reported in Vietnam we advocate an urgent review of antimicrobial use in the Vietnamese healthcare system.

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.

Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels

2006 ◽  
Vol 26 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Si Sun ◽  
Jo Han Gan ◽  
Jennifer J. Paynter ◽  
Stephen J. Tucker
Keyword(s):  
Functional Properties ◽  
Functional Characterization ◽  
Unicellular Eukaryote ◽  
Channel Blockers ◽  
E Coli ◽  
Inwardly Rectifying Potassium Channels ◽  
Genes Encoding ◽  
Functionally Diverse ◽  
Inwardly Rectifying

Our understanding of the mammalian inwardly rectifying family of K+ channels (Kir family) has recently been advanced by X-ray crystal structures of two homologous prokaryotic orthologs (KirBac1.1 and KirBac3.1). However, the functional properties of these KirBac channels are still poorly understood. To address this problem, we cloned and characterized genes encoding KirBac orthologs from a wide variety of different prokaryotes and a simple unicellular eukaryote. The functional properties of these KirBacs were then examined by growth complementation in a K+ uptake-deficient strain of Escherichia coli (TK2420). Whereas some KirBac genes exhibited robust growth complementation, others either did not complement or showed temperature-dependent complementation including KirBac1.1 and KirBac3.1. In some cases, KirBac expression was also toxic to the growth of E. coli. The KirBac family exhibited a range of sensitivity to the K+ channel blockers Ba2+ and Cs+ as well as differences in their ability to grow on very low-K+ media, thus demonstrating major differences in their permeation properties. These results reveal the existence of a functionally diverse superfamily of microbial KirBac genes and present an excellent resource for the structural and functional analysis of this class of K+ channels. Furthermore, the complementation assay used in this study provides a simple and robust method for the functional characterization of a range of prokaryotic K+ channels that are difficult to study by traditional methods.

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