Low Level Of Sequence Similarity Research Articles

Molecular and Mechanistic Characterization of PddB, the First PLP-Independent 2,4-Diaminobutyric Acid Racemase Discovered in an Actinobacterial D-Amino Acid Homopolymer Biosynthesis.

We recently disclosed that the biosynthesis of antiviral γ-poly-D-2,4-diaminobutyric acid (poly-D-Dab) in Streptoalloteichus hindustanus involves an unprecedented cofactor independent stereoinversion of Dab catalyzed by PddB, which shows weak homology to diaminopimelate epimerase (DapF). Enzymological properties and mechanistic details of this enzyme, however, had remained to be elucidated. Here, through a series of biochemical characterizations, structural modeling, and site-directed mutageneses, we fully illustrate the first Dab-specific PLP-independent racemase PddB and further provide an insight into its evolution. The activity of the recombinant PddB was shown to be optimal around pH 8.5, and its other fundamental properties resembled those of typical PLP-independent racemases/epimerases. The enzyme catalyzed Dab specific stereoinversion with a calculated equilibrium constant of nearly unity, demonstrating that the reaction catalyzed by PddB is indeed racemization. Its activity was inhibited upon incubation with sulfhydryl reagents, and the site-directed substitution of two putative catalytic Cys residues led to the abolishment of the activity. These observations provided critical evidence that PddB employs the thiolate-thiol pair to catalyze interconversion of Dab isomers. Despite the low levels of sequence similarity, a phylogenetic analysis of PddB indicated its particular relevance to DapF among PLP-independent racemases/epimerases. Secondary structure prediction and 3D structural modeling of PddB revealed its remarkable conformational analogy to DapF, which in turn allowed us to predict amino acid residues potentially responsible for the discrimination of structural difference between diaminopimelate and its specific substrate, Dab. Further, PddB homologs which seemed to be narrowly distributed only in actinobacterial kingdom were constantly encoded adjacent to the putative poly-D-Dab synthetase gene. These observations strongly suggested that PddB could have evolved from the primary metabolic DapF in order to organize the biosynthesis pathway for the particular secondary metabolite, poly-D-Dab. The present study is on the first molecular characterization of PLP-independent Dab racemase and provides insights that could contribute to further discovery of unprecedented PLP-independent racemases.

The structure of a major surface antigen SAG19 from Eimeria tenella unifies the Eimeria SAG family

In infections by apicomplexan parasites including Plasmodium, Toxoplasma gondii, and Eimeria, host interactions are mediated by proteins including families of membrane-anchored cysteine-rich surface antigens (SAGs) and SAG-related sequences (SRS). Eimeria tenella causes caecal coccidiosis in chickens and has a SAG family with over 80 members making up 1% of the proteome. We have solved the structure of a representative E. tenella SAG, EtSAG19, revealing that, despite a low level of sequence similarity, the entire Eimeria SAG family is unified by its three-layer αβα fold which is related to that of the CAP superfamily. Furthermore, sequence comparisons show that the Eimeria SAG fold is conserved in surface antigens of the human coccidial parasite Cyclospora cayetanensis but this fold is unrelated to that of the SAGs/SRS proteins expressed in other apicomplexans including Plasmodium species and the cyst-forming coccidia Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. However, despite having very different structures, Consurf analysis showed that Eimeria SAG and Toxoplasma SRS families each exhibit marked hotspots of sequence hypervariability that map to their surfaces distal to the membrane anchor. This suggests that the primary and convergent purpose of the different structures is to provide a platform onto which sequence variability can be imposed.

A method for making alignments of related protein sequences that share very little similarity; shark interleukin 2 as an example.

An optimized alignment of related protein sequences helps to see their important shared features and to deduce their phylogenetic relationships. At low levels of sequence similarity, there are no suitable computer programs for making the best possible alignment. This review summarizes some guidelines for how in such instances, nevertheless, insightful alignments can be made. The method involves, basically, the understanding of molecular family features at both the protein and intron-exon level, and the collection of many related sequences so that gradual differences may be observed. The method is exemplified by identifying and aligning interleukin 2 (IL-2) and related sequences in Elasmobranchii (sharks/rays) and coelacanth, as other authors have expressed difficulty with their identification. From the point of general immunology, it is interesting that the unusual long "leader" sequence of IL-15, already known in other species, is even more impressively conserved in cartilaginous fish. Furthermore, sequence comparisons suggest that IL-2 in cartilaginous fish has lost its ability to bind an IL-2Rα/15Rα receptor chain, which would prohibit the existence of a mechanism for regulatory T cell regulation identical to mammals.

Rhizobium panacihumi sp. nov., an isolate from ginseng-cultivated soil, as a potential plant growth promoting bacterium.

A novel bacterial strain designated DCY116T was isolated from ginseng-cultivated soil in Gochang-gun, Republic of Korea. Strain DCY116T, belongs to the genus Rhizobium, and is closely related to Rhizobium yantingense H66T (98.3%), Neorhizobium huautlense S02T (98.2%), Rhizobium soli DS-42T (98.1%), Rhizobium smilacinae PTYR-5T (97.9%), and Neorhizobium alkalisoli CCBAU 01393T (97.9%) based on 16S rRNA gene sequence analysis. Analysis of the housekeeping genes atpD, recA, and glnII showed low levels of sequence similarity (96.8%) between strain DCY116T and other closely related species. Strain DCY116T was Gram-stain negative, motile by peritrichous flagella, rod-shaped, strictly aerobic, catalase- and oxidase-positive. Q-10 was the predominant ubiquinone. The major cellular fatty acids were identified as C16:0 and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and an unknown lipid (L1-3). Genomic DNA G + C content of strain DCY116T was determined to be 57.2mol%. DNA-DNA homology values between strain DCY116T and closely related species of the genus Rhizobium were lower than 40%. Strain DCY116T produced indole-3-acetic acid, siderophores, and was able to solubilize phosphate as a potential plant growth promoting bacterium. In conclusion, the results of this study support strain DCY116T as a novel species of the genus Rhizobium, for which the name Rhizobium panacihumi is proposed. The type strain is DCY116T (= KCTC 62017T = JCM 32251T).

Improving insect control protein activity for GM crops: A case study demonstrating that increased target insect potency can be achieved without impacting mammalian safety

Many insect-protected crops express insecticidal crystal (Cry) proteins derived from the soil bacterium Bacillus thuringiensis (Bt), including both naturally-occurring Cry proteins and chimeric Cry proteins created through biotechnology. The Cry51Aa2 protein is a naturally-occurring Cry protein that was modified to increase its potency and expand its insect activity spectrum through amino acid sequence changes. The improved Cry51Aa2 variant, Cry51Aa2.834_16, and other developmental variants belong to the ETX_MTX2 family of proteins but share a low level of sequence similarity to other members of this family. This similarity is largely localized to the pore-forming and oligomerization protein domains, while sequence divergence is observed within the head domain that confers receptor binding specificity. The intact Cry51Aa2.834_16 protein was heat labile at temperatures ≥55 °C, and was rapidly degraded after exposure to the gastrointestinal protease pepsin. No acute oral toxicity was observed in mice for three protein variants of Cry51Aa2, including Cry51Aa2.834_16, at doses exceeding 1000 mg/kg body weight. The weight-of-evidence therefore supports the conclusion of safety for Cry51Aa2.834_16 and demonstrates that amino acid sequence modifications can be used to substantially increase insecticidal activity of a protein without an increased hazard to mammals.

Rhizobium gei sp. nov., a bacterial endophyte of Geum aleppicum.

A bacterial strain, designated as ZFJT-2T, was isolated from the stem of Geum aleppicum Jacq. collected from Taibai Mountain in Shaanxi Province, north-west China. Cells of strain ZFJT-2T were Gram-stain-negative, strictly aerobic, rod-shaped and motile by means of a single polar flagellum. The major fatty acids were summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0, 11-methyl C18 : 1ω7c and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), and the DNA G+C content was 58.3 mol% (HPLC). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ZFJT-2T was a member of the genus Rhizobium and was most closely related to Rhizobium giardinii KACC 10720T (98.6 % similarity) and Rhizobium herbae CCBAU 83011T (98.5 %). The low levels of sequence similarity found between the atpD, recA and glnII gene sequences of strain ZFJT-2T and those of recognized species of the genus Rhizobium (no more than 94.4, 87.2 and 89.5 %, respectively) indicated that it may represent a separate species of the genus Rhizobium. The DNA-DNA relatedness values for strain ZFJT-2T with respect to R. giardinii KACC 10720T and R. herbae CCBAU 83011T were 17.6 and 41.9 %, respectively. On the basis of phenotypic, phylogenetic and genotypic data, strain ZFJT-2T is considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium gei sp. nov. is proposed. The type strain is ZFJT-2T (=CCTCC AB 2013015T=KCTC 32301T=LMG 27603T).

Rhizobium yantingense sp. nov., a mineral-weathering bacterium.

A Gram-stain-negative, rod-shaped bacterial strain, H66(T), was isolated from the surfaces of weathered rock (purple siltstone) found in Yanting, Sichuan Province, PR China. Cells of strain H66(T) were motile with peritrichous flagella. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain H66(T) belongs to the genus Rhizobium. It is closely related to Rhizobium huautlense SO2(T) (98.1 %), Rhizobium alkalisoli CCBAU 01393(T) (98.0 %) and Rhizobium cellulosilyticum ALA10B2(T) (98.0 %). Analysis of the housekeeping genes, recA, glnII and atpD, showed low levels of sequence similarity (<92.0 %) between strain H66(T) and other recognized species of the genus Rhizobium. The predominant components of the cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0. The G+C content of strain H66(T) was 60.3 mol%. Strain H66(T) is suggested to be a novel species of the genus Rhizobium based on the low levels of DNA-DNA relatedness (ranging from 14.3 % to 40.0 %) with type strains of species of the genus Rhizobium and on its unique phenotypic characteristics. The namehttp://dx.doi.org/10.1601/nm.1279Rhizobium yantingense sp. nov. is proposed for this novel species. The type strain is H66(T) ( = CCTCC AB 2014007(T) = LMG 28229(T)).

Rhizobium lemnae sp. nov., a bacterial endophyte of Lemna aequinoctialis.

Bacterial strain L6-16(T) was isolated from Lemna aequinoctialis. Cells were Gram-stain-negative, rod-shaped and motile with monopolar flagella. The phylogenetic analysis of its nearly complete 16S rRNA gene sequence revealed that strain L6-16(T) was a member of the genus Rhizobium. Its closest relative was Rhizobium tarimense PL-41(T) with a 16S rRNA gene sequence similarity value of 98.3%. Sequence similarity analysis of the housekeeping recA and atpD genes showed low levels of sequence similarity (<93.9%) between strain L6-16(T) and other species of the genus Rhizobium. Strain L6-16(T) was able to grow between pH 5 and 11 (optimum 7.0) and at temperatures ranging from 20 to 41 °C (optimum 30 °C). It tolerated NaCl up to 1 % (w/v) (optimum 0.5%). C18 : 1ω7c and/or C18 : 1ω6c (summed feature 8; 79.5%) were found as predominant cellular fatty acids. The DNA G+C content of strain L6-16(T) was 58.1 mol% (Tm). Based on low levels of DNA-DNA relatedness, strain L6-16(T) was distinct from members of phylogenetically related species including R. tarimense PL-41(T) (38.3 ± 0.8%), Rhizobium rosettiformans W3(T) (6.9 ± 0.4%) and Rhizobium pseudoryzae J3-A127(T) (12.3 ± 0.6 %). Strain L6-16(T) was unable to nodulate the roots of Phaseolus vulgaris, and nodC and nifH genes were not detected. The results obtained from phylogenetic analyses, phenotypic characterization and DNA-DNA hybridization indicated that strain L6-16(T) represents a novel species of the genus Rhizobium, for which the name Rhizobium lemnae sp. nov. is proposed. The type strain is L6-16(T) ( = NBRC 109339(T) = BCC 55143(T)).

Rhizobium paknamense sp. nov., isolated from lesser duckweeds (Lemna aequinoctialis)

A Gram-stain-negative, rod-shaped bacterium was isolated and designated strain L6-8(T) during a study of endophytic bacterial communities in lesser duckweed (Lemna aequinoctialis). Cells of strain L6-8(T) were motile with peritrichous flagella. The analysis of the nearly complete 16S rRNA gene sequence indicated that strain L6-8(T) was phylogenetically related to species of the genus Rhizobium. Its closest relatives were Rhizobium borbori DN316(T) (97.6 %), Rhizobium oryzae Alt 505(T) (97.3 %) and Rhizobium pseudoryzae J3-A127(T) (97.0 %). The sequence similarity analysis of housekeeping genes recA, glnII, atpD and gyrB showed low levels of sequence similarity (<91.5 %) between strain L6-8(T) and other species of the genus Rhizobium with validly published names. The pH range for growth was 4.0-9.0 (optimum 6.0-7.0), and the temperature range for growth was 20-45 °C (optimum 30 °C). Strain L6-8(T) tolerated NaCl up to 2 % (w/v) (optimum 1 % NaCl). The predominant components of cellular fatty acids were C19 : 0 cyclo ω8c (31.32 %), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 25.39 %) and C16 : 0 (12.03 %). The DNA G+C content of strain L6-8(T) was 60.4 mol% (Tm). nodC and nifH were not amplified in strain L6-8(T). DNA-DNA relatedness between strain L6-8(T) and R. borbori DN316(T), R. oryzae Alt505(T) and R. pseudoryzae J3-A127(T) was between 11.2 and 18.3 %. Based on the sequence similarity analyses, phenotypic, biochemical and physiological characteristics and DNA-DNA hybridization, strain L6-8(T) could be readily distinguished from its closest relatives and represents a novel species of the genus Rhizobium, for which the name Rhizobium paknamense sp. nov. is proposed. The type strain is L6-8(T) ( = NBRC 109338(T) = BCC 55142(T)).

Vibrio inhibens sp. nov., a novel bacterium with inhibitory activity against Vibrio species

Strain BFLP-10(T), isolated from faeces of wild long-snouted seahorses (Hippocampus guttulatus), is a Gram-negative, motile and facultatively anaerobic rod. This bacterium produces inhibitory activity against Vibrio species. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BFLP-10(T) was a member of the genus Vibrio and was most closely related to Vibrio owensii (99%), Vibrio communis (98.9%), Vibrio sagamiensis (98.9%) and Vibrio rotiferianus (98.4%). However, multilocus sequence analysis using gyrB, pyrH, recA and topA genes revealed low levels of sequence similarity (<91.2%) with these closely related species. In addition, strain BFLP-10(T) could be readily differentiated from other closely related species by several phenotypic properties and fatty acid profiles. The G+C content of the DNA was 45.6 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BFLP-10(T) represents a novel species within the genus Vibrio, for which the name Vibrio inhibens sp. nov. is proposed. The type strain is BFLP-10(T) (=CECT 7692(T)=DSM 23440(T)).

Circumscribing the Conformational Peptide Epitope Landscape

The development of vaccines for new and re-emerging pathologies and infections is based on the ability to define immunogenic epitopes. An immunogenic B-cell peptide epitope is a specific restricted antigen region that is capable of eliciting a humoral immune response and of combining with a specific site on antibodies. Using a number of experimental models and based on data from several literature reports, we identified low levels of sequence similarity to the host proteome as one of the main factors modulating the B-cell epitope repertoire in the humoral immune response. In point of fact, a low level of sequence identity to the host proteins is a common denominator unifying the composite, disparate assembly of linear peptide B-cell epitopes that has been experimentally validated and described in the literature. Here, we explore the proteomic similarity of conformational epitopes experimentally validated and described in published reports. Again, discontinuous epitopic structures formed by non-contiguous amino acid residues were found to define peptide sequences with a low level of similarity to the host. The present meta-analysis adds further significance to the immunological low-similarity theory and its clinical implications. Potentially, low-similarity peptide epitopes pave the way for novel effective vaccines in cancer, autoimmunity, and infectious diseases.

HIV infection and HERV expression: a review

The human genome contains multiple copies of retrovirus genomes known as endogenous retroviruses (ERVs) that have entered the germ-line at some point in evolution. Several of these proviruses have retained (partial) coding capacity, so that a number of viral proteins or even virus particles are expressed under various conditions. Human ERVs (HERVs) belong to the beta-, gamma-, or spuma- retrovirus groups. Endogenous delta- and lenti- viruses are notably absent in humans, although endogenous lentivirus genomes have been found in lower primates. Exogenous retroviruses that currently form a health threat to humans intriguingly belong to those absent groups. The best studied of the two infectious human retroviruses is the lentivirus human immunodeficiency virus (HIV) which has an overwhelming influence on its host by infecting cells of the immune system. One HIV-induced change is the induction of HERV transcription, often leading to induced HERV protein expression. This review will discuss the potential HIV-HERV interactions.Several studies have suggested that HERV proteins are unlikely to complement defective HIV virions, nor is HIV able to package HERV transcripts, probably due to low levels of sequence similarity. It is unclear whether the expression of HERVs has a negative, neutral, or positive influence on HIV-AIDS disease progression. A positive effect was recently reported by the specific expression of HERVs in chronically HIV-infected patients, which results in the presentation of HERV-derived peptides to CD8+ T-cells. These cytotoxic T-cells were not tolerant to HERV peptides, as would be expected for self-antigens, and consequently lysed the HIV-infected, HERV-presenting cells. This novel mechanism could control HIV replication and result in a low plasma viral load. The possibility of developing a vaccination strategy based on these HERV peptides will be discussed.

Sequence–structure relationships in yeast mRNAs

It is generally accepted that functionally important RNA structure is more conserved than sequence due to compensatory mutations that may alter the sequence without disrupting the structure. For small RNA molecules sequence–structure relationships are relatively well understood. However, structural bioinformatics of mRNAs is still in its infancy due to a virtual absence of experimental data. This report presents the first quantitative assessment of sequence–structure divergence in the coding regions of mRNA molecules based on recently published transcriptome-wide experimental determination of their base paring patterns. Structural resemblance in paralogous mRNA pairs quickly drops as sequence identity decreases from 100% to 85–90%. Structures of mRNAs sharing sequence identity below roughly 85% are essentially uncorrelated. This outcome is in dramatic contrast to small functional non-coding RNAs where sequence and structure divergence are correlated at very low levels of sequence similarity. The fact that very similar mRNA sequences can have vastly different secondary structures may imply that the particular global shape of base paired elements in coding regions does not play a major role in modulating gene expression and translation efficiency. Apparently, the need to maintain stable three-dimensional structures of encoded proteins places a much higher evolutionary pressure on mRNA sequences than on their RNA structures.

Towards defining the complement of mammalian WFDC-domain-containing proteins

WFDC (whey/four-disulfide core)-domain-containing proteins are defined by the possession of one or more 40-50 amino acid domains that include eight conserved cysteine residues linked by four characteristic intramolecular disulfide bonds. Some also contain other structural domains, whereas in many the WFDC-domain is the only domain present. The WFDC-domain is not limited to mammals but is widespread across all lineages. There is increasing evidence to suggest that mammalian WFDC-domain-containing proteins are undergoing rapid molecular evolution and as might be expected they exhibit low levels of sequence similarity coupled with multiple examples of species-specific gene acquisition and gene loss. The characteristic structural domain (that is generally encoded by a single exon) makes these proteins relatively easy to identify in databases. This review will outline the repertoire of such domains within the mouse, but similar principles can be applied to the identification of all proteins within individual species.

Distant cousins: genomic and sequence diversity within the BPI fold-containing (BPIF)/PLUNC protein family

PLUNC (palate, lung and nasal epithelium clone) proteins make up the largest branch of the BPI (bactericidal/permeability-increasing protein)/LBP (lipopolysaccharide-binding protein) family of lipid-transfer proteins. PLUNCs make up one of the most rapidly evolving mammalian protein families and exhibit low levels of sequence similarity coupled with multiple examples of species-specific gene acquisition and gene loss. Vertebrate genomes contain multiple examples of genes that do not meet our original definition of what is required to be a member of the PLUNC family, namely conservation of exon numbers/sizes, overall protein size, genomic location and the presence of a conserved disulfide bond. This suggests that evolutionary forces have continued to act on the structure of this conserved domain in what are likely to be functionally important ways.

Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome

Functional screening for lipolytic enzymes at low temperatures resulted in the isolation of the novel cold-active esterases, EstM-N1 and EstM-N2, from a metagenomic DNA library of arctic soil samples. EstM-N1 and EstM-N2 were 395 and 407 amino acids in length, respectively, and showed the highest similarity to class C β-lactamases. However, they shared a relatively low level of sequence similarity (30%) with each other. Phylogenetic analysis of bacterial lipolytic enzymes confirmed that EstM-N1 and EstM-N2 belonged to family VIII of bacterial esterases/lipases. The (His)(6)-tagged esterases were purified to about 99% homogeneity from the soluble fraction of recombinant Escherichia coli cultures. The purified EstM-N1 and EstM-N2 retained more than 50% of maximal activity in the temperature range of 0-35 °C, with optimal temperatures of 20 °C and 30 °C, respectively. Both enzymes preferred the short acyl chains of p-nitrophenyl esters and exhibited very narrow substrate specificity, indicating that they are typical esterases. The β-lactamase activity of EstM-N1 and EstM-N2 was also detected and reached about 31% and 13% of the positive control enzyme, Bacillus cereus β-lactamase, respectively. These first cold-active esterases belonging to family VIII are expected to be useful for potential biotechnological applications as interesting biocatalysts.

The Crystal Structure of the C2A Domain of Otoferlin Reveals an Unconventional Top Loop Region

Otoferlin (Otof), whose genetic mutations cause profound deafness in humans, is a protein composed of at least six C2 domains, which are known as Ca2+-binding and phospholipid-binding regions. Mammalian ferlin proteins are proposed to act in membrane fusion events, with Otof being specifically required for exocytosis in auditory hair cells. Ferlin C2 domains exhibit a rather low level of sequence similarity to those of synaptotagmins, protein kinase C isoforms, or phospholipases. Here, we report the crystal structure of the N-terminal C2 domain of Otof (C2A) at 1.95-Å resolution. In contrast to previous predictions, we found that this C2 domain is complete with eight β-strands. Comparing the structure of Otof C2A to those of other C2 domains revealed one top loop in Otof to be significantly shorter. This results in a depression of the surface, which is positively charged for the Otof C2A domain, and contrasts with the head-like protrusion surrounded by a negatively charged “neck” typically found in other C2 domains. Isothermal titration calorimetry and circular dichroism spectroscopy studies confirmed that Otof C2A is unable to bind Ca2+, while the synaptotagmin-1 C2A domain exhibited Ca2+ binding under the same conditions. Furthermore, floatation assays revealed a failure of Otof C2A to bind to phospholipid membranes. Accordingly, no positively charged β-groove-like surface structure, which is known to bind phosphatidylinositol-4,5-bisphosphate in other C2 domains, was found at the respective position in Otof C2A. Taken together, these data demonstrate that the Otof C2A domain differs structurally and functionally from other C2 domains.

Genomic Evidence for Elements of a Programmed Cell Death Pathway In Plasmodium: Exploiting Programmed Parasite Death for Malaria Control?

AbstractAbstract 4226Malaria, caused by species of the Plasmodium parasite, remains a major global health burden. To combat the disease, new areas of investigation are constantly developed with a view to understanding the biology of the organism and identifying potential drug targets. A new line of enquiry has emerged concerning the occurrence of parasite programmed cell death (PCD); however, there are conflicting experimental reports regarding the presence and phenotype of PCD in Plasmodium species. Furthermore, very little genomic evidence exists for a PCD pathway in this genus with only caspase-like domains being detected. One reason for the limited availability of genomic data may be the unusual characteristics of the P. falciparum genome, such as the extreme AT bias. In this study we examined the complete genome sequences of four Plasmodium species (P. falciparum, P. vivax, P. yoelii and P. knowlesi) for evidence of a p53-dependant PCD pathway. This pathway is well characterized in animals, and elements of the molecular machinery have been identified in protozoa. In addition, p53-like responses leading to apoptosis (a common PCD phenotype) have been demonstrated in phylogenetically diverse eukaryotes, including animals, protozoa, green algae and plants, suggesting that the origin of p53-initiated PCD is evolutionary ancient leading us to believe that a similar genomic toolkit may exist in Plasmodium. A detailed analysis of the four Plasmodium genomes was performed using an array of computational approaches, which included standard homology methods, phylogenetics, structural models and a novel evolutionary rate-based alignment algorithm FIRE (Functional Inference using the Rates of Evolution), which was developed to identify homologous and analogous genes in organisms with unusual genomes, such as P. falciparum, and hence low sequence similarity. Homology methods uncovered key elements of a classical PCD pathway, including ATM, MDM2, CR6 and three peptidase C14 (catalytic caspase) domains in each of the four Plasmodium genomes. Phylogenetic analysis demonstrated that the peptidase C14 (caspase) domains are evolutionary ancient and cluster with other PCD-related caspases suggesting that they are involved in a PCD pathway as opposed to other cellular functions that may use similar domains. In addition, highly sensitive hidden Markov models retrieved 15 sequences with low similarity to the eukaryotic p53 DNA-binding domain (DBD). Further analysis revealed that two P. falciparum sequences (accession numbers PFE1120w and PF11_0091 in the Plasmodium database www.PlasmoDB.org version 6.5) may be p53 DBD-like sequences. Both have similar evolutionary rates across codons, as well as anti-parallel beta sheet structures with Greek key topology, which are a characteristic feature of known p53 DBDs. Whether either of these proteins has functional significance for the PCD pathway in P. falciparum requires empirical verification. However, these data and the existence of p53-like activity in plants and algae, suggest that this pathway has an ancient origin and provide the first evidence for a p53-dependant PCD pathway in malaria parasites. The presence of this genomic toolkit raises questions regarding the potential role of this pathway in the P. falciparum lifecycle. The phenomenon of programmed parasite death may have adaptive significance and proteins in the PCD pathway may potentially be sufficiently different from the human orthologs, given the low level of sequence similarity. These findings could thus open a new line of investigation for novel malaria drug targets. Disclosures:No relevant conflicts of interest to declare.

Vibrio stylophorae sp. nov., isolated from the reef-building coral Stylophora pistillata

A bacterial strain designated KTW-12(T) was isolated from a reef-building coral in Kenting, Taiwan, and was characterized using a polyphasic taxonomic approach. Strain KTW-12(T) was Gram-negative, semi-transparent, slightly curved rod-shaped, and non-motile. Growth occurred at 15-35 °C (optimum, 30 °C), at pH 6.0-9.0 (optimum, pH 7.0-8.0), and with 0.5-6.0 % NaCl (optimum, 2 %). The major cellular fatty acids were summed feature 3 (C₁₆:₁ω7c and/or C₁₆:₁ω6c), C₁₄:₀ and C₁₆:₀. The DNA G+C content was 47.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain KTW-12(T) was most closely related to Vibrio porteresiae MSSRF30(T), with 94.8 % gene sequence similarity. Further multilocus sequence analysis using rpoA, recA and pyrH genes also revealed low levels of sequence similarity (74.6-85.0 %) with all species of the genus Vibrio with validly published names. A multigene phylogenetic tree using concatenated sequences of the four genes (16S rRNA, rpoA, recA and pyrH) elucidated that strain KTW-12(T) occupied a distinct phylogenetic position, forming a long branch that was not clustered with any other known species of the genus Vibrio. Strain KTW-12(T) differed from V. porteresiae MSSRF30(T) in the ability to reduce nitrate to nitrite, hydrolysis of chitin, fermentation of sorbitol and production of arginine dihydrolase, valine arylamidase, cystine arylamidase and N-acetyl-β-glucosaminidase. On the basis of phenotypic, chemotaxonomic and phylogenetic distinctiveness, strain KTW-12(T) should be classified as representing a novel species, for which the name Vibrio stylophorae sp. nov. is proposed. The type strain is KTW-12(T) ( = BCRC 80105(T) = LMG 25357(T)).

Analogy-based protein structure prediction: III. Optimizing the combination of the substitution matrix and pseudopotentials used to align protein sequences with spatial structures

A general and fast method for maximizing the “recognition ability” of a linear combination of an arbitrary number of various methods used to recognize protein structures and produce sequence-to-structure alignments for the structurally analogous proteins is described. It is shown that, at a low level of sequence similarity, the optimal combination of methods displays a significantly higher recognition ability than each method alone; the leading role in this combination is played by (1) pseudopotentials of long-range interactions, (2) matrices of secondary structure similarity, and (3) amino acid substitution matrices. In the case of a high sequence similarity, substitution matrices play the leading and practically the sole role in the optimal combination, although the addition of pseudopotentials of long-range interactions and matrices of secondary structure similarity somewhat increases the recognition ability of the combined method.