Comprehensive in silico structural-functional analysis of Enterobacter GH19 class I chitinase (chiRAM) gene: cloning and heterologous expression

I. Background. Present study aims to clone and express the gene-encoding chitinase / GH19 family from Enterobacter sp. in E.coli with in silico sequence analyses.. II. Methods and results. The putative open reading frame of GH19 chitinase from Enterobacter sp. strain EGY1 was cloned and expressed into pGEM-T and pET-28a + vectors, respectively using a degenerate primer. The isolated nucleotide sequence (1821 bp, Genbank accession no.: MK533791.2) was translated to chiRAM protein (606 amino acids, UniProt accession no.: A0A4D6J2L9). chiRAM in silico protein sequence analysis revealed GH19 class I chitinase: N-terminus signal peptide (Met1-Ala23), catalytic domain (Val83-Glu347 & catalytic triad Glu149, Glu171, Ser218), proline-rich hinge (Pro414 -Pro450), (polycystic kidney disease protein motif (Gly 465-Ser 533), C-terminus chitin-binding domain (Ala553- Glu593), and class I conserved motifs (NYNY and AQETGG). Three dimensional model was constructed by LOMETS MODELLER, PDB template: 2dkvA (Oryza sativa L. japonica class I chitinase). Recombinant chiRAM was overexpressed as inclusion bodies (IBs) (~ 72kDa; SDS-PAGE) in 1.0 mM IPTG induced E.coli BL21 (DE3) Rosetta at room temperature, 18 hrs post induction. Optimized expression yielded active chiRAM with 1.974 U/mL ± 0.0002, on shrimp colloidal chitin (SCC), in induced E.coli BL21 (DE3) Rosetta cells growing in SB medium. LC-MS/MS identified the 72 kDa band in the soluble fraction with 52.3% coverage sequence exclusive to Enterobacter cloacae chitinase/GH19 (WP_063869339.1). III. Conclusions. Despite the successful cloning and expression of chiRAM of Enterobacter sp. in E.coli with an appreciable chitinase activity, prospective studies would focus on minimizing IBs to facilitate chiRAM purification and characterization.

A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia

Background Polo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation. Methods To assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites Results After incubating trophozoites with 5 μM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation. Conclusions These data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.

The True Host/s of Picobirnaviruses

Picobirnaviruses (PBVs) are bisegmented double-stranded RNA viruses that have been detected in a wide variety of animal species including invertebrates and in environmental samples. Since PBVs are ubiquitous in feces/gut contents of humans and other animals with or without diarrhea, they were considered as opportunistic enteric pathogens of mammals and avian species. However, the virus remains to be propagated in animal cell cultures, or in gnotobiotic animals. Recently, the classically defined prokaryotic motif, the ribosomal binding site sequence, has been identified upstream of putative open reading frame/s in PBV and PBV-like sequences from humans, various animals, and environmental samples, suggesting that PBVs might be prokaryotic viruses. On the other hand, based on the detection of some novel PBV-like RNA-dependent RNA polymerase sequences that use the alternative mitochondrial genetic code (that of mold or invertebrates) for translation, and principal component analysis of codon usage bias for these sequences, it has been proposed that PBVs might be fungal viruses with a lifestyle reminiscent of mitoviruses. These contradicting observations warrant further studies to ascertain the true host/s of PBVs, which still remains controversial. In this minireview, we have focused on the various findings that have raised a debate on the true host/s of PBVs.

First Report on Detection and Genetic Diversity of Picobirnaviruses in the Small Indian Mongoose (Herpestes auropunctatus)

Picobirnaviruses (PBVs), family Picobirnaviridae, are bi-segmented, double-stranded RNA viruses. PBVs are considered opportunistic enteric pathogens. Gene segment-1 of PBV encodes the capsid protein, whilst gene segment-2 codes for RNA-dependent RNA polymerase (RdRp). Based on differences in gene segment-2, PBVs are classified into genogroup-I (GI) and GII. Although PBVs have been detected in a wide variety of host species, there are no reports on PBVs from mongoose so far. We report here high rates of detection (35.36%, 29/82) of GI PBVs in fecal samples from the small Indian mongoose (Herpestes auropunctatus) on the Caribbean island of St. Kitts. Applying a combination of a non-specific primer-based amplification method and conventional RT-PCR using a newly designed primer targeting the 3′-untranslated region (UTR), we could amplify and sequence the complete/nearly complete gene segment-2 of eight mongoose PBV strains. Except for a single strain, the gene segment-2 of the remaining mongoose PBV strains contained the putative open reading frame encoding the RdRp. The gene segment-2/putative RdRps of the mongoose PBV strains retained various features that are conserved in other PBVs (5′- and 3′-terminal nucleotide sequences, bacterial ribosomal binding site sequence in 5′-UTR, and the three domains in putative RdRps). On the other hand, phylogenetic analysis and sequence identities of the putative RdRps revealed high genetic diversity among the mongoose PBV strains and with those of PBVs from other host species. To our knowledge, this is the first report on detection and genetic diversity of PBVs from the mongoose, expanding the host range of PBVs and providing vital insights into the various features and evolution of putative RdRps of PBVs in a new host species.

Detection of ALDH3B2 in Human Placenta

Aldehyde dehydrogenase 3B2 (ALDH3B2) gene contains a premature termination codon, which can be skipped or suppressed resulting in full-length protein expression. Alternatively, the longest putative open reading frame starting with the second in-frame start codon would encode short isoform. No unequivocal evidence of ALDH3B2 expression in healthy human tissues is available. The aim of this study was to confirm its expression in human placenta characterized by the highest ALDH3B2 mRNA abundance. ALDH3B2 DNA and mRNA were sequenced. The expression was investigated using western blot. The identity of the protein was confirmed using mass spectrometry (MS). The predicted tertiary and quaternary structures, subcellular localization, and phosphorylation sites were assessed using bioinformatic analyses. All DNA and mRNA isolates contained the premature stop codon. In western blot analyses, bands corresponding to the mass of full-length protein were detected. MS analysis led to the identification of two unique peptides, one of which is encoded by the nucleotide sequence located upstream the second start codon. Bioinformatic analyses suggest cytoplasmic localization and several phosphorylation sites. Despite premature stop codon in DNA and mRNA sequences, full-length ALDH3B2 was found. It can be formed as a result of premature stop codon readthrough, complex phenomenon enabling stop codon circumvention.

Structure of GTP cyclohydrolase I from Listeria monocytogenes, a potential anti-infective drug target

A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (K m = 53 µM; v max = 180 nmol mg−1 min−1). The protein was crystallized from 1.3 M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4 Å (R free = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D 5-symmetric decamer with ten topologically equivalent active sites. Screening a small library of about 9000 compounds afforded several inhibitors with IC50 values in the low-micromolar range. Several inhibitors had significant selectivity with regard to human GTP cyclohydrolase I. Hence, GTP cyclohydrolase I may be a potential target for novel drugs directed at microbial infections, including listeriosis, a rare disease with high mortality.

Complete genome sequence and evolution analysis of Psychrobacter sp. YP14 from Gammaridea Gastrointestinal Microbiota of Yap Trench

Psychrobacter sp. YP14, a moderately psychrophilic bacterium belonging to the class Gammaproteobacteria, was isolated from Gammaridea Gastrointestinal Microbiota of Yap Trench. The strain has one circular chromosome of 2,895,311 bp with a 44.66% GC content, consisting of 2333 protein-coding genes, 53 tRNA genes and 9 rRNA genes. Four plasmids were completely assembled and their sizes were 13,712 bp, 19711 bp, 36270 bp, 8194 bp, respectively. In particular, a putative open reading frame (ORF) for dienelactone hydrolase (DLH) related to degradation of chlorinated aromatic hydrocarbons. To get an better understanding of the evolution of Psychrobacter sp. YP14 in this genus, six Psychrobacter strains (G, PRwf-1, DAB_AL43B, AntiMn-1,P11G5, P2G3), with publicly available complete genome, were selected and comparative genomics analysis were performed among them. The closest phylogenetic relationship was identified between strains G and K5 based on 16s gene and ANI (average nucleotide identity) values. Analysis of the pan-genome structure found that YP14 has fewer COG clusters associated with transposons and prophage which indicates fewer sequence rearrangements compared with PRwf-1. Besides, stress response-related genes of strain YP14 demonstrates that it has less strategies to cope with extreme environment, which is consistent with its intestinal habitat. The difference of metabolism and strategies coped with stress response of YP14 are more conducive to the study of microbial survival and metabolic mechanisms in deep sea environment.

The Human CytomegalovirusUL116Gene Encodes an Envelope Glycoprotein Forming a Complex with gH Independently from gL

ABSTRACTHuman cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and is the leading viral cause of birth defects after congenital infection. HCMV infection relies on the recognition of cell-specific receptors by one of the viral envelope glycoprotein complexes. Either the gH/gL/gO or the gH/gL/UL128/UL130/UL131A (Pentamer) complex has been found to fulfill this role, accounting for HCMV entry into almost all cell types. We have studied theUL116gene product, a putative open reading frame identified byin silicoanalysis and predicted to code for a secreted protein. Virus infection experiments in mammalian cells demonstrated that UL116 is expressed late in the HCMV replication cycle and is a heavily glycosylated protein that first localizes to the cellular site of virus assembly and then inserts into the virion envelope. Transient-transfection studies revealed that UL116 is efficiently transported to the plasma membrane when coexpressed with gH and that gL competes with UL116 for gH binding. Further evidence for gH/UL116 complex formation was obtained by coimmunoprecipitation experiments on both transfected and infected cells and biochemical characterization of the purified complex. In summary, our results show that the product of theUL116gene is an HCMV envelope glycoprotein that forms a novel gH-based complex alternative to gH/gL. Remarkably, the gH/UL116 complex is the first herpesvirus gH-based gL-less complex.IMPORTANCEHCMV infection can cause severe disease in immunocompromised adults and infants infectedin utero. The dissection of the HCMV entry machinery is important to understand the mechanism of viral infection and to identify new vaccine antigens. The gH/gL/gO and gH/gL/UL128/UL130/UL131 (Pentamer) complexes play a key role in HCMV cell entry and tropism. Both complexes are formed by an invariant gH/gL scaffold on which the other subunits assemble. Here, we show that theUL116gene product is expressed in infected cells and forms a heterodimer with gH. The gH/UL116 complex is carried on the infectious virions, although in smaller amounts than gH/gL complexes. No gH/UL116/gL ternary complex formed in transfected cells, suggesting that the gH/UL116 complex is independent from gL. This new gH-based gL-free complex represents a potential target for a protective HCMV vaccine and opens new perspectives on the comprehension of the HCMV cell entry mechanism and tropism.

Mytilus galloprovincialis Carbonic Anhydrase II: Activity and cDNA Sequence Analysis

The esterase activity of carbonic anhydrase (CA) was investigated in mussels sampled at 24 locations along the Croatian coast of Adriatic Sea. The gills were the target tissue because the respiratory, ionic transport and pH regulatory enzyme function of CA and its potential usage as biomarkers of environmental pollution was the main topic. Total esterase activity was measured in cytosolic fraction by colorimetric end-point reaction using p-nitrophenyl acetate as enzyme substrate. CA activity was estimated by the same enzymatic reaction using acetazolamide as a specific CA inhibitor. The results of total esterase activities in winter (March; all sites average value 0.137±0.057) were lower than determined for summer season (August; 0.153±0.036) at almost all investigated locations. CA activities determined in gills of mussel sampled in winter ranged from 1.75% to 24.65% of total esterase activities and in summer samples were lower (0.83 to 13.45%). Although recent research showed potential application of CA activity in bioassay and biomarker in pollution studies, further research is needed. Here we report a short-term simple colorimetric microplate method which can be applied for analyses of large numbers of samples. Furthermore in this study we characterized full length coding sequence (cDNA) of M. galloprovincialis carbonic anhydrase II (CAII). The CAII cDNA (with the 5’ and 3’ untranslated regions) is 1317 bp long. The putative open reading frame encodes a polypeptide of 256 amino acids, with a theoretical pI/Mw 5.87/28.416 kDa and conserved domains (active site and zinc binding site).

Sonorensin: an Antimicrobial Peptide, Belonging to the Heterocycloanthracin Subfamily of Bacteriocins, from a New Marine Isolate, Bacillus sonorensis MT93

ABSTRACTMarine environments are the greatest fronts of biodiversity, representing a resource of unexploited or unknown microorganisms and new substances having potential applications. Among microbial products, antimicrobial peptides (AMPs) have received great attention recently due to their applications as food preservatives and therapeutic agents. A new marine soil isolate producing an AMP was identified asBacillus sonorensisbased on 16S rRNA gene sequence analysis. It produced an AMP that showed a broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The peptide, named sonorensin, was purified to homogeneity using a combination of chromatographic techniques. The intact molecular mass of the purified peptide, 6,274 Da, as revealed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF), was in agreement with Tricine-SDS-PAGE analysis. A PCR array of primers was used to identify AMP structural genes, which allowed the successful amplification of the related genes from strain MT93. The putative open reading frame of sonorensin was amplified, cloned into the pET-32a(+) vector, expressed as a thioredoxin (Trx) fusion protein inEscherichia coli, and then purified. Sequence alignment analysis revealed that the bacteriocin being reported could belong to new subfamily of bacteriocins, heterocycloanthracin. The peptide indicated its potential as a biocontrol agent or food antimicrobial agent, due to its antimicrobial activity against bacteria such asListeria monocytogenesandStaphylococcus aureus. This is the first report of the production, purification, and characterization of wild-type and recombinant bacteriocin byB. sonorensisand the first bacteriocin of the heterocycloanthracin subfamily to be characterized.