Comparative Sequence Analysis Research Articles

Streptomyces solincola sp. nov., isolated from soil in Malaysia.

A urease-producing Gram-stain-positive actinobacterium, designated strain T5T, was isolated from a soil sample collected at a highway hillslope in Selangor, Malaysia. The strain was found to produce pale yellowish-pink aerial mycelia with smooth long chain spores and extensively branched light yellowish-pink substrate mycelia on oatmeal agar. Strain T5T grew at 15-37 °C, pH 6-11, and tolerated up to 9 % (w/v) NaCl, with optimal growth occurring at 28 °C, pH 6-9 and without NaCl. The whole-cell sugar hydrolysate of strain T5T contained galactose, glucose and ribose. The ll-diaminopimelic acid isomer was detected in the cell wall. Diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were found to be the predominant polar lipids. The main fatty acids were anteiso-C17 : 0, iso-C16 : 0, anteiso-C15 : 0 and iso-C14 : 0. Comparative analysis of the 16S rRNA gene sequences indicated that strain T5T belonged to Streptomyces of the family Streptomycetaceae with the highest 16S rRNA gene sequence similarity to Streptomyces lichenis LCR6-01T (99.0 %). The overall genome relatedness indices revealed that the closest related species was S. lichenis LCR6-01T with 89.4 % average nucleotide identity and 33.7 % digital DNA-DNA hybridization. Phylogeny analyses showed that strain T5T was closely related to Streptomyces fradiae, Streptomyces lavendofoliae, Streptomyces lichenis, Streptomyces roseolilacinus and Streptomyces somaliensis. Based on these polyphasic data, strain T5T represents a novel species, for which the name Streptomyces solincola sp. nov. is proposed. The type strain is T5T (=TBRC 5137T= DSM 42166T).

Structural analysis of the pseudaminic acid synthase PseI from Campylobacter jejuni

Campylobacter jejuni PseI is a pseudaminic acid synthase that condenses the 2,4-diacetamido-2,4,6-trideoxy-l-altrose sugar (6-deoxy AltdiNAc) and phosphoenolpyruvate to generate pseudaminic acid, a sialic acid-like 9-carbon backbone α-keto sugar. Pseudaminic acid is conjugated to cell surface proteins and lipids and plays a key role in the mobility and virulence of C. jejuni and other pathogenic bacteria. To provide insights into the catalytic mechanism of PseI, we performed a structural study on PseI. PseI forms a two-domain structure and assembles into a domain-swapped homodimer. The PseI dimer has two cavities, each of which accommodates a metal ion using conserved histidine residues. A comparative analysis of structures and sequences suggests that the cavity of PseI functions as an active site that binds the 6-deoxy AltdiNAc and phosphoenolpyruvate substrates and mediates their condensation. Furthermore, we propose the substrate binding-induced structural rearrangement of PseI and predict 6-deoxy AltdiNAc recognition residues that are specific to PseI.

Fine-mapping and candidate gene analysis of qFL-c10-1 controlling fiber length in upland cotton (Gossypium hirsutum L.).

A fiber length QTL, qFL-c10-1, was fine-mapped to a 96.5-kb region containing one gene that has not been characterized in plants. Fiber length is an important component of cotton fiber quality, which is associated with other quality properties such as fiber strength, fiber maturity, and fineness. In our previous studies, a stable QTL qFL-c10-1 controlling fiber length had been identified on chromosome A10 in an upland cotton recombinant inbred line (RIL) population from a cross between Jimian5 and DH962. To fine-map qFL-c10-1, an F2 population with 1081 individual plants from a cross between a recombinant line DJ61 and Jimian5 was established. Using linkage analysis and progeny recombination experiment, qFL-c10-1 was mapped into a 96.5-kb genomic region that just contained one proper transcript Ghir_A10G022020 (described as GhFL10), an undescribed gene in plants. One 214-bp deletion was identified in the promoter region of DJ61 compared with Jimian5. Quantitative real-time PCR (qRT-PCR) and comparative analysis of parental sequences suggested that GhFL10 was the most promising candidate gene for qFL-c10-1. According to RNA-seq, yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC), we speculate that GhFL10 interacts with NF-YA transcription factors to negatively regulate fiber elongation.

Role of Klebsiella pneumoniae Type VI secretion system (T6SS) in long-term gastrointestinal colonization

Type VI secretion systems (T6SS), recently described in hypervirulent K. pneumoniae (hvKp) strains, are involved in bacterial warfare but their role in classical clinical strains (cKp) has been little investigated. In silico analysis indicated the presence of T6SS clusters (from zero to four), irrespective of the strains origin or virulence, with a high prevalence in the K. pneumoniae species (98%). In the strain CH1157, two T6SS-apparented pathogenicity islands were detected, T6SS-1 and -2, harboring a phospholipase-encoding gene (tle1) and a potential new effector-encoding gene named tke (Type VI Klebsiella effector). Tle1 expression in Escherichia coli periplasm affected cell membrane permeability. T6SS-1 isogenic mutants colonized the highest gastrointestinal tract of mice less efficiently than their parental strain, at long term. Comparative analysis of faecal 16S sequences indicated that T6SS-1 impaired the microbiota richness and its resilience capacity. Oscillospiraceae family members could be specific competitors for the long-term gut establishment of K. pneumoniae.

Comprehensive collection of genes and comparative analysis of full-length transcriptome sequences from Japanese larch (Larix kaempferi) and Kuril larch (Larix gmelinii var. japonica)

BackgroundJapanese larch (Larix kaempferi) is an economically important deciduous conifer species that grows in cool-temperate forests and is endemic to Japan. Kuril larch (L. gmelinii var. japonica) is a variety of Dahurian larch that is naturally distributed in the Kuril Islands and Sakhalin. The hybrid larch (L. gmelinii var. japonica × L. kaempferi) exhibits heterosis, which manifests as rapid juvenile growth and high resistance to vole grazing. Since these superior characteristics have been valued by forestry managers, the hybrid larch is one of the most important plantation species in Hokkaido. To accelerate molecular breeding in these species, we collected and compared full-length cDNA isoforms (Iso-Seq) and RNA-Seq short-read, and merged them to construct candidate gene as reference for both Larix species. To validate the results, candidate protein-coding genes (ORFs) related to some flowering signal-related genes ​were screened from the reference sequences, and the phylogenetic relationship with closely related species was elucidated.ResultsUsing the isoform sequencing of PacBio RS ll and the de novo assembly of RNA-Seq short-read sequences, we identified 50,690 and 38,684 ORFs in Japanese larch and Kuril larch, respectively. BUSCO completeness values were 90.5% and 92.1% in the Japanese and Kuril larches, respectively. After comparing the collected ORFs from the two larch species, a total of 19,813 clusters, comprising 22,571 Japanese larch ORFs and 22,667 Kuril larch ORFs, were contained in the intersection of the Venn diagram. In addition, we screened several ORFs related to flowering signals (SUPPRESSER OF OVEREXPRESSION OF CO1: SOC1, LEAFY: LFY, FLOWERING Locus T: FT, CONSTANCE: CO) from both reference sequences, and very similar found in other species.ConclusionsThe collected ORFs will be useful as reference sequences for molecular breeding of Japanese and Kuril larches, and also for clarifying the evolution of the conifer genome and investigating functional genomics.

Comparative full genome sequence analysis of wild-type and chicken embryo origin vaccine-like infectious laryngotracheitis virus field isolates from Canada.

Infectious laryngotracheitis (ILT), caused by infectious laryngotracheitis virus (ILTV), occurs sporadically in poultry flocks in Canada. Live attenuated chicken embryo origin (CEO) vaccines are being used routinely to prevent and control ILTV infections. However, ILT outbreaks still occur since vaccine strains could revert to virulence in the field. In this study, 7 Canadian ILTV isolates linked to ILT outbreaks across different time in Eastern Canada (Ontario; ON and Quebec; QC) were whole genome sequenced. Phylogenetic analysis confirmed the close relationship between the ON isolates and the CEO vaccines, whereas the QC isolates clustered with strains previously known as CEO revertant and wild-type ILTVs. Recombination network analysis of ILTV sequences revealed clear evidence of historical recombination between ILTV strains circulating in Canada and other geographical regions. The comparison of ON CEO clustered and QC CEO revertant clustered isolates with the LT Blen® CEO vaccine reference sequence showed amino acid differences in 5 and 12 open reading frames (ORFs), respectively. Similar analysis revealed amino acid differences in 32 ORFs in QC wild-type isolates. Compared to all CEO vaccine strains in the public domain, the QC wild-type isolates showed 15 unique mutational sites leading to amino acid changes in 13 ORFs. Our outcomes add to the knowledge of the molecular mechanisms behind ILTV genetic variance and provide genetic markers between wild-type and vaccine strains.

Screening of immune-related genes against bacterial infection in Ostrinia furnacalis (Lepidoptera: Crambidae)

Ostrinia furnacalis GuenĂŠe is an important pest of maize. The current use of microbial agents to control O. furnacalis have been relatively successful. However, upon infestation with microorganisms, O. furnacalis initiates an innate immune response to defend itself against foreign invaders. Therefore, understanding the immune mechanisms in O. furnacalis is important to ensure a more efficient use of microbial agents for the control of this pest. In the present study, a gram-positive bacterium, Staphylococcus aureus, and a gram-negative bacterium, Escherichia coli, were used to induce an immune response in O. furnacalis. Transcriptomic sequencing was then used to screen for genes that were significantly differentially expressed after microbial induction and sequence comparison and phylogenetic analysis to identify immune-related genes the expression of which was significantly induced. The results show a large number of genes were differentially expressed in O. furnacalis after microbial induction, the majority of which were immune-related genes. The expression of some immune-related genes such as those encoding antimicrobial peptides, peptidoglycan recognition proteins and serine protease inhibitors were strongly induced. Overall, our study increases the understanding of the mechanism of the innate immune response in O. furnacalis and provides a theoretical basis for better biological control using microbial agents.

Characterization of internal transcribed spacer-1 and apical membrane antigen-1 sequences provides insights into the genetic diversity of Eimeria tenella strains.

Coccidiosis is a major recurring problem in the poultry industry and is caused by infection of one or more of the seven Eimeria species known to infect chickens, with Eimeria tenella among the best studied and economically important. Studies on the genetic diversity of E. tenella strains is essential for the development of universally acceptable diagnostic markers and vaccines against the disease. Eimeria tenella internal transcribed spacer-1 (ITS-1) and apical membrane antigen-1 (AMA-1) sequences from different parts of the world are available in the public domain and therefore provide suitable comparative markers for genetic diversity study. In this study, the ITS-1 and AMA-1 sequence diversity of two local E. tenella strains, namely EtNSN6 and EtSGR6 were characterized. Both ITS-1 and AMA-1 sequences for EtNSN6 and EtSGR6 were retrieved by mapping to their respective genome sequences generated using next generation sequencing. Multiple sequence alignment of the ITS-1 and AMA-1 sequences with selected homologous sequences revealed the presence of a total of five and 13 single nucleotide polymorphisms (SNPs) respectively. All SNPs appeared to occur at random and did not show any unique pattern based on geographical regions while no insertions and deletions (indels) was found to occur in the aligned sequences. However, unique bases that defined certain strains were detected. Phylogenetics analyses performed with Maximum Parsimony (MP) and Maximum Likelihood (ML) methods revealed similar topology for the internal groups with all the E. tenella ITS-1 and AMA-1 sequences grouped in the same clade supported by high bootstrap confidence. This confirmed that both EtNSN6 and EtSGR6 samples are E. tenella strains. Sequence comparison and phylogenetics analyses further suggest the possibility of low genetic diversity among E. tenella strains.

Transcriptomic Identification of a Unique Set of Nodule-Specific Cysteine-Rich Peptides Expressed in the Nitrogen-Fixing Root Nodule of Astragalus sinicus.

Legumes in the inverted repeat-lacking clade (IRLC) each produce a unique set of nodule-specific cysteine-rich (NCR) peptides, which act in concert to determine the terminal differentiation of nitrogen-fixing bacteroid. IRLC legumes differ greatly in their numbers of NCR and sequence diversity. This raises the significant question how bacteroid differentiation is collectively controlled by the specific NCR repertoire of an IRLC legume. Astragalus sinicus is an IRLC legume that forms indeterminate nodules with its microsymbiont Mesorhizobium huakuii 7653R. Here, we performed transcriptome analysis of root and nodule samples at 3, 7, 14, 28 days postinoculation with M. huakuii 7653R and its isogenic ∆bacA mutant. BacA is a broad-specificity peptide transporter required for the host-derived NCRs to target rhizobial cells. A total of 167 NCRs were identified in the RNA transcripts. Comparative sequence and electrochemical analysis revealed that A. sinicus NCRs (AsNCRs) are dominated by a unique cationic group (termed subgroup C), whose mature portion is relatively long (>60 amino acids) and phylogenetically distinct and possessing six highly conserved cysteine residues. Subsequent functional characterization showed that a 7653R variant harboring AsNCR083 (a representative of subgroup C AsNCR) displayed significant growth inhibition in laboratory media and formed ineffective white nodules on A. sinicus with irregular symbiosomes. Finally, bacterial two-hybrid analysis led to the identification of GroEL1 and GroEL3 as the molecular targets of AsNCR067 and AsNCR076. Together, our data contribute to a systematic understanding of the NCR repertoire associated with the A. sinicus and M. huakuii symbiosis. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Prevalence and Genomic Sequence Analysis of Domestic Cat Hepadnavirus in the United States.

Hepadnaviruses are partially double-stranded DNA viruses that infect a variety of species. The prototypical virus in this family is the human hepatitis B virus, which chronically infects approximately 400 million people worldwide and is a risk factor for progressive liver disease and liver cancer. The first hepadnavirus isolated from carnivores was a domestic cat hepadnavirus (DCH), initially identified in Australia and subsequently detected in cats in Europe and Asia. As with all characterized hepadnaviruses so far, DCH infection has been associated with hepatic disease in its host. Prevalence of this infection in the United States has not been explored broadly. Thus, we utilized conventional and quantitative PCR to screen several populations of domestic cats to estimate DCH prevalence in the United States. We detected DCH DNA in 1 out of 496 animals (0.2%) in the U.S. cohort. In contrast, we detected circulating DCH DNA in 7 positive animals from a cohort of 67 domestic cats from Australia (10.4%), consistent with previous studies. The complete consensus genome of the U.S. DCH isolate was sequenced by Sanger sequencing with overlapping PCR products. An in-frame deletion of 157 bp was identified in the N-terminus of the core open reading frame. The deletion begins at the direct repeat 1 sequence (i.e., the 5′ end of the expected double-stranded linear DNA form), consistent with covalently closed circular DNA resultant from illegitimate recombination described in other hepadnaviruses. Comparative genome sequence analysis indicated that the closest described relatives of the U.S. DCH isolate are those previously isolated in Italy. Motif analysis supports DCH using NTCP as an entry receptor, similar to human HBV. Our work indicates that chronic DCH prevalence in the U.S. is likely low compared to other countries.

388 Young Scholar Award Talk: Increasing Efficiency of Finishing Beef Production: Optimizing Starch Digestion, Improving Flaking Quality, and Reducing Methane Emissions

Abstract The objectives of my dissertation research are to develop strategies to increase the efficiency of finishing beef production by optimizing the site and extent of starch digestion, improving flaking quality and consistency, and reducing methane emissions. Post-flaking sampling and handling techniques such as sifting steam-flaked corn samples with a sieve (P < 0.01), air equilibration time (P < 0.01), and storage temperature during shipping (P < 0.01) all influenced enzymatic starch availability of steam-flaked corn. Inducing starch retrogradation of steam-flaked corn decreased the rate (P < 0.01), but not the extent (P = 0.80), of ruminal dry matter degradation and thus, energetic efficiency was predicted to increase by up to 3.4% because of increased postruminal flow and digestion of starch in the small intestine. If producing steam-flaked corn is not feasible for smaller capacity feedlots, finely ground corn has a similar ruminal starch solubility to steamflaked corn (38.1% vs. 44.4, respectively, P > 0.05), suggesting an alternative processing method to increase feeding value in finishing diets. Duodenal infusion of glucoamylase (fungal origin) increased (P = 0.02) small intestinal starch disappearance in steers, while duodenal infusion of porcine α-amylase did not (P = 0.41), supporting the hypothesis that small intestinal carbohydrases are the primary hydrolytic limit to small intestinal starch digestion. Comparative sequencing analyses revealed a missing region of the stalk region of the sucrase-isomaltase protein sequence (AA 47-60) which is important for intracellular sorting of the sucrase subunit to the apical membrane. The absence of this region of the protein likely explains the absence of intestinal sucrase activity and quantitative limitations of starch digestion in the ruminant small intestine. Feeding decoquinate with Yucca schidigera extract for 7-d decreased (P < 0.01) ruminal acetate proportion and tended to increase ruminal (P = 0.08) propionate proportion, suggesting potential for hydrogen shifts which could contribute to reductions in methane emissions with longer feeding periods. The findings of my dissertation research provide: 1) post-flaking sampling and handling recommendations for commercial feedlots and laboratories to improve estimates of starch availability and flaking consistency, 2) a mechanistic target (sucrase-isomaltase) for limitations in small intestinal starch digestion, 3) practical feeding strategies to shift the site of starch digestion to the small intestine to increase feed efficiency (retrogradation), 4) new processing alternatives for small feedlot producers (fine ground corn inclusion), and 5) new information on how combinations of feed additives could potentially decrease methane production in feedlot cattle.

Antisense pairing and SNORD13 structure guide RNA cytidine acetylation.

N4-acetylcytidine (ac4C) is an RNA nucleobase found in all domains of life. The establishment of ac4C in helix 45 (h45) of human 18S ribosomal RNA (rRNA) requires the combined activity of the acetyltransferase NAT10 and the box C/D snoRNA SNORD13. However, the molecular mechanisms governing RNA-guided nucleobase acetylation in humans remain unexplored. After applying comparative sequence analysis and site-directed mutagenesis to provide evidence that SNORD13 folds into three main RNA helices, we report two assays that enable the study of SNORD13-dependent RNA acetylation in human cells. First, we demonstrate that ectopic expression of SNORD13 rescues h45 in a SNORD13 knockout cell line. Next, we show that mutant snoRNAs can be used in combination with nucleotide resolution ac4C sequencing to define structure and sequence elements critical for SNORD13 function. Finally, we develop a second method that reports on the substrate specificity of endogenous NAT10-SNORD13 via mutational analysis of an ectopically expressed pre-rRNA substrate. By combining mutational analysis of these reconstituted systems with nucleotide resolution ac4C sequencing, our studies reveal plasticity in the molecular determinants underlying RNA-guided cytidine acetylation that is distinct from deposition of other well-studied rRNA modifications (e.g., pseudouridine). Overall, our studies provide a new approach to reconstitute RNA-guided cytidine acetylation in human cells as well as nucleotide resolution insights into the mechanisms governing this process.

Genomic Analysis of LEA Genes in Carica papaya and Insight into Lineage-Specific Family Evolution in Brassicales.

Late embryogenesis abundant (LEA) proteins comprise a diverse superfamily involved in plant development and stress responses. This study presents a first genome-wide analysis of LEA genes in papaya (Carica papaya L., Caricaceae), an economically important tree fruit crop widely cultivated in the tropics and subtropics. A total of 28 members were identified from the papaya genome, which belong to eight families with defined Pfam domains, i.e., LEA_1 (3), LEA_2 (4), LEA_3 (5), LEA_4 (5), LEA_5 (2), LEA_6 (2), DHN (4), and SMP (3). The family numbers are comparable to those present in Ricinus communis (Euphorbiaceae, 28) and Moringa oleifera (Moringaceae, 29), but relatively less than that found in Moringa oleifera (Cleomaceae, 39) and Arabidopsis thaliana (Brassicaceae, 51), implying lineage-specific evolution in Brassicales. Indeed, best-reciprocal-hit-based sequence comparison and synteny analysis revealed the presence of 29 orthogroups, and significant gene expansion in Tarenaya and Arabidopsis was mainly contributed by whole-genome duplications that occurred sometime after their split with the papaya. Though a role of transposed duplication was also observed, tandem duplication was shown to be a key contributor in gene expansion of most species examined. Further comparative analyses of exon-intron structures and protein motifs supported fast evolution of this special superfamily, especially in Arabidopsis. Transcriptional profiling revealed diverse expression patterns of CpLEA genes over various tissues and different stages of developmental fruit. Moreover, the transcript level of most genes appeared to be significantly regulated by drought, cold, and salt stresses, corresponding to the presence of cis-acting elements associated with stress response in their promoter regions. These findings not only improve our knowledge on lineage-specific family evolution in Brassicales, but also provide valuable information for further functional analysis of LEA genes in papaya.

Molecular and genetic detection of infectious laryngeotrachitis disease virus in broiler farms after a disease outbreak in Egypt.

Infectious laryngotracheitis (ILT) is a viral respiratory illness in poultry that causes massive financial losses. This research aimed to isolate and identify the ILT virus in suspected outbreaks of broiler flocks in Egypt during 2020-2021, besides investigating its genetic link with other circulating strains. Real-time-PCR was used to test 57 samples taken from unvaccinated broiler farms. Ten samples are positive for ILTV, and the virus is being isolated in SPF chicken embryos. The Sanger sequencing was used to conduct (partial) sequencing of the infected cell protein4 gene (ICP4) for eight isolates. Phylogenetic analysis conducted Maximum Likelihood, comparative sequencing analysis of ICP4 of strains under study with vaccination ILT reference strains reveled that all isolates were clustered into two major groups. The (OM291843and OM291846) clustered together with the chicken embryo origin vaccine strains (IV and V group). The remaining six strains belong to the TCO vaccine(I, II and III group). The total sequence similarity between the strains under study and the various Egyptian strains varied from (97 to 100%) while the similarity with TCO or chicken embryo origin -vaccine strains ranged from (95to 100%). There were no deletions detected in the 272-283-bp region of the ICP4 gene. Detection of arginine to methionine substitutions at position 180 (R180M) and change of Serine to Asparagine at position 227 (S227N) in the (OM291843 and OM291846) which were previously described in chicken embryo origin -vaccine strains. This reveals that field strains may have evolved from vaccine strains, notably identification of non-synonymous substitutions which might be linked to the virulence strains' attenuation. Finally, independent of geographical distribution, both chicken embryo origin-vaccine-like and TCO-Vaccine-like virus strains were circulating in Egyptian non-vaccinated broiler flocks in 2020 and 2021. Despite their genetic differences, both viruses caused significant illnesses in the field.

Comparative analysis of genome-encoded viral sequences reveals the evolutionary history of flavivirids (family Flaviviridae).

Flavivirids (family Flaviviridae) are a group of positive-strand ribonucleic acid (RNA) viruses that pose serious risks to human and animal health on a global scale. Here, we use flavivirid-derived deoxyribonucleic acid (DNA) sequences, identified in animal genomes, to reconstruct the long-term evolutionary history of family Flaviviridae. We demonstrate that flavivirids are >100 million years old and show that this timing can be combined with dates inferred from co-phyletic analysis to produce a cohesive overview of their evolution, distribution, and diversity wherein the main flavivirid subgroups originate in early animals and broadly co-diverge with major animal phyla. In addition, we reveal evidence that the 'classical flaviviruses' of vertebrates, most of which are transmitted via blood-feeding arthropod vectors, originally evolved in haematophagous arachnids and later acquired the capacity to be transmitted by insects. Our findings imply that the biological properties of flavivirids have been acquired gradually over the course of animal evolution. Thus, broad-scale comparative analysis will likely reveal fundamental insights into their biology. We therefore published our results via an open, extensible, database (Flavivirid-GLUE), which we constructed to facilitate the wider utilisation of genomic data and evolution-related domain knowledge in flavivirid research.

Probiotic potentials of the silkworm gut symbiont Enterococcus casseliflavusECB140, a promising L-tryptophan producer living inside the host.

L-tryptophan is an essential aromatic amino acid for the growth and development of animals. Studies about enteric L-tryptophan-producing bacteria are scarce. In this report, we characterized the probiotic potential of Enterococcus casseliflavus ECB140, focusing on its L-tryptophan production abilities. ECB140 strain was isolated from the silkworm gut and can survive under strong alkaline environmental conditions. Bacterial colonization traits (motility and biofilm) were examined and showed that only ECB140 produced flagellum and strong biofilms compared with other Enterococcus strains. Comparative genome sequence analyses showed that only ECB140 possessed a complete route for L-tryptophan synthesis among all 15 strains. High-performance liquid chromatography and qRT-PCR confirmed the capability of ECB140 to produce L-tryptophan. Besides, the genome also contains the biosynthesis pathways of several other essential amino acids, such as phenylalanine, threonine, valine, leucine, isoleucine and lysine. These results indicate that ECB140 has the ability to survive passage through the gut and could act as a candidate probiotic. The study describes a novel, natural silkworm gut symbiont capable of producing L-tryptophan. Enterococcus casseliflavus ECB140 physical and genomic attributes offer possibilities for its colonization and provide L-tryptophan for lepidopteran insects.

Comparative genomic characterization and screening of regulatory regions of casein gene family in Bos taurus and Bubalus bubalis

Both cattle and buffalo are rich genetic resource, raised for dairy, meat and draft purposes and possess great economic significance globally. Genomic characterization is imperative to explore inherent potential of animal species for targeted improvement in performance traits. This study aimed to perform comparative genomic characterization and sequence analysis of casein genes family (CSN1S1, CSN1S2, CSN2 and CSN3) in Bos taurus and Bubalus bubalis. Phylogenetic, motif, conserved domain and gene structure analyses discovered evolutionarily conserved nature of these genes in cattle and buffalo. Results indicated that casein proteins were thermostable and hydrophilic, although αs1, β, and κ caseins showed acidic behavior except for αs2-casein that was somewhat basic. The present study revealed lower distribution of transcription binding (TFB) sites particularly CAP, GATA, HSF2 and LMO2COM in Bubalus bubalis as compared to Bos taurus which might have potential effects on the transcription and translational efficiencies of casein genes in Bos taurus. However, functional studies are required to evaluate the effects of these TFB sites on regulation of casein genes expression in Bos taurus and Bubalus bubalis.

Molecular explorations of the Leishmania donovani 6-phosphogluconolactonase enzyme, a key player in the pentose phosphate pathway

The enzymes of the pentose phosphate pathway are vital to survival in kinetoplastids. The second step of the pentose phosphate pathway involves hydrolytic cleavage of 6-phosphogluconolactone to 6-phosphogluconic acid by 6- phosphogluconolactonase (6PGL). In the present study, Leishmania donovani 6PGL (Ld6PGL) was cloned and overexpressed in bacterial expression system. Comparative sequence analysis revealed the conserved sequence motifs, functionally and structurally important residues in 6PGL family. In silico amino acid substitution study and interacting partners of 6PGL were predicted. The Ld6PGL enzyme was found to be active in the assay and in the parasites. Specificity was confirmed by Western blot analysis. The ∼30 kDa protein was found to be a dimer in MALDI, glutaraldehyde crosslinking and size exclusion chromatography studies. Kinetic analysis and structural stability studies of Ld6PGL were performed with denaturants and at varied temperature. Computational 3D Structural modelling of Ld6PGL elucidates that it has a similar α/β hydrolase fold structural topology as in other members of 6PGL family. The three loops are found in extended form when the structure is compared with the human 6PGL (Hs6PGL). Further, enzyme substrate binding mode and its mechanism were investigated using the molecular docking and molecular simulation studies. Interesting dynamics action of substrate 6-phosphogluconolactone was observed into active site during MD simulation. Interesting differences were observed between host and parasite enzyme which pointed towards its potential to be explored as an antileishmanial drug target. This study forms the basis for further analysis of the role of Ld6PGL in combating oxidative stress in Leishmania.

Crystal Structure of the Recombination Mediator Protein RecO from Campylobacter jejuni and Its Interaction with DNA and a Zinc Ion.

Homologous recombination is involved in repairing DNA damage, contributing to maintaining the integrity and stability of viral and cellular genomes. In bacteria, the recombination mediator proteins RecO and RecR are required to load the RecA recombinase on ssDNA for homologous recombination. To structurally and functionally characterize RecO, we determined the crystal structure of RecO from Campylobacter jejuni (cjRecO) at a 1.8 Å resolution and biochemically assessed its capacity to interact with DNA and a metal ion. cjRecO folds into a curved rod-like structure that consists of an N-terminal domain (NTD), C-terminal domain (CTD), and Zn2+-binding domain (ZnD). The ZnD at the end of the rod-like structure coordinates three cysteine residues and one histidine residue to accommodate a Zn2+ ion. Based on an extensive comparative analysis of RecO structures and sequences, we propose that the Zn2+-binding consensus sequence of RecO is CxxC…C/HxxC/H/D. The interaction with Zn2+ is indispensable for the protein stability of cjRecO but does not seem to be required for the recombination mediator function. cjRecO also interacts with ssDNA as part of its biological function, potentially using the positively charged patch in the NTD and CTD. However, cjRecO displays a low ssDNA-binding affinity, suggesting that cjRecO requires RecR to efficiently recognize ssDNA for homologous recombination.

Isolation of a diazinon-degrading strain Sphingobium sp. DI-6 and its novel biodegradation pathway.

Diazinon is one of the most widely used organophosphate insecticides, one that is frequently detected in the environment. In this study, a diazinon-degrading bacterium, DI-6, previously isolated from diazinon-contaminated soil in China has been subsequently identified as Sphingobium sp. on the basis of its physiological and biochemical characteristics, as well as by virtue of a comparative analysis of 16S rRNA gene sequences. This strain is capable of using diazinon as its sole carbon source for growth and was able to degrade 91.8% of 100 mg L–1 diazinon over a 60-h interval. During the degradation of diazinon, the following seven metabolites were captured and identified by gas chromatography/mass spectrometry (GC–MS) analysis: diazoxon, diazinon aldehyde, isopropenyl derivative of diazinon, hydroxyethyl derivative of diazinon, diazinon methyl ketone, O-[2-(1-hydroxyethyl)-6-methylpyrimidin-4-yl] O-methyl O-hydrogen phosphorothioate, and O-(6-methyl pyrimidin-4-yl) O,O-dihydrogen phosphorothioate. Based on these metabolites, a novel microbial biodegradation pathway of diazinon by Sphingobium sp. DI-6 is proposed. This research provides potentially useful information for the application of the DI-6 strain in bioremediation of diazinon-contaminated environments.