scholarly journals A Novel Thermostable Keratinase from Deinococcus geothermalis with Potential Application in Feather Degradation

2021 ◽  
Vol 11 (7) ◽  
pp. 3136
Author(s):  
Yin Tang ◽  
Leizhou Guo ◽  
Mingming Zhao ◽  
Yuan Gui ◽  
Jiahui Han ◽  
...  
Keyword(s):  
Thermal Stability ◽  
High Efficiency ◽  
In Silico Analysis ◽  
Maximum Activity ◽  
Feather Degradation ◽  
Good Tolerance ◽  
E Coli ◽  
Thermophilic Species ◽  
Mature Domain ◽  
Silico Analysis

Keratinase can specifically attack disulfide bridges in keratin to convert them from complex to simplified forms. Keratinase thermal stability has drawn attention to various biotechnological industries. In this study, a keratinase DgeKer was identified from a slightly thermophilic species, D. geothermalis. The in silico analysis showed that DgeKer is composed of signal peptide, N-terminal propeptide, mature domain, and C-terminal extension. DgeKer and its C-terminal extension-truncated enzyme (DgeKer-C) were cloned and expressed in E. coli. The purified DgeKer and DgeKer-C showed maximum activity at 70 °C and pH 9–The thermal stability assay (60 °C) showed that the half-life value of DgeKer and DgeKer-C were 103.45 min and 169.10 min, respectively. DgeKer and DgeKer-C were stable at the range of pH from 9 to 11 and showed good tolerance to some metal ions, surfactants and organic solvent. Furthermore, DgeKer could degrade feathers at 70 °C for 60 min. However, the medium became turbid with obvious softening of barbules after being treated with DgeKer-C, which might be due to C-terminal extension. In summary, a thermostable keratinase DgeKer with high efficiency degradation of feathers may have great potential in industry.

scholarly journals Two Distinct Modes of Lysis Regulation in Campylobacter Fletchervirus and Firehammervirus Phages

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1247
Author(s):  
Athina Zampara ◽  
Stephen J. Ahern ◽  
Yves Briers ◽  
Lone Brøndsted ◽  
Martine Camilla Holst Sørensen
Keyword(s):  
Signal Peptide ◽  
In Silico ◽  
Protein Domains ◽  
In Silico Analysis ◽  
Cell Lysis ◽  
E Coli ◽  
Sec Pathway ◽  
Gene Functions ◽  
Lytic Genes ◽  
Silico Analysis

Campylobacter phages are divided into two genera; Fletchervirus and Firehammervirus, showing only limited intergenus homology. Here, we aim to identify the lytic genes of both genera using two representative phages (F352 and F379) from our collection. We performed a detailed in silico analysis searching for conserved protein domains and found that the predicted lytic genes are not organized into lysis cassettes but are conserved within each genus. To verify the function of selected lytic genes, the proteins were expressed in E. coli, followed by lytic assays. Our results show that Fletchervirus phages encode a typical signal peptide (SP) endolysin dependent on the Sec-pathway for translocation and a holin for activation. In contrast, Firehammervirus phages encode a novel endolysin that does not belong to currently described endolysin groups. This endolysin also uses the Sec-pathway for translocation but induces lysis of E. coli after overexpression. Interestingly, co-expression of this endolysin with an overlapping gene delayed and limited cell lysis, suggesting that this gene functions as a lysis inhibitor. These results indicate that Firehammervirus phages regulate lysis timing by a yet undescribed mechanism. In conclusion, we found that the two Campylobacter phage genera control lysis by two distinct mechanisms.

scholarly journals Haemophilus influenzae HP1 Bacteriophage Encodes a Lytic Cassette with a Pinholin and a Signal-Arrest-Release Endolysin

2020 ◽  
Vol 21 (11) ◽  
pp. 4013
Author(s):  
Monika Adamczyk-Popławska ◽  
Zuzanna Tracz-Gaszewska ◽  
Przemysław Lasota ◽  
Agnieszka Kwiatek ◽  
Andrzej Piekarowicz
Keyword(s):  
Haemophilus Influenzae ◽  
Gene Product ◽  
Transmembrane Protein ◽  
In Silico Analysis ◽  
Viable Cell ◽  
Cell Lysis ◽  
Gene Products ◽  
E Coli ◽  
Myoviridae Family ◽  
Silico Analysis

HP1 is a temperate bacteriophage, belonging to the Myoviridae family and infecting Haemophilus influenzae Rd. By in silico analysis and molecular cloning, we characterized lys and hol gene products, present in the previously proposed lytic module of HP1 phage. The amino acid sequence of the lys gene product revealed the presence of signal-arrest-release (SAR) and muraminidase domains, characteristic for some endolysins. HP1 endolysin was able to induce lysis on its own when cloned and expressed in Escherichia coli, but the new phage release from infected H. influenzae cells was suppressed by inhibition of the secretion (sec) pathway. Protein encoded by hol gene is a transmembrane protein, with unusual C-out and N-in topology, when overexpressed/activated. Its overexpression in E. coli did not allow the formation of large pores (lack of leakage of β-galactosidase), but caused cell death (decrease in viable cell count) without lysis (turbidity remained constant). These data suggest that lys gene encodes a SAR-endolysin and that the hol gene product is a pinholin. HP1 SAR-endolysin is responsible for cell lysis and HP1 pinholin seems to regulate the cell lysis and the phage progeny release from H. influenzae cells, as new phage release from the natural host was inhibited by deletion of the hol gene.

scholarly journals Mobilization of the nonconjugative virulence plasmid from hypervirulent Klebsiella pneumoniae

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Yanping Xu ◽  
Jianfeng Zhang ◽  
Meng Wang ◽  
Meng Liu ◽  
Guitian Liu ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
In Silico ◽  
In Silico Analysis ◽  
Conjugative Plasmid ◽  
Virulence Plasmid ◽  
E Coli ◽  
Virulence Plasmids ◽  
Carbapenem Resistant ◽  
Helper Plasmids ◽  
Silico Analysis

Abstract Background Klebsiella pneumoniae, as a global priority pathogen, is well known for its capability of acquiring mobile genetic elements that carry resistance and/or virulence genes. Its virulence plasmid, previously deemed nonconjugative and restricted within hypervirulent K. pneumoniae (hvKP), has disseminated into classic K. pneumoniae (cKP), particularly carbapenem-resistant K. pneumoniae (CRKP), which poses alarming challenges to public health. However, the mechanism underlying its transfer from hvKP to CRKP is unclear. Methods A total of 28 sequence type (ST) 11 bloodstream infection-causing CRKP strains were collected from Ruijin Hospital in Shanghai, China, and used as recipients in conjugation assays. Transconjugants obtained from conjugation assays were confirmed by XbaI and S1 nuclease pulsed-field gel electrophoresis, PCR detection and/or whole-genome sequencing. The plasmid stability of the transconjugants was evaluated by serial culture. Genetically modified strains and constructed mimic virulence plasmids were employed to investigate the mechanisms underlying mobilization. The level of extracellular polysaccharides was measured by mucoviscosity assays and uronic acid quantification. An in silico analysis of 2608 plasmids derived from 814 completely sequenced K. pneumoniae strains available in GenBank was performed to investigate the distribution of putative helper plasmids and mobilizable virulence plasmids. Results A nonconjugative virulence plasmid was mobilized by the conjugative plasmid belonging to incompatibility group F (IncF) from the hvKP strain into ST11 CRKP strains under low extracellular polysaccharide-producing conditions or by employing intermediate E. coli strains. The virulence plasmid was mobilized via four modes: transfer alone, cotransfer with the conjugative IncF plasmid, hybrid plasmid formation due to two rounds of single-strand exchanges at specific 28-bp fusion sites or homologous recombination. According to the in silico analysis, 31.8% (242) of the putative helper plasmids and 98.8% (84/85) of the virulence plasmids carry the 28-bp fusion site. All virulence plasmids carry the origin of the transfer site. Conclusions The nonconjugative virulence plasmid in ST11 CRKP strains is putatively mobilized from hvKP or E. coli intermediates with the help of conjugative IncF plasmids. Our findings emphasize the importance of raising public awareness of the rapid dissemination of virulence plasmids and the consistent emergence of hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) strains.

Camelus dromedarius glucose transporter 4: in silico analysis, cloning, expression, purification and characterisation in E. coli

2017 ◽  
Vol 123 (4) ◽  
pp. 254-264
Author(s):  
Anwar Ahmed ◽  
Mohammed Arshad ◽  
Ajamaluddin Malik ◽  
Shama Parveen ◽  
Abdulrahman M. Alsenaidy
Keyword(s):  
In Silico ◽  
Glucose Transporter ◽  
In Silico Analysis ◽  
Camelus Dromedarius ◽  
Glucose Transporter 4 ◽  
E Coli ◽  
Silico Analysis

scholarly journals In Silico Analysis, Cloning and Expression of Recombinant CD166 in E. coli BL21 (DE3) as a Marker for Detection and Treatment of Colorectal Cancer

2017 ◽  
Vol 06 (01) ◽  
Author(s):  
Vahid Marmari ◽  
Habibollah Mahmoodzadeh ◽  
Hassan Dana ◽  
Ghanbar Mahmoodi Chalbatani ◽  
Ali Mazraeh ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
In Silico ◽  
In Silico Analysis ◽  
Cloning And Expression ◽  
E Coli ◽  
Silico Analysis

scholarly journals Human carriage of cefotaxime-resistant Escherichia coli in North-East India: an analysis of STs and associated resistance mechanisms

2019 ◽  
Author(s):  
Deepjyoti Paul ◽  
Dmitriy Babenko ◽  
Mark A Toleman
Keyword(s):  
Escherichia Coli ◽  
Core Genome ◽  
In Silico Analysis ◽  
Carbapenem Resistance ◽  
Resistance Mechanisms ◽  
E Coli ◽  
North East India ◽  
North East ◽  
The City ◽  
Silico Analysis

Abstract Objectives To determine the prevalence of Escherichia coli STs and associated resistance mechanisms carried by the community in North-East India. Methods E. coli (108) were isolated from sewage collected from 19 sites across the city of Silchar by plating on MacConkey agar with/without selection (50 mg/L cefotaxime). Species identification was confirmed by MALDI-TOF MS for 82 isolates. Common resistance mechanisms were determined by WGS of pooled E. coli isolates. PFGE combined with specific probes determined the presence of common resistance mechanisms in all isolates. Phylotypes, multilocus STs, core-genome multilocus STs, resistance genes and virulence genes were determined by in silico analysis of 38 genomes. Results and conclusions Analysis of isolates collected without selection (n = 33) indicated that cefotaxime resistance in E. coli was 42% (14/33) and estimated meropenem resistance at 9%. The remaining 58% (19/33) were additionally susceptible to ampicillin, trimethoprim, ciprofloxacin and aminoglycosides. The most common ST among the cefotaxime-resistant E. coli was ST167 (29%), followed by ST410 (17%) and ST648 (10%). E. coli ST131 was absent from the collection. Sixty-three isolates were resistant to cefotaxime and harboured blaCTX-M-15 [54% (34/63)] or blaCMY-42 [46% (29/63)], of which 10% (6/63) harboured both genes. Carbapenem resistance was due to blaNDM-5, found in 10/63 cefotaxime-resistant isolates, and/or blaOXA-181, found in 4/63 isolates. NDM-5 was encoded by IncX3 and/or IncFII plasmids and CMY-42 was mostly encoded by IncI plasmids. NDM-5 appears to have replaced NDM-1 in this region and CMY-42 appears to be in the process of replacing CTX-M-15.

scholarly journals The transcriptional activator of the bfp operon in EPEC (PerA) interacts with the RNA polymerase alpha subunit

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Cristina Lara-Ochoa ◽  
Fabiola González-Lara ◽  
Luis E. Romero-González ◽  
Juan B. Jaramillo-Rodríguez ◽  
Sergio I. Vázquez-Arellano ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Virulence Genes ◽  
Phenotypic Expression ◽  
In Silico Analysis ◽  
Alpha Subunit ◽  
E Coli ◽  
Enterocyte Effacement ◽  
Silico Analysis ◽  
Activate Gene

AbstractEnteropathogenic E. coli virulence genes are under the control of various regulators, one of which is PerA, an AraC/XylS-like regulator. PerA directly promotes its own expression and that of the bfp operon encoding the genes involved in the biogenesis of the bundle-forming pilus (BFP); it also activates PerC expression, which in turn stimulates locus of enterocyte effacement (LEE) activation through the LEE-encoded regulator Ler. Monomeric PerA directly binds to the per and bfp regulatory regions; however, it is not known whether interactions between PerA and the RNA polymerase (RNAP) are needed to activate gene transcription as has been observed for other AraC-like regulators. Results showed that PerA interacts with the alpha subunit of the RNAP polymerase and that it is necessary for the genetic and phenotypic expression of bfpA. Furthermore, an in silico analysis shows that PerA might be interacting with specific alpha subunit amino acids residues highlighting the direction of future experiments.

scholarly journals Calcium/calmodulin inhibition of the Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase provides a possible link between calcium and brassinosteroid signalling

2012 ◽  
Vol 443 (2) ◽  
pp. 515-523 ◽  
Author(s):  
Man-Ho Oh ◽  
Hyoung Seok Kim ◽  
Xia Wu ◽  
Steven D. Clouse ◽  
Raymond E. Zielinski ◽  
...  
Keyword(s):  
Synthetic Peptide ◽  
In Silico Analysis ◽  
Cytoplasmic Domain ◽  
Dependent Manner ◽  
Receptor Kinase ◽  
Plant Growth And Development ◽  
E Coli ◽  
Tyrosine Residues ◽  
Silico Analysis

The receptor kinase BRI1 (BRASSINOSTEROID-INSENSITIVE 1) is a key component in BR (brassinosteroid) perception and signal transduction, and has a broad impact on plant growth and development. In the present study, we demonstrate that Arabidopsis CaM (calmodulin) binds to the recombinant cytoplasmic domain of BRI1 in a Ca2+-dependent manner in vitro. In silico analysis predicted binding to Helix E of the BRI1 kinase subdomain VIa and a synthetic peptide based on this sequence interacted with Ca2+/CaM. Co-expression of CaM with the cytoplasmic domain of BRI1 in Escherichia coli strongly reduced autophosphorylation of BRI1, in particular on tyrosine residues, and also reduced the BRI1-mediated transphosphorylation of E. coli proteins on tyrosine, threonine and presumably serine residues. Several isoforms of CaM and CMLs (CaM-like proteins) were more effective (AtCaM6, AtCaM7 and AtCML8, where At is Arabidopsis thaliana) than others (AtCaM2, AtCaM4 and AtCML11) when co-expressed with BRI1 in E. coli. These results establish a novel assay for recombinant BRI1 transphosphorylation activity and collectively uncover a possible new link between Ca2+ and BR signalling.

scholarly journals Discovery of a Kojibiose Hydrolase by Analysis of Specificity-Determining Correlated Positions in Glycoside Hydrolase Family 65

Molecules ◽  
2021 ◽  
Vol 26 (20) ◽  
pp. 6321
Author(s):  
Emma De De Beul ◽  
Alana Jongbloet ◽  
Jorick Franceus ◽  
Tom Desmet
Keyword(s):  
Glycoside Hydrolase ◽  
In Silico Analysis ◽  
High Specificity ◽  
Glycoside Hydrolase Family ◽  
E Coli ◽  
Specificity Determining Positions ◽  
Correlated Mutations ◽  
Enzyme Families ◽  
Hydrolase Family ◽  
Silico Analysis

The Glycoside Hydrolase Family 65 (GH65) is an enzyme family of inverting a-glucoside phosphorylases and hydrolases that currently contains 10 characterized enzyme specificities. However, its sequence diversity has never been studied in detail. Here, an in-silico analysis of correlated mutations was performed, revealing specificity-determining positions that facilitate annotation of the family’s phylogenetic tree. By searching these positions for amino acid motifs that do not match those found in previously characterized enzymes from GH65, several clades that may harbor new functions could be identified. Three enzymes from across these regions were expressed in E. coli and their substrate profile was mapped. One of those enzymes, originating from the bacterium Mucilaginibacter mallensis, was found to hydrolyze kojibiose and a-1,2-oligoglucans with high specificity. We propose kojibiose glucohydrolase as the systematic name and kojibiose hydrolase or kojibiase as the short name for this new enzyme. This work illustrates a convenient strategy for mapping the natural diversity of enzyme families and smartly mining the ever-growing number of available sequences in the quest for novel specificities.

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