Environmental Microbiology

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.

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.

Summary Studies were conducted in opposing gradients of oxygen and sulfide in microslide capillaries to (i) characterize the chemical microenvironment preferred by Candidatus Arcobacter sulfidicus, a highly motile, sulfur‐oxidizing bacterium that produces sulfur in filamentous form, and (ii) to develop a model describing the mechanism of filamentous‐sulfur formation. The highly motile microorganisms are microaerophilic, with swarms effectively aggregating within oxic‐anoxic interfaces by exhibiting a chemotactic response. The position of the band was found to be largely independent of the sulfide concentration as it always formed at the oxic‐anoxic interface. Flux calculations based on steady state gradients of oxygen and sulfide indicate that sulfide is incompletely oxidized to sulfur, in line with the formation of filamentous sulfur by these organisms. It is proposed that Candidatus Arcobacter sulfidicus effectively competes with other sulfur‐oxidizing bacteria in the environment by being able to tolerate higher concentrations of hydrogen sulfide (1–2 mM) and by possessing the ability to grow at very low oxygen concentrations (1–10 μM). The formation of mat‐like structures from filamentous sulfur appears to be a population mediated effort allowing these organisms to effectively colonize environments characterized by high sulfide, low oxygen and dynamic fluid movement.

Summary Accumulating evidence supports a role for Chlamydia ‐related organisms as emerging pathogens for human and animals. Assessment of their pathogenicity requires strain availability, at least for animal models and serological studies. As these obligate intracellular species are able to grow inside amoebae, we used co‐culture with Acanthamoeba castellanii in an attempt to recover new Chlamydia ‐related species from river water. We isolated two strains from eight water samples. The first strain is a new Parachlamydia acanthamoebae strain that differs from previously described isolates by only two bases in the complete 16S rRNA gene sequence. The second isolate is the first representative of a new Chlamydiales family, as demonstrated by genetic and phylogenetic analyses of the 16S rRNA, 23S rRNA, ADP/ATP translocase and RnpB encoding genes. Using fluorescent in situ hybridization and electron microscopy, we demonstrated that it grows in high numbers in amoebae, where it exhibits a Chlamydia ‐like developmental cycle with reticulate bodies and star‐like elementary bodies. Based on these results, we propose to name this new species ‘ Criblamydia sequanensis ’. This work confirmed that amoebal co‐culture is a relevant method to isolate new chlamydiae, and that it can be successfully applied to ecosystems colonized with a complex microbial community.

Summary Twelve strains of filamentous bacteria morphologically identified as ‘ Microthrix parvicella ’ were isolated from industrial activated sludge wastewater treatment plants. 16S rRNA gene sequences analysis showed that these strains were all closely related to ‘ Candidatus Microthrix parvicella’. Six of them, however, had a 16S rRNA gene similarity of only 95.7% and 96.7% to ‘ Candidatus Microthrix parvicella’ suggesting the presence of a new species. The name ‘ Candidatus Microthrix calida’ is proposed for this new microorganism. The physiological properties of these six isolates supported the description of a new taxon. The ‘ Candidatus Microthrix calida’ strains produced thin filaments (0.3–0.7 µm diameter), they did not grow on the media supporting the growth of ‘ Candidatus Microthrix parvicella’ and could be cultivated at higher temperature (up to 36.5°C). Preliminary data on substrate uptake were obtained by microautoradiography on pure culture. Two new fluorescence in situ hybridization (FISH) probes, Mpa‐T1‐1260 specific for ‘ Candidatus Microthrix calida’ and Mpa‐all‐1410 targeting both Microthrix species, were designed. The presence of Microthrix spp. was investigated in 114 activated sludge plants. ‘ Microthrix parvicella ’ morphotype was detected in 23% of the analysed samples and FISH analysis revealed that ‘ Candidatus Microthrix calida’ was present in 5% of them. The remaining ‘ M. parvicella ’ filaments were positive with probe Mpa‐all‐1410 but could not all be identified as ‘ Candidatus Microthrix parvicella’ suggesting the presence of more hitherto undescribed biodiversity within this morphotype.

Summary Previously uncultured nitrite‐oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira ‐like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospira ‐like bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 µg ml −1 . The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA‐targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira ‐like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as ‘ Candidatus Nitrospira defluvii’.

Summary The X‐bacteria which initiated organismic association with the D strain of Amoeba proteus in 1966 as parasites have changed to obligate endosymbionts on which the host depends for survival. Owing to the difficulty in cultivating the bacteria in vitro , the identity of X‐bacteria has not been determined. As the life cycle of X‐bacteria is similar to that of Legionella spp. in soil amoebae, we applied the polymerase chain reaction method with specific primers aimed at Legionella spp. for the detection and cloning of 16S rRNA gene. The identity and intracellular localization of the endosymbiont were confirmed by the application of a specific fluorescently labelled 16S rRNA‐targeted probe. In addition we cloned RNA polymerase β‐subunit gene ( rpoB ) of X‐bacteria by genomic library tagging. A phylogenetic analysis of the 16S rRNA gene placed the bacterium within a unique monophyletic group containing all other members of the genus Legionella . Phylogeny from rpoB and mip genes further confirmed the taxonomic context of X‐bacteria to be a Legionella sp. In all three phylogenic analyses, X‐bacterium was placed apart from Legionella ‐like amoebal pathogens present in soil amoebae. Thus, we propose the name ‘ Candidatus Legionella jeonii’ sp. nov. for the endosymbiotic X‐bacteria in Amoeba proteus .

Summary Two phylogenetically distinct marine strains producing transparent exopolymers (TEP), designated HTCC2155 T and HTCC2160, were cultivated from Oregon coast seawater by dilution to extinction in a high throughput culturing format. When cultured in low‐nutrient seawater media, these strains copiously produced Alcian Blue‐stainable viscous TEP. Growing cells were attached to each other by the TEP in a three dimensional network. Polymerase chain reaction employing 16S rDNA primers specific for the novel isolates indicated that they are indigenous to the water column of the Atlantic and Pacific oceans. The abundance of the isolates as determined by 16S rRNA dot blots, however, indicated that they are less than 1% of the total bacterial community. In phylogenetic analyses, the strains consistently formed a new phylum‐level lineage within the domain Bacteria , together with members of the candidate phylum VadinBE97, which consists of Victivallis, the first cultured genus in the candidate phylum, and 16S rRNA gene clones from DNA extracted from marine or anaerobic terrestrial habitats. Five putative subgroups were delineated within this phylum‐level lineage, including a marine group and an anaerobic group. The isolates are Gram negative, strictly aerobic, chemoheterotrophic, and facultatively oligotrophic sphere‐shaped bacteria. The DNA G+C content of strain HTCC2155 T was 48.3 mol% and the genome size was 2.9 mb. It is proposed from these observations that the strains be placed into a new genus and a new species named Lentisphaera araneosa (type strain HTCC2155 T  = ATCC BAA‐859 T  = KCTC 12141 T ) gen. nov., sp. nov., the cultured marine representative of the Lentisphaerae phyl. nov., and the phylum be divided into two novel orders named the Lentisphaerales ord. nov. and the Victivallales ord. nov.

Summary ‘ Aegyptianella ranarum ’ (order Rickettsiales), an ultrastructurally defined small, Gram‐negative rod, is known to replicate in the red blood cells of frogs. Heretofore, this bacterium has not been characterized genetically. We cloned and sequenced the 16S rRNA (1310 bp) and gyrB (718 bp) genes of ‘ A. ranarum ’ from a Canadian frog blood specimen. In situ hybridization (with an ‘ A. ranarum ’ 16S rRNA gene polymerase chain reaction product as probe) and electron microscopy confirmed that ‘ A. ranarum ’ forms cytoplasmic inclusions in frog erythrocytes. blast comparisons with GenBank 16S rRNA and gyrB sequences showed that both ‘ A. ranarum ’ genes were most similar (91% and 67% identity) to those of Chryseobacterium meningosepticum , a bacterium in the family Flavobacteriaceae . In contrast, ‘ A. ranarum ’ 16S rRNA shared only 61% identity with Aegyptianella pullorum . Phylogenetic analyses of these genes using phylip supported ‘ A. ranarum ’ as a member of Flavobacteriaceae, but suggested that its cladistic sibling may be Bergeyella zoohelcum or Weeksella virosa , rather than C. meningosepticum. We propose to classify ‘ Aegyptianella ranarum ’ as ‘ Candidatus Hemobacterium ranarum’ in the family Flavobacteriaceae . Our results provide a starting point for studies of related intraerythrocytic bacterial infections in frogs.