Extracellular Ionic Locks Determine Variation in Constitutive Activity and Ligand Potency between Species Orthologs of the Free Fatty Acid Receptors FFA2 and FFA3

Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs, it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations, marked variation in ligand-independent constitutive activity was identified using a [³⁵S]GTPγS assay. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity in this assay, whereas the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the second extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity and in most cases also yielded corresponding changes in SCFA potency.     

Mycobacterium tuberculosis Rv2179c Protein Establishes a New Exoribonuclease Family with Broad Phylogenetic Distribution

Ribonucleases (RNases) maintain the cellular RNA pool by RNA processing and degradation. In many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), the enzymes mediating several central RNA processing functions are still unknown. Here, we identify the hypothetical Mtb protein Rv2179c as a highly divergent exoribonuclease. Although the primary sequence of Rv2179c has no detectable similarity to any known RNase, the Rv2179c crystal structure reveals an RNase fold. Active site residues are equivalent to those in the DEDD family of RNases, and Rv2179c has close structural homology to Escherichia coli RNase T. Consistent with the DEDD fold, Rv2179c has exoribonuclease activity, cleaving the 3′ single-strand overhangs of duplex RNA. Functional orthologs of Rv2179c are prevalent in actinobacteria and found in bacteria as phylogenetically distant as proteobacteria. Thus, Rv2179c is the founding member of a new, large RNase family with hundreds of members across the bacterial kingdom.     

Characterization and evaluation of an arbitrary primed Polymerase Chain Reaction (PCR) product for the specific detection of Brucella species

Laboratory detection of Brucella is based largely on bacterial isolation and phenotypic characterization. These methods are lengthy and labor-intensive and have been associated with a heightened risk of laboratory-acquired infection. Antibody based indirect detection methods also suffer from limitations in proper diagnosis of the organism. To overcome these problems, nucleic acid amplification has been explored for rapid detection and confirmation of the presence of Brucella spp. PCR-based diagnostics is useful for screening large populations of livestock to identify infected individuals and confirms the presence of the pathogen. Random Amplification of Polymorphic DNA (RAPD) was performed and identified a 1.3 kb PCR fragment specifically amplifiable from DNA isolated from Brucella. A BLAST search revealed no significant homology with the reported sequences from species other than the members of Brucella. The isolated fragment seems to be a part of d-alanine–d-alanine ligase gene in Brucella sp. Translational BLAST revealed certain degree of homology of this sequence with orthologs of this gene reported from other microbial species at the deduced amino acid level. The sequence information was used to develop PCR based assays to detect Brucella sp. from various samples. The minimum detection limit of Brucella from blood and milk samples spiked with Brucella DNA was found to be 1 ng/ml and 10 ng/ml, respectively. In conclusion, we demonstrated that the PCR based detection protocol was successfully used for the detection of Brucella from various organs and spiked samples of diseased sheep. Diagnosis of Brucellosis by PCR based method reported in this study is relatively rapid, specific and simple.     

Genome sequencing of two Neorhizobium galegae strains reveals a noeT gene responsible for the unusual acetylation of the nodulation factors

Background

The species Neorhizobium galegae comprises two symbiovars that induce nodules on Galega plants. Strains of both symbiovars, orientalis and officinalis, induce nodules on the same plant species, but fix nitrogen only in their own host species. The mechanism behind this strict host specificity is not yet known. In this study, genome sequences of representatives of the two symbiovars were produced, providing new material for studying properties of N. galegae, with a special interest in genomic differences that may play a role in host specificity.

Results

The genome sequences confirmed that the two representative strains are much alike at a whole-genome level. Analysis of orthologous genes showed that N. galegae has a higher number of orthologs shared with Rhizobium than with Agrobacterium. The symbiosis plasmid of strain HAMBI 1141 was shown to transfer by conjugation under optimal conditions. In addition, both sequenced strains have an acetyltransferase gene which was shown to modify the Nod factor on the residue adjacent to the non-reducing-terminal residue. The working hypothesis that this gene is of major importance in directing host specificity of N. galegae could not, however, be confirmed.

Conclusions

Strains of N. galegae have many genes differentiating them from strains of Agrobacterium, Rhizobium and Sinorhizobium. However, the mechanism behind their ecological difference is not evident. Although the final determinant for the strict host specificity of N. galegae remains to be identified, the gene responsible for the species-specific acetylation of the Nod factors was identified in this study. We propose the name noeT for this gene to reflect its role in symbiosis.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-500) contains supplementary material, which is available to authorized users.     

Detection of Mycobacterium bovis and Mycobacterium tuberculosis from Cattle: Possible Public Health Relevance

Mycobacterium bovis and Mycobacterium tuberculosis infect both animals and humans. The disease epidemiology by these agents differs in developed and developing countries due to the differences in the implementation of the prevention and control strategies. The present study describes the detection of M. bovis and M. tuberculosis from specimens of lungs and pulmonary lymph nodes of four cattle died in an organized herd of 183 cattle in the state of Himachal Pradesh, India, with inconclusive skin test results. Identification and distinction of these closely related mycobacterial species was done by PCR-RFLP targeting hsp65 gene followed by spacer oligonucleotide typing. Mixed infection of M. bovis and M. tuberculosis was detected in one cattle.     

Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus,Medicago, and Phaseolus

Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution.     

Identification,characterization, and utilization of single copy genes in 29 angiosperm genomes

Background

Single copy genes are common across angiosperm genomes. With the sufficiently high quality sequenced genomes, the identification of large-scale single copy genes among multiple species is possible. Although some characteristics have been reported, our study provides novel insights into single copy genes.

Results

We identified single copy genes across 29 angiosperm genomes. A significant negative correlation was found between the number of duplicate blocks and the number of single copy genes. We found that a considerable number of single copy genes are located in organelles, showing a preference for binding and catalytic activity. The analysis of effective number of codons (Nc) illustrates that single copy genes have a stronger codon bias than non-single copy genes in eudicots. The relative high expression level of single copy genes was partially confirmed by the RNA-seq data, rather than the Codon Adaptation Index (CAI). Unlike in most other species, a strongly negatively correlation occurs between Nc and GC3 among single copy genes in grass genomes. When compared to all non-single copy genes, single copy genes indicate more conservation (as indicated by Ka and Ks values). But our alternative splicing (AS) results reveal that selective constraints are weaker in single copy genes than in low copy family genes (1–10 in-paralogs) and stronger than high copy family genes (>10 in-paralogs). Using concatenated shared single copy genes, we obtained a well-resolved phylogenetic tree. With the addition of intron sequences, the branch support is improved, but striking incongruences are also evident. Therefore, it is noteworthy that inclusion of intron sequences seems more appropriate for the phylogenetic reconstruction at lower taxonomic levels.

Conclusions

Our analysis provides insight into the evolutionary characteristics of single copy genes across 29 angiosperm genomes. The results suggest that there are key differences in evolutionary constraints between single copy genes and non-single copy genes. And to some extent, these evolutionary constraints show some species-specific differences, especially between eudicots and monocots. Our preliminary evidence also suggests that the concatenated shared single copy genes are well suited for use in resolving phylogenetic relationships.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-504) contains supplementary material, which is available to authorized users.     

Annotation of hypothetical proteins orthologous in Pongo abelii and Sus scrofa

A hypothetical protein is predicted to be expressed from an open reading frame without known experimental evidence of translation. They constitute a substantial fraction of proteomes. Domain extraction from these hypothetical sequences helps to search for protein coding genes for protein structural and functional annotation. We describe the analysis of prediction data in a sequence dataset of hypothetical protein orthologs of Pongo abelii (orangutan) and Sus scrofa (pig). It should be noted that these orangutan-pig orthologs are also non-homologous to human proteins. These predicted data find application in the genome wide annotation of proteins in poorly understood genomes.

Abbreviations

PDB - Protein Data Bank, DEG - Database of Essential Genes, CDD - Conserved Domain Database, IUCN - International Union for Conservation of Nature.     

In vitro propagation of Rudraksha (Elaeocarpus sphaericus (Gaertn.) K. Schum): a biotechnological approach for conservation

The species Elaeocarpus sphaericus (Rudraksha) is a religious, medicinally important threatened tree of India. An efficient micropropagation protocol has been developed from nodal explants of this plant species collected from north-east India for large scale production of planting material at favourable sites within the country. Best shoot initiation occurred in MS medium supplemented with 2.2μM BA+2.2μM Kn in combination. Addition of Casein Hydrolysate (CH) (100mg/L) increased the shoot number. Microshoots excised and subcultured in 2.0μM BA further enhanced growth and multiplication. The shoot cultures were maintained in this concentration for 2years with subculturing at 6weeks interval. MS medium containing 5.0μM NAA was most effective for rooting. Successfully acclimatized plants (80%) showed normal growth under suitable habitat conditions.     

Evolutionary analysis of PHLPP1 gene in humans and non-human primates

The chromosome 18q22-23 region has been shown to be implicated in bipolar disorder (BPAD) by several studies. PHLPP1 gene, in the locus (chromosome 18q22-23), is involved in circadian pathways and bears modules like ‘PH domain and leucine rich repeat protein phosphatase’. This gene also contains a polyglutamine (CAG or PolyQ) repeat motif at the carboxyl terminal end. A comparative analysis of the PolyQ repeats of the PHLPP1 gene in humans, non-human primates and other species has been attempted in order to investigate the possible significance of repeat length as seen in other triplet-repeat associated diseases. Sequencing of the CAG repeat in humans and in non-human primates revealed that the CAG repeat is not polymorphic in humans; whereas, in other species it shows an area of high variability, both in length and sequence composition. Despite the conservation of circadian clock components in different species, there is remarkable diversity in the protein structure, regulation and biochemical functions of the circadian orthologs. These can be due to specific adaptations in accordance with the physiology of the particular species providing a species-specific biological advantage.     

Human GABARAP can restore autophagosome biogenesis in a C. elegans lgg-1 mutant

We recently described in C. elegans embryos, the acquisition of specialized functions for orthologs of yeast Atg8 (e.g., mammalian MAP1LC3/LC3) in allophagy, a selective and developmentally regulated autophagic process. During the formation of double-membrane autophagosomes, the ubiquitin-like Atg8/LC3 proteins are recruited to the membrane through a lipidation process. While at least 6 orthologs and paralogs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GABARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. During allophagy, LGG-1 acts upstream of LGG-2 and is essential for autophagosome biogenesis, whereas LGG-2 facilitates their maturation. We demonstrated that LGG-2 directly interacts with the HOPS complex subunit VPS-39, and mediates the tethering between autophagosomes and lysosomes, which also requires RAB-7. In the present addendum, we compared the localization of autophagosomes, endosomes, amphisomes, and lysosomes in vps-39, rab-7, and lgg-2 depleted embryos. Our results suggest that lysosomes interact with autophagosomes or endosomes through a similar mechanism. We also performed a functional complementation of an lgg-1 null mutant with human GABARAP, its closer homolog, and showed that it localizes to autophagosomes and can rescue LGG-1 functions in the early embryo.     

Sequence conservation among orthologous vomeronasal type 1 receptor-like (ora) genes does not support the differential tuning hypothesis in Salmonidae

Salmon utilize olfactory cues to guide natal stream homing during spawning migrations. Both inorganic and biogenic chemicals have been proposed as odorants that might be used by salmon during homing. In this study, we used genomic DNA sequence data from nine salmonid species to compare nucleotide identities for orthologous main olfactory receptor (mOR) genes with nucleotide identities for orthologous vomeronasal type 1-like (ora) receptor genes. We found that orthologs for both classes of olfactory receptor genes (mORs and Oras) appear to be highly conserved among species. Our findings do not support the differential tuning hypothesis in Salmonidae, which predicts higher sequence conservation for mORs than ora. We did, however, find convincing evidence for site-specific positive selection acting on paralogous main olfactory receptor genes.     

Two newly recognized species of Hemidactylus (Squamata,Gekkonidae) from the Arabian Peninsula?and Sinai,Egypt

A recent molecular phylogeny of the Arid clade of the genus Hemidactylus revealed that the recently described H. saba and two unnamed Hemidactylus species from Sinai, Saudi Arabia and Yemen form a well-supported monophyletic group within the Arabian radiation of the genus. The name ‘Hemidactylus saba species group’ is suggested for this clade. According to the results of morphological comparisons and the molecular analyses using two mitochondrial (12S and cytb) and four nuclear (cmos, mc1r, rag1, rag2) genes, the name Hemidactylus granosus Heyden, 1827 is resurrected from the synonymy of H. turcicus for the Sinai and Saudi Arabian species. The third species of this group from Yemen is described formally as a new species H. ulii sp. n. The phylogenetic relationships of the members of ‘Hemidactylus saba species group’ are evaluated and the distribution and ecology of individual species are discussed.     

Meloidogyne trifoliophila n. sp. (Nemata: Meloidogynidae), a Parasite of Clover from Tennessee

Meloidogyne trifoliophila n. sp. is described from white clover collected at Ames Plantation, Fayette County, Tennessee. The perineal pattern is rounded, with long, smooth striae and rounded arch, and without distinct lateral lines or perivulval striae. The female stylet is 12.6-15.5 μm long, the excretory pore is level with or up to one stylet length posterior to the stylet knobs, and the vulva is subterminal. The posterior terminus is weakly protuberant. The male lateral field is composed of approximately eight repeatedly broken or forked incisures. The male stylet is 17.0-18.9 μm long, the stylet knobs are rounded and sloping, gradually merging with the shaft, and the head region consists of one large annule. Second-stage juveniles are 357-400 μm long, with a stylet length of 11.9-13.6 μm and one head annule. The tail tapers to a slender tip. This new species is similar to M. graminicola and M. triticoryzae but differs from them in perineal pattern and lateral field morphology, and numerous morphometric characters.