Comparative Genomics Analysis of Streptococcus Isolates from the Human Small Intestine Reveals their Adaptation to a Highly Dynamic Ecosystem

The human small-intestinal microbiota is characterised by relatively large and dynamic Streptococcus populations. In this study, genome sequences of small-intestinal streptococci from S. mitis, S. bovis, and S. salivarius species-groups were determined and compared with those from 58 Streptococcus strains in public databases. The Streptococcus pangenome consists of 12,403 orthologous groups of which 574 are shared among all sequenced streptococci and are defined as the Streptococcus core genome. Genome mining of the small-intestinal streptococci focused on functions playing an important role in the interaction of these streptococci in the small-intestinal ecosystem, including natural competence and nutrient-transport and metabolism. Analysis of the small-intestinal Streptococcus genomes predicts a high capacity to synthesize amino acids and various vitamins as well as substantial divergence in their carbohydrate transport and metabolic capacities, which is in agreement with observed physiological differences between these Streptococcus strains. Gene-specific PCR-strategies enabled evaluation of conservation of Streptococcus populations in intestinal samples from different human individuals, revealing that the S. salivarius strains were frequently detected in the small-intestine microbiota, supporting the representative value of the genomes provided in this study. Finally, the Streptococcus genomes allow prediction of the effect of dietary substances on Streptococcus population dynamics in the human small-intestine.     

Finding of Bov-B LINE retroelement in parthenogenetic and bisexual lizard species of the genus Darevskia (Lacertidae)

The Bov-B LINE retrotransposon was first discovered in Ruminantia and was long considered to be specific for this order. Later, this mobile element was described in snakes and some lizard species. Analysis of phylogenetic relationships of Bov-B LINE elements from different ruminants, snakes, and lizard species led to the suggestion on horizontal transfer of this retrotransposon from Squamata to Ruminantia. In the Squamata group, Bov-B LINE element was found in all snakes and some lizard species examined. The element was not detected in the genomes of some species of the genera Lacerta and Podarcis. In the present study, using PCR amplification and sequencing of PCR products, Bov-B LINE element was identified in the genomes of parthenogenetic and bisexual species of the genus Darevskia (Lacertidae), as well as in such species as Lacerta agilis and Zootoca vivipara, where this retrotransposon had not been not detected before.     

Quantitative Tracing, by Taq Nuclease Assays, of a Synechococcus Ecotype in a Highly Diversified Natural Population

Quantitative Taq nuclease assays (TNAs) (TaqMan PCR), nested PCR in combination with denaturing gradient gel electrophoresis (DGGE), and epifluorescence microscopy were used to analyze the autotrophic picoplankton (APP) of Lake Constance. Microscopic analysis revealed dominance of phycoerythrin (PE)-rich Synechococcus spp. in the pelagic zone of this lake. Cells passing a 3-μm-pore-size filter were collected during the growth period of the years 1999 and 2000. The diversity of PE-rich Synechococcus spp. was examined using DGGE to analyze GC-clamped amplicons of a noncoding section of the 16S-23S intergenic spacer in the ribosomal operon. In both years, genotypes represented by three closely related PE-rich Synechococcus strains of our culture collection dominated the population, while other isolates were traced sporadically or were not detected in their original habitat by this method. For TNAs, primer-probe combinations for two taxonomic levels were used, one to quantify genomes of all known Synechococcus-type cyanobacteria in the APP of Lake Constance and one to enumerate genomes of a single ecotype represented by the PE-rich isolate Synechococcus sp. strain BO 8807. During the growth period, genome numbers of known Synechococcus spp. varied by 2 orders of magnitude (2.9 × 10³ to 3.1 × 10⁵ genomes per ml). The ecotype Synechococcus sp. strain BO 8807 was detected in every sample at concentrations between 1.6 × 10¹ and 1.3 × 10⁴ genomes per ml, contributing 0.02 to 5.7% of the quantified cyanobacterial picoplankton. Although the quantitative approach taken in this study has disclosed several shortcomings in the sampling and detection methods, this study demonstrated for the first time the extensive internal dynamics that lie beneath the seemingly arbitrary variations of a population of microbial photoautotrophs in the pelagic habitat.     

Identification and annotation of noncoding RNAs in Saccharomycotina

The importance of ncRNAs in biological processes makes their annotation an essential component of any genome-sequencing project. The identification of ncRNAs in genomes requires specific expertise and tools that are distinct from the traditional protein gene annotation tools. Here, we describe the assembly of two automatic annotation pipelines, integrating publicly available tools, for homology and de novo ncRNA search in genomes. We applied both pipelines to 10 Saccharomycotina genomes and were able to find and annotate 693 ncRNA genes, corresponding to 81% of the ncRNAs expected for those genomes assuming the number of ncRNAs in Saccharomyces cerevisiae (86) as a reference. Several new ncRNAs, not yet known in the Saccharomycotina clade, were also detected. The results show the feasibility of automatic search for ncRNAs in full genomes and the utility of such approaches in large multi-genome sequencing and annotation projects.     

Persistence and Expression of the Herpes Simplex Virus Genome in the Absence of Immediate-Early Proteins

The immediate-early (IE) proteins of herpes simplex virus (HSV) function on input genomes and affect many aspects of host cell metabolism to ensure the efficient expression and regulation of the remainder of the genome and, subsequently, the production of progeny virions. Due to the many and varied effects of IE proteins on host cell metabolism, their expression is not conducive to normal cell function and viability. This presents a major impediment to the use of HSV as a vector system. In this study, we describe a series of ICP4 mutants that are defective in different subsets of the remaining IE genes. One mutant, d109, does not express any of the IE proteins and carries a green fluorescent protein (GFP) transgene under the control of the human cytomegalovirus IE promoter (HCMVIEp). d109 was nontoxic to Vero and human embryonic lung (HEL) cells at all multiplicities of infection tested and was capable of establishing persistent infections in both of these cell types. Paradoxically, the genetic manipulations that were required to eliminate toxicity and allow the genome to persist in cells for long periods of time also dramatically lowered the level of transgene expression. Efficient expression of the HCMVIEp-GFP transgene in the absence of ICP4 was dependent on the ICP0 protein. In d109-infected cells, the level of transgene expression was very low in most cells but abundant in a small subpopulation of cells. However, expression of the transgene could be induced in cells containing quiescent d109 genomes weeks after the initial infection, demonstrating the functionality of the persisting genomes.     

Detection of virulence-related genes in <Emphasis Type="Italic">Lactococcus garvieae</Emphasis> and their expression in response to different conditions

Lactococcus garvieae has emerged as an important zoonotic pathogen. However, information regarding mechanisms and factors related to its pathogenicity is lacking. In the present study, we investigated the distribution and functionality of genes related to virulence factors in L. garvieae strains isolated from different niches (diseased fish, humans, meat and dairy products, vegetables), using both post-genomic and genotypic analysis. Putative genes encoding hemolysin, fibronectin-binding protein, and penicillin acylase were detected in all analyzed genomes/strains. Their expression was significantly induced by bile salt stress. Putative genes encoding bile salt hydrolase were found in a few strains from dairy and human sources, as well as the mobilizable tet genes. Finally, all genomes possessed a folate gene cluster, in which mutations in the dihydropteroate synthase gene (folP) could be related to sulfonamide resistance. To the best of our knowledge, this is the first study aimed to explore the pathogenic potential of L. garvieae through the analysis of numerous L. garvieae genomes/strains, coming from different sources. This approach allowed the detection of virulence-related genes not yet investigated in the species and the study of their expression after exposure to different environmental stresses. The results obtained suggest a virulence potential in some L. garvieae strains that can be exploited for survival in the human gastrointestinal tract.     

Human Adenoviruses and Coliphages in Urban Runoff-Impacted Coastal Waters of Southern California

A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.     

Identification of Two Novel Members of the Tentative Genus Wukipolyomavirus in Wild Rodents

Two novel polyomaviruses (PyVs) were identified in kidney and chest-cavity fluid samples of wild bank voles (Myodes glareolus) and common voles (Microtus arvalis) collected in Germany. All cloned and sequenced genomes had the typical PyV genome organization, including putative open reading frames for early regulatory proteins large T antigen and small T antigen on one strand and for structural late proteins (VP1, VP2 and VP3) on the other strand. Virus-like particles (VLPs) were generated by yeast expression of the VP1 protein of both PyVs. VLP-based ELISA and large T-antigen sequence-targeted polymerase-chain reaction investigations demonstrated signs of infection of these novel PyVs in about 42% of bank voles and 18% of common voles. In most cases only viral DNA, but not VP1-specific antibodies were detected. In additional animals exclusively VP1-specific antibodies, but no viral DNA was detected, indicative for virus clearance. Phylogenetic and clustering analysis including all known PyV genomes placed novel bank vole and common vole PyVs amongst members of the tentative Wukipolymavirus genus. The other known four rodent PyVs, Murine PyV and Hamster PyV, and Murine pneumotropic virus and Mastomys PyV belong to different phylogenetic clades, tentatively named Orthopolyomavirus I and Orthopolyomavirus II, respectively. In conclusion, the finding of novel vole-borne PyVs may suggest an evolutionary origin of ancient wukipolyomaviruses in rodents and may offer the possibility to develop a vole-based animal model for human wukipolyomaviruses.     

The characterization of complete mitochondrial genome and phylogenetic relationship within Rattus genus (Rodentia: Muridae)

The genus Rattus is one of the main pest genus of rodent. Most species of the genus carry all kinds of pathogenic bacteria to human being. They are traditionally considered to be a least understood group. The complete mitochondrial genome of the White-Footed Indochinese Rat, Rattus nitidus was determined in this study. The characterization of mitochondrial genomes of Rattus genus was also analyzed based on comprehensive comparison. The result of evolutionary patterns of protein-coding genes (PCGs) suggested purifying selection was the predominant evolutionary forces in the mitochondrial genomes of Rattus genus. The NADH dehydrogenase 4 gene (ND4) showed a highly elevated Ka/Ks ratio compared to the other protein-coding genes, which indicated ND4 was most likely under relaxed selection pressure. Phylogenetic analysis provided a well-supported outline of Rattus genus, and revealed two groups in the genus. R. nitidus had a sister relationship with R. norvegicus.     

Development of SSR markers and identification of major quantitative trait loci controlling shelling percentage in cultivated peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Key message

A total of 204,439 SSR markers were developed in diploid genomes, and 25 QTLs for shelling percentage were identified in a RIL population across 4 years including five consistent QTLs.

Abstract

Cultivated peanut (Arachis hypogaea L.) is an important grain legume providing edible oil and protein for human nutrition. Genome sequences of its diploid ancestors, Arachis duranensis and A. ipaensis, were reported, but their SSRs have not been well exploited and utilized hitherto. Shelling percentage is an important economic trait and its improvement has been one of the major objectives in peanut breeding programs. In this study, the genome sequences of A. duranensis and A. ipaensis were used to develop SSR markers, and a mapping population (Yuanza 9102 × Xuzhou 68-4) with 195 recombinant inbred lines was used to map QTLs controlling shelling percentage. The numbers of newly developed SSR markers were 84,383 and 120,056 in the A. duranensis and A. ipaensis genomes, respectively. Genotyping of the mapping population was conducted with both newly developed and previously reported markers. QTL analysis using the phenotyping data generated in Wuhan across four consecutive years and genotyping data of 830 mapped loci identified 25 QTLs with 4.46–17.01% of phenotypic variance explained in the four environments. Meta-analysis revealed five consistent QTLs that could be detected in at least two environments. Notably, the consistent QTL cqSPA09 was detected in all four environments and explained 10.47–17.01% of the phenotypic variance. The segregation in the progeny of a residual heterozygous line confirmed that the cpSPA09 locus had additive effect in increasing shelling percentage. These consistent and major QTL regions provide opportunity not only for further gene discovery, but also for the development of functional markers for breeding.

     

Identification and characterization of expressed retrotransposons in the genome of the Paracoccidioides species complex

Background

Species from the Paracoccidioides complex are thermally dimorphic fungi and the causative agents of paracoccidioidomycosis, a deep fungal infection that is the most prevalent systemic mycosis in Latin America and represents the most important cause of death in immunocompetent individuals with systemic mycosis in Brazil. We previously described the identification of eight new families of DNA transposons in Paracoccidioides genomes. In this work, we aimed to identify potentially active retrotransposons in Paracoccidioides genomes.

Results

We identified five different retrotransposon families (four LTR-like and one LINE-like element) in the genomes of three Paracoccidioides isolates. Retrotransposons were present in all of the genomes analyzed. P. brasiliensis and P. lutzii species harbored the same retrotransposon lineages but differed in their copy numbers. In the Pb01, Pb03 and Pb18 genomes, the number of LTR retrotransposons was higher than the number of LINE-like elements, and the LINE-like element RtPc5 was transcribed in Paracoccidioides lutzii (Pb01) but could not be detected in P. brasiliensis (Pb03 and Pb18) by semi-quantitative RT-PCR.

Conclusion

Five new potentially active retrotransposons have been identified in the genomic assemblies of the Paracoccidioides species complex using a combined computational and experimental approach. The distribution across the two known species, P. brasiliensis and P. lutzii, and phylogenetics analysis indicate that these elements could have been acquired before speciation occurred. The presence of active retrotransposons in the genome may have implications regarding the evolution and genetic diversification of the Paracoccidioides genus.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1564-7) contains supplementary material, which is available to authorized users.     

Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways

Malaria, a disease affecting humans and other animals, is caused by a protist of the genus Plasmodium. At the intraerythrocytic stage, the parasite synthesizes a high amount of phospholipids through a bewildering number of pathways. In the human Plasmodium falciparum species, a plant-like pathway that relies on serine decarboxylase and phosphoethanolamine N-methyltransferase activities diverts host serine to provide additional phosphatidylcholine and phosphatidylethanolamine to the parasite. This feature of parasitic dependence toward its host was investigated in other Plasmodium species. In silico analyses led to the identification of phosphoethanolamine N-methyltransferase gene orthologs in primate and bird parasite genomes. However, the gene was not detected in the rodent P. berghei, P. yoelii, and P. chabaudi species. Biochemical experiments with labeled choline, ethanolamine, and serine showed marked differences in biosynthetic pathways when comparing rodent P. berghei and P. vinckei, and human P. falciparum species. Notably, in both rodent parasites, ethanolamine and serine were not significantly incorporated into phosphatidylcholine, indicating the absence of phosphoethanolamine N-methyltransferase activity. To our knowledge, this is the first study to highlight a crucial difference in phospholipid metabolism between Plasmodium species. The findings should facilitate efforts to develop more rational approaches to identify and evaluate new targets for antimalarial therapy.     

Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean

Background

The soybean-Bradyrhizobium symbiosis can be highly efficient in fixing nitrogen, but few genomic sequences of elite inoculant strains are available. Here we contribute with information on the genomes of two commercial strains that are broadly applied to soybean crops in the tropics. B. japonicum CPAC 15 (=SEMIA 5079) is outstanding in its saprophytic capacity and competitiveness, whereas B. diazoefficiens CPAC 7 (=SEMIA 5080) is known for its high efficiency in fixing nitrogen. Both are well adapted to tropical soils. The genomes of CPAC 15 and CPAC 7 were compared to each other and also to those of B. japonicum USDA 6^T and B. diazoefficiens USDA 110^T.

Results

Differences in genome size were found between species, with B. japonicum having larger genomes than B. diazoefficiens. Although most of the four genomes were syntenic, genome rearrangements within and between species were observed, including events in the symbiosis island. In addition to the symbiotic region, several genomic islands were identified. Altogether, these features must confer high genomic plasticity that might explain adaptation and differences in symbiotic performance. It was not possible to attribute known functions to half of the predicted genes. About 10% of the genomes was composed of exclusive genes of each strain, but up to 98% of them were of unknown function or coded for mobile genetic elements. In CPAC 15, more genes were associated with secondary metabolites, nutrient transport, iron-acquisition and IAA metabolism, potentially correlated with higher saprophytic capacity and competitiveness than seen with CPAC 7. In CPAC 7, more genes were related to the metabolism of amino acids and hydrogen uptake, potentially correlated with higher efficiency of nitrogen fixation than seen with CPAC 15.

Conclusions

Several differences and similarities detected between the two elite soybean-inoculant strains and between the two species of Bradyrhizobium provide new insights into adaptation to tropical soils, efficiency of N₂ fixation, nodulation and competitiveness.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-420) contains supplementary material, which is available to authorized users.     

Molecular diversity in the primary and secondary gene pools of genus Oryza

The objective of the present investigation was to assess the genetic relationships among the species of Oryza that belong to the primary gene pool (sativa complex) and the secondary gene pool (officinalis complex) using three marker systems such as RAPDs, ISSRs and SSRs. A total of 432 clear and reproducible bands were amplified from 18 RAPD primers; 113 bands were detected from 8 ISSR primers and 78 alleles were found to be amplified across the Oryza species from 13 SSR primer pairs. All the three dendrograms constructed, using UPGMA from the genetic similarity matrices based on the three marker data sets, were similar in their groupings. In all the three trees, two accessions of Oryza sativa formed an exclusive group indicating its genomic differentiation from its wild ancestors through the process of domestication. Distinctness between the wild species of the sativa and officinalis complexes was evident in all the trees derived from different markers. The groupings obtained among the species of the sativa complex were in perfect concordance with the species relationships established through classical crossability and cytogenetic analysis. This study has brought out some information on the species relationship between the diploid and tetraploid genomes of the officinalis complex possessing BB, CC and DD genomes. The higher level of similarity observed between the species possessing C and D genomes supports the view of many earlier authors that these two genomes might have originated from a single hybridization event. The results of this study also show that the diploid species possessing C genomes such as Oryza officinalis, Oryza rhizomatis and Oryza eichingeri are distinct from their allotetraploid counterparts possessing BBCC and CCDD genomes indicating a wider genomic differentiation in their evolutionary process.