Some Aspects of the Physiology of the Nyctotherus velox, a Commensal Ciliated Protozoon Taken from the Hindgut of the Tropical Millipede Archispirostreptus gigas.

In this paper, the growth requirements, fermentation pattern, and hydrolytic enzymatic activities of anaerobic ciliates collected from the hindgut of the African tropical millipede Archispirostreptus gigas are described. Single-cell molecular analysis showed that ciliates from the millipede hindgut could be assigned to the Nyctotherus velox and a new species named N. archispirostreptae n. sp. The ciliate N. velox can grow in vitro with unspecified prokaryotic populations and various plant polysaccharides (rice starch-RS, xylan, crystalline cellulose20-CC, carboxymethylcellulose-CMC, and inulin) or without polysaccharides (NoPOS) in complex reduced medium with soluble supplements (peptone, glucose, and vitamins). Specific catalytic activity (nkat/g of protein) of α amylase of 300, xylanase of 290, carboxymethylcellulase of 190, and inulinase of 170 was present in the crude protein extract of N. velox. The highest in vitro dry matter digestibility was observed in RS and inulin after 96 h of fermentation. The highest methane concentration was observed in xylan and inulin substrates. The highest short-chain fatty acid concentration was observed in RS, inulin, and xylan. In contrast, the highest ammonia concentration was observed in NoPOS, CMC, and CC. The results indicate that starch is the preferred substrate of the N. velox. Hydrolytic enzyme activities of N. velox showed that the ciliates contribute to the fermentation of plant polysaccharides in the gut of millipedes.

New adenoviruses from new primate hosts - growing diversity reveals taxonomic weak points.

The knowledge of the closest human relatives of human adenoviruses (AdVs) such as adenoviruses found in nonhuman primates is still limited, despite the growing importance of adenoviruses in vaccine development, gene and cancer therapy. We examined 153 stool samples of 17 non-human primate species and detected adenoviral DNA sequences of DNA polymerase (DPOL) gene in 54 samples (35%), originating from 12 out of 17 primate species. We further sequenced 15 hexon gene fragments and based on the phylogenetic analysis we propose two new provisional species SAdV-H and SAdV-I. Our study shows extensive diversity of adenoviral strains forming separate clades often from closely related host species from old world monkeys suggesting the existence of new species of AdVs and shows the necessity for clear ICTV guidelines for final establishment of so far provisional AdV species.

Characterization of tet(Y)-carrying LowGC plasmids exogenously captured from cow manure at a conventional dairy farm.

Manure from dairy farms has been shown to contain diverse tetracycline resistance genes that are transferable to soil. Here, we focus on conjugative plasmids that may spread tetracycline resistance at a conventional dairy farm. We performed exogenous plasmid isolation from cattle feces using chlortetracycline for transconjugant selection. The transconjugants obtained harbored LowGC-type plasmids and tet(Y). A representative plasmid (pFK2-7) was fully sequenced and this was compared with previously described LowGC plasmids from piggery manure-treated soil and a GenBank record from Acinetobacter nosocomialis that we also identified as a LowGC plasmid. The pFK2-7 plasmid had the conservative backbone typical of LowGC plasmids, though this region was interrupted with an insert containing the tet(Y)-tet(R) tetracycline resistance genes and the strA-strB streptomycin resistance genes. Despite Acinetobacter populations being considered natural hosts of LowGC plasmids, these plasmids were not found in three Acinetobacter isolates from the study farm. The isolates harbored tet(Y)-tet(R) genes in identical genetic surroundings as pFK2-7, however, suggesting genetic exchange between Acinetobacter and LowGC plasmids. Abundance of LowGC plasmids and tet(Y) was correlated in manure and soil samples from the farm, indicating that LowGC plasmids may be involved in the spread of tet(Y) in the environment.

Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques.

A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

Detection of Diverse Novel Bat Astrovirus Sequences in the Czech Republic.

Astroviruses are a major cause of gastroenteritis in humans and animals. Recently, novel groups of astroviruses were identified in apparently healthy insectivorous bats. We report the detection of diverse novel astrovirus sequences in nine different European bat species: Eptesicus serotinus, Hypsugo savii, Myotis emarginatus, M. mystacinus, Nyctalus noctula, Pipistrellus nathusii or P. pygmaeus, P. pipistrellus, Vespertilio murinus, and Rhinolophus hipposideros. In six bat species, astrovirus sequences were detected for the first time. One astrovirus strain detected in R. hipposideros clustered phylogenetically with Chinese astrovirus strains originating from bats of the families Rhinolophidae and Hipposideridae. All other Czech astrovirus sequences from vesper bats formed, together with one Hungarian sequence, a separate monophyletic lineage within the bat astrovirus group. These findings provide new insights into the molecular epidemiology, ecology, and prevalence of astroviruses in European bat populations.

Full-length genome analysis of Calovo strains of Batai orthobunyavirus (Bunyamwera serogroup): implications to taxonomy.

Batai virus (BATV) is a poorly studied arthropod-borne virus belonging to the genus Orthobunyavirus (Bunyamwera serogroup) within the family Bunyaviridae. It has been associated with human influenza-like febrile illness in several Asian, African, and European countries. Calovo virus (CVOV), isolated in 1960 in Slovakia, has been classified as BATV based on high antigenic similarity, and since then both CVOV and BATV were used as synonyms. In order to fully clarify the phylogenetic relationships between CVOV, BATV, and other members of the Bunyamwera serogroup, we performed whole genome sequencing of four CVOV strains isolated in Europe and phylogenetic analyses of all related viruses. The nucleocapsid protein, encoded by the S genomic segment, contains 233 amino acids, 60 of which, putatively critical for protein function, are conserved. Within the CVOV polyprotein encoded by the M genomic segment, putative cleavage sites, N-glycosylation sites, and seven transmembrane regions were identified. The RNA-dependent RNA polymerase, encoded by the L genome segment, exhibits conservation of the three regions known to be conserved among bunyavirus and arenavirus L proteins. Phylogenetic analyses of all three genomic segments of selected orthobunyaviruses clearly revealed that European and Asian/African strains of BATV are phylogenetically different and form two distinct lineages, indicating the existence of two different genotypes of BATV, tentatively named European genotype (with CVOV as a type strain) and Afro-Asian genotype (with BATV as a type strain) of BATV.

Anaplasma infections in ticks and reservoir host from Slovakia.

Anaplasma phagocytophilum is a worldwide distributed bacterium with a significant medical and veterinary importance. It grows within the phagosome of infected neutrophils and is responsible for human granulocytic anaplasmosis (HGA), tick-borne fever (TBF) of small ruminants and cattle, canine and equine granulocytic anaplasmosis, but infects also a great variety of wildlife species. Wild ungulates and rodents are considered reservoirs of infection in natural foci. The objective of this study was to determine the spectrum of animal species involved in the circulation of A. phagocytophilum in Slovakia and to analyze the variability of obtained nucleotide sequences, in order to determine whether genotypes from Slovakia cluster according to host-species or geographical location. Several animal species and vector ticks were screened for the presence of members of the family Anaplasmataceae using PCR based methods. Additional data on the molecular evidence of Anaplasma ovis and Candidatus Neoehrlichia mikurensis are presented. These pathogens were detected in tested sheep flocks and rodents with the mean infection rates of 8.16% and 10.75%, respectively. A. phagocytophilum was genotyped by 16S rRNA and groEL gene sequencing. Bacterial DNA was confirmed in questing ixodid ticks, in domesticated canine, wild rodents and several species of wild ungulates. In European isolates, 16S rRNA gene does not seem to be an appropriate locus for the analyses of heterogeneity as it is too conservative. Similarly, 16S rRNA isolates from our study did not reveal any polymorphisms. All isolates were identical in overlapped region and showed identity with sequences from ticks, horses or ruminants previously isolated elsewhere in the world. On the other hand, the groESL heat shock operon is widely used for determination of diversity and the analyses have already revealed considerable degree of heterogeneity. Tested ungulates were infected with A. phagocytophilum to a considerable extent. High proportions of red and roe deer tested positive and the rates of infection reached over 60.0%. GroEL sequences from canine, wild ungulates and ticks from Slovakia clustered within a clade together with isolates from horses, humans, wild ungulates and ticks from Slovakia or elsewhere in the world. Sequences from rodents clustered apart from those obtained from wild ungulates, ticks and humans. These results suggest that European rodents do not harbour A. phagocytophilum strains with strong zoonotic potential such as those from United States.

The variability of the large genomic segment of Tahyna orthobunyavirus and an all-atom exploration of its anti-viral drug resistance.

Tahyna virus (TAHV), a member of the Bunyaviridae family (California complex), is an important but neglected human mosquito-borne pathogen. The virus genome is composed of three segments, i.e., small (S), medium (M), and large (L). Previous studies on genetic variability of viruses within the California complex were focused on S and M segments, but the L segment remains relatively unstudied. To assess the genetic variation and the relation to virus phenotype we analyzed the L segment sequences of biologically diverse TAHV strains isolated in the Czech Republic and Slovakia. Phylogenetic analysis covering all available sequences of the L segment of TAHV clearly revealed two distinguished lineages, tentatively named as "European" and "Asian". The L segment strains within the European lineage are highly conserved (identity 99.3%), whilst Asian strains are more genetically diverse (identity 97%). Based on sequence comparison with other bunyaviruses, several non-synonymous nucleotide substitutions unique for TAHV in the L segment were identified. We also identified specific residue substitutions in the endonuclease domain of TAHV compared with the La Crosse virus. Since the endonuclease domain of the La Crosse virus has been resolved, we employed an all energy landscape algorithm to analyze the ligand migration of a viral polymerase inhibitor. This allowed us to demonstrate, at the atomic level, that this viral polymerase inhibitor randomly explored the specific residue substitutions in the endonuclease domain of the TAHV L segment.

Candidatus Sodalis melophagi sp. nov.: phylogenetically independent comparative model to the tsetse fly symbiont Sodalis glossinidius.

Bacteria of the genus Sodalis live in symbiosis with various groups of insects. The best known member of this group, a secondary symbiont of tsetse flies Sodalis glossinidius, has become one of the most important models in investigating establishment and evolution of insect-bacteria symbiosis. It represents a bacterium in the early/intermediate state of the transition towards symbiosis, which allows for exploring such interesting topics as: usage of secretory systems for entering the host cell, tempo of the genome modification, and metabolic interaction with a coexisting primary symbiont. In this study, we describe a new Sodalis species which could provide a useful comparative model to the tsetse symbiont. It lives in association with Melophagus ovinus, an insect related to tsetse flies, and resembles S. glossinidius in several important traits. Similar to S. glossinidius, it cohabits the host with another symbiotic bacterium, the bacteriome-harbored primary symbiont of the genus Arsenophonus. As a typical secondary symbiont, Candidatus Sodalis melophagi infects various host tissues, including bacteriome. We provide basic morphological and molecular characteristics of the symbiont and show that these traits also correspond to the early/intermediate state of the evolution towards symbiosis. Particularly, we demonstrate the ability of the bacterium to live in insect cell culture as well as in cell-free medium. We also provide basic characteristics of type three secretion system and using three reference sequences (16 S rDNA, groEL and spaPQR region) we show that the bacterium branched within the genus Sodalis, but originated independently of the two previously described symbionts of hippoboscoids. We propose the name Candidatus Sodalis melophagi for this new bacterium.

Multiple origins of endosymbiosis within the Enterobacteriaceae (γ-Proteobacteria): convergence of complex phylogenetic approaches.

BACKGROUND: The bacterial family Enterobacteriaceae gave rise to a variety of symbiotic forms, from the loosely associated commensals, often designated as secondary (S) symbionts, to obligate mutualists, called primary (P) symbionts. Determination of the evolutionary processes behind this phenomenon has long been hampered by the unreliability of phylogenetic reconstructions within this group of bacteria. The main reasons have been the absence of sufficient data, the highly derived nature of the symbiont genomes and lack of appropriate phylogenetic methods. Due to the extremely aberrant nature of their DNA, the symbiotic lineages within Enterobacteriaceae form long branches and tend to cluster as a monophyletic group. This state of phylogenetic uncertainty is now improving with an increasing number of complete bacterial genomes and development of new methods. In this study, we address the monophyly versus polyphyly of enterobacterial symbionts by exploring a multigene matrix within a complex phylogenetic framework. RESULTS: We assembled the richest taxon sampling of Enterobacteriaceae to date (50 taxa, 69 orthologous genes with no missing data) and analyzed both nucleic and amino acid data sets using several probabilistic methods. We particularly focused on the long-branch attraction-reducing methods, such as a nucleotide and amino acid data recoding and exclusion (including our new approach and slow-fast analysis), taxa exclusion and usage of complex evolutionary models, such as nonhomogeneous model and models accounting for site-specific features of protein evolution (CAT and CAT+GTR). Our data strongly suggest independent origins of four symbiotic clusters; the first is formed by Hamiltonella and Regiella (S-symbionts) placed as a sister clade to Yersinia, the second comprises Arsenophonus and Riesia (S- and P-symbionts) as a sister clade to Proteus, the third Sodalis, Baumannia, Blochmannia and Wigglesworthia (S- and P-symbionts) as a sister or paraphyletic clade to the Pectobacterium and Dickeya clade and, finally, Buchnera species and Ishikawaella (P-symbionts) clustering with the Erwinia and Pantoea clade. CONCLUSIONS: The results of this study confirm the efficiency of several artifact-reducing methods and strongly point towards the polyphyly of P-symbionts within Enterobacteriaceae. Interestingly, the model species of symbiotic bacteria research, Buchnera and Wigglesworthia, originated from closely related, but different, ancestors. The possible origins of intracellular symbiotic bacteria from gut-associated or pathogenic bacteria are suggested, as well as the role of facultative secondary symbionts as a source of bacteria that can gradually become obligate maternally transferred symbionts.

New defensins from hard and soft ticks: similarities, differences, and phylogenetic analyses.

Despite the importance of ticks as vectors of disease very little is known about their immune system. Antimicrobial peptides, including defensins (phylogenetically ancient antibacterial peptides) are major components of innate immunity in ticks that have been shown to provide protection against gram-negative and gram-positive bacteria, fungi, viruses and protozoan parasites. With the aim of studying the evolution of the genes involved in tick defense, we identified the preprodefensin genes from four Ornithodoros tick species (O. papillipes: isoforms A, B, and D; O. tartakovskyi and O. puertoricensis: isoforms A and B; O. rostratus: isoform A) and from two Dermacentor tick species (D. reticulatus and D. marginatus: one isoform) not previously described. Phylogenetic analyses revealed that Ornithodoros defensin isoforms (A, B, C, and D) form 4 separate clades, while hard tick defensins are divided into several branches based on particular tick species.

Evolution of the apicoplast and its hosts: from heterotrophy to autotrophy and back again.

The photosynthetic origin of apicomplexan parasites was proposed upon the discovery of a reduced non-photosynthetic plastid termed the apicoplast in their cells. Although it is clear that the apicoplast has evolved through a secondary endosymbiosis, its particular origin within the red or green plastid lineage remains controversial. The recent discovery of Chromera velia, the closest known photosynthetic relative to apicomplexan parasites, sheds new light on the evolutionary history of alveolate plastids. Here we review our knowledge on the evolutionary history of Apicomplexa and particularly their plastids, with a focus on the pathway by which they evolved from free-living heterotrophs through photoautotrophs to omnipresent obligatory intracellular parasites. New sequences from C. velia (histones H2A, H2B; GAPDH, TufA) and phylogenetic analyses are also presented and discussed here.

A photosynthetic alveolate closely related to apicomplexan parasites.

Many parasitic Apicomplexa, such as Plasmodium falciparum, contain an unpigmented chloroplast remnant termed the apicoplast, which is a target for malaria treatment. However, no close relative of apicomplexans with a functional photosynthetic plastid has yet been described. Here we describe a newly cultured organism that has ultrastructural features typical for alveolates, is phylogenetically related to apicomplexans, and contains a photosynthetic plastid. The plastid is surrounded by four membranes, is pigmented by chlorophyll a, and uses the codon UGA to encode tryptophan in the psbA gene. This genetic feature has been found only in coccidian apicoplasts and various mitochondria. The UGA-Trp codon and phylogenies of plastid and nuclear ribosomal RNA genes indicate that the organism is the closest known photosynthetic relative to apicomplexan parasites and that its plastid shares an origin with the apicoplasts. The discovery of this organism provides a powerful model with which to study the evolution of parasitism in Apicomplexa.