Publications

4366

Summary Many reports have stated that flagellated protists in termite guts harbour ectosymbiotic spirochetes on their cell surface. In this study, we describe another bristle‐like ectosymbiont affiliated with the order Bacteroidales . The 16S rRNA phylotype Rs‐N74 predominates among Bacteroidales clones obtained from the gut of the termite Reticulitermes speratus . An Rs‐N74 phylotype‐specific probe was designed in this study and used for detection of the corresponding bacteria in the gut by fluorescence in situ hybridization (FISH) analysis. Surprisingly, the signals were detected specifically from the bristle‐like ‘appendages’ of various flagellate species belonging to the genus Dinenympha ; these ‘appendages’ had been believed to be spirochetal ectosymbionts or structures of the protists. The Rs‐N74 bacteria attached to the cell surface of the protists by a tip and coexisted with the spirochetal ectosymbionts. An electron micrograph revealed their morphology to be similar to a typical Bacteroidales bacterium. This bacterium is proposed to represent a novel genus and species, ‘ Candidatus Symbiothrix dinenymphae’, phylogenetically affiliated with a cluster consisting exclusively of uncultured strains from termite guts. A Bacteroidales ‐specific probe for FISH further revealed that this type of symbiosis exists also in various other protists, including parabasalids and oxymonads, and is widespread in termite guts.

Summary Recombination is an important process in microbial evolution. Rates of recombination with extracellular DNA matter because models of microbial population structure are profoundly influenced by the degree to which recombination is occurring within the population. Low rates of recombination may be sufficient to ensure the lateral propagation of genes that have a high selective advantage without disrupting the clonal pattern of inheritance for other genes. High rates of recombination potentially can obscure clonal patterns, leading to linkage equilibrium, and give microbial populations a population genetic structure more akin to sexually interbreeding eukaryotic populations. We examined eight loci from nine strains of candidatus Pelagibacter ubique (SAR11), isolated from a single 2L niskin sample of natural seawater, for evidence of genetic recombination between strains. The Shimodaira–Hasegawa test revealed significant phylogenetic incongruence in seven of the genes, indicating that frequent recombination obscures phylogenetic signals from the linear inheritance of genes in this population. Statistical evidence for intragenic recombination was found for six loci. An informative sites matrix showed extensive evidence for a widespread breakdown of linkage disequilibrium. Although the mechanisms of genetic transfer in native SAR11 populations are unknown, we measured recombination rates, ρ , that are much higher than point mutation rates, θ , as a source of genetic diversity in this clade. The eukaryotic model of species sharing a common pool of alleles is more apt for this SAR11 population than a strictly clonal model of inheritance in which allelic diversity is controlled by periodic selection.

AbstractA new TaqMan minor groove binding (MGB) probe and new PCR conditions were designed for quick, specific and sensitive detection of ‘Candidatus Phytoplasma mali’. The new probe can distinguish a single mismatch between ‘Ca. P. mali’ and ‘Candidatus Phytoplasma prunorum’, this constituting an improvement over a previously published method. In our study, the relative position of the mismatch in the MGB probe influenced greatly the specificity of detection. Our new real‐time PCR protocol was able to detect one plasmid copy in water and 100 copies in healthy plant DNA extracts. The sensitivity of this new real‐time PCR method, three other real‐time PCR protocols and a conventional PCR with fU5/rU3 primers was compared. Periwinkles and MM106 rootstocks were grafted with infected material and surveyed over time by symptom observation, conventional PCR and real‐time PCR. Phytoplasma infection was detected by symptom observation in all periwinkles within 4 months and in 75% of the MM106 rootstocks by the end of 7 months. Conventional PCR detected phytoplasma infection in all periwinkles within 4 months and in all MM106 rootstocks within 7 months. Best results were obtained by our real‐time PCR, which detected phytoplasma infection in all grafted plants within 3 months after inoculation.

Summary The biodegradation rate of chlorophenols in the environment seems to be limited by a competitive mechanism of O‐methylation which produces chloroanisoles with a high potential of being bioconcentrated in living organisms. In this work we report for the first time the isolation of three soil bacterial strains able to efficiently degrade 2,4,6‐trichloroanisole (2,4,6‐TCA). These strains were identified as Xanthomonas retroflexus INBB4, Pseudomonas putida INBP1 and Acinetobacter radioresistens INBS1. In these isolates 2,4,6‐TCA was efficiently metabolized in a minimal medium containing methanol and 2,4,6‐TCA as the only carbon sources, with a concomitant release of 3 mol of chloride ion from 1 mol of 2,4,6‐TCA, indicating complete dehalogenation of 2,4,6‐TCA. 2,4,6‐trichlorophenol (2,4,6‐TCP) was identified as a degradative intermediate, indicating that 2,4,6‐TCA underwent O‐demethylation as the first step in the biodegradation process. 2,4,6‐TCP was further transformed into 2,6‐dichloro‐ para ‐hydroquinone (2,6‐DCHQ) and subsequently mineralized. The degradation of chloroanisoles could improve the overall biodegradation of chlorophenols in the environment, because those chlorophenols previously biomethylated might also be later biodegraded. Xanthomonas retroflexus INBB4 has two O‐demethylation systems: one is an oxygenase‐type demethylase, and the other is a tetrahydrofolate (THF)‐dependent O ‐demethylase. On the contrary O‐demethylation of 2,4,6‐TCA in P. putida INBP1 is just catalysed by an oxygenase‐type NADH/NADPH‐dependent O ‐demethylase, whereas in A. radioresistens INBS1 a THF‐dependent O ‐demethylase activity was detected.

AbstractBackground:  The spectrum of human non‐pylori Helicobacter infections is expanding, with species such as H. heilmannii and H. felis occasionally being associated with gastritis. However, the existence of non‐pylori Helicobacter colonization in asymptomatic subjects has not been evaluated. The aim of this study was to investigate whether Helicobacter species other than pylori are present in the upper digestive tract of asymptomatic human subjects.Materials and methods:  A Helicobacteraceae‐specific semi‐nested polymerase chain reaction (PCR) assay was used to detect Helicobacter‐like organisms in the upper digestive tract of 91 Venezuelan volunteers (aged 18–68 years, 41 females, 50 males). Species were identified by denaturing gradient gel electrophoresis analysis and sequencing of the PCR products.Results:  We detected DNA sharing 99–100% sequence identity in over 300–400 bp with the 16S rRNA genes of H. pylori, H. cetorum, and Candidatus Wolinella africanus in 76%, 16%, and 15% of the subjects, respectively. Multiple colonization was documented in 10% of the subjects: H. cetorum and Candidatus W. africanus (4%), H. pylori and Candidatus W. africanus (4%), and H. pylori and H. cetorum (2%).Conclusions:  Our results suggest that non‐pylori Helicobacteraceae colonization is relatively common in the Venezuelan asymptomatic population. This is the first report documenting the presence of H. cetorum DNA in the human upper digestive tract, and the second report of the recently discovered Candidatus W. africanus.

ABSTRACT We investigated the fine-scale population structure of the “ Candidatus Accumulibacter” lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene ( ppk1 ) as a genetic marker. We retrieved fragments of “ Candidatus Accumulibacter” 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the “ Candidatus Accumulibacter” lineage. Sequences from at least five clades of “ Candidatus Accumulibacter” were recovered by ppk1 -targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using “ Candidatus Accumulibacter”-specific 16S rRNA and “ Candidatus Accumulibacter” clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total “ Candidatus Accumulibacter” lineage and the relative distributions and abundances of the five “ Candidatus Accumulibacter” clades. The qPCR-based estimation of the total “ Candidatus Accumulibacter” fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1 , demonstrating the power of ppk1 as a genetic marker for detection of all currently defined “ Candidatus Accumulibacter” clades. The relative distributions of “ Candidatus Accumulibacter” clades varied among different EBPR systems and also temporally within a system. Our results suggest that the “ Candidatus Accumulibacter” lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.

ABSTRACTPointed, rod-shaped bacteria colonizing the cuticular surface of the hindgut of the terrestrial isopod crustaceanPorcellio scaber(Crustacea: Isopoda) were investigated by comparative 16S rRNA gene sequence analysis and electron microscopy. The results of phylogenetic analysis, and the absence of a cell wall, affiliated these bacteria with the classMollicutes, within which they represent a novel and deeply branched lineage, sharing less than 82.6% sequence similarity to knownMollicutes. The lineage has been positioned as a sister group to the clade comprising theSpiroplasmagroup, theMycoplasma pneumoniaegroup, and theMycoplasma hominisgroup. The specific signature sequence was identified and used as a probe in in situ hybridization, which confirmed that the retrieved sequences originate from the attached rod-shaped bacteria from the hindgut ofP. scaberand made it possible to detect these bacteria in their natural environment. Scanning and transmission electron microscopy revealed a spherically shaped structure at the tapered end of the rod-shaped bacteria, enabling their specific and exclusive attachment to the tip of the cuticular spines on the inner surface of the gut. Specific adaptation to the gut environment, as well as phylogenetic positioning, indicate the long-term association and probable coevolution of the bacteria and the host. Taking into account their pointed, rod-shaped morphology and their phylogenetic position, the name “CandidatusBacilloplasma” has been proposed for this new lineage of bacteria specifically associated with the gut surface ofP. scaber.

ABSTRACT “ Candidatus Accumulibacter phosphatis” is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes ( nirK and nirS ) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that “ Ca . Accumulibacter phosphatis” has nirS . Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of “ Ca . Accumulibacter phosphatis.” The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that “ Ca . Accumulibacter phosphatis” behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.