Applied Microbiology and Biotechnology

To characterize potentially important surface-exposed proteins of the phytoplasma causing chrysanthemum yellows (CY), new primers were designed based on the conserved regions of 3 membrane protein genes of the completely sequenced onion yellows and aster yellows witches’ broom phytoplasmas and were used to amplify CY DNA. The CY genes secY, amp, and artI, encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively, were cloned and completely sequenced. Alignment of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16S rDNA-based classification, while amp sequences were highly variable within the ‘Candidatus Phytoplasma asteris’. Five CY partial sequences were cloned into the pRSetC expression vector, and 3 of the encoded protein fragments (Amp 64/651, Amp 64/224, ArtI 131/512) were expressed as fusion antigens for the production of CY-specific polyclonal antibodies (A416 against Amp 64/224; A407 against ArtI 131/512). A416 recognized, in Western blots, the full-length Amp from CY-infected plants (periwinkle, daisy) and insect vectors ( Euscelidius variegatus , Macrosteles quadripunctulatus ). A416 also reacted to European aster yellows, to primula yellows phytoplasmas, to northern Italian strains of ‘Ca. Phytoplasma asteris’ from lettuce and gladiolus, but it did not react to American aster yellows phytoplasma.

ABSTRACT In this study we analyzed the membrane lipid composition of “ Candidatus Nitrosopumilus maritimus,” the only cultivated representative of the cosmopolitan group I crenarchaeota and the only mesophilic isolate of the phylum Crenarchaeota . The core lipids of “ Ca . Nitrosopumilus maritimus” consisted of glycerol dialkyl glycerol tetraethers (GDGTs) with zero to four cyclopentyl moieties. Crenarchaeol, a unique GDGT containing a cyclohexyl moiety in addition to four cyclopentyl moieties, was the most abundant GDGT. This confirms unambiguously that crenarchaeol is synthesized by species belonging to the group I.1a crenarchaeota. Intact polar lipid analysis revealed that the GDGTs have hexose, dihexose, and/or phosphohexose head groups. Similar polar lipids were previously found in deeply buried sediments from the Peru margin, suggesting that they were in part synthesized by group I crenarchaeota.

ABSTRACT The identity and ecophysiology of a group of uncultured protein-hydrolyzing epiphytic rods attached to filamentous bacteria in activated sludge from nutrient removal plants were investigated by using the full-cycle rRNA approach combined with microautoradiography and histochemical staining. The epiphytic group consists of three closely related clusters, each containing 11 to 16 clones. The closest related cultured isolate is the type strain Haliscomenobacter hydrossis (ATCC 27775) (<87% similarity) in the family Saprospiraceae of the phylum Bacteroidetes . Oligonucleotide probes at different hierarchical levels were designed for each cluster and used for ecophysiological studies. All three clusters behaved similarly in their physiology and were specialized in protein hydrolysis and used amino acids as energy and carbon sources. They were not involved in denitrification. No storage of polyphosphate and polyhydroxyalkanoates was found. They all colonized probe-defined filamentous bacteria belonging to the phyla Chloroflexi , Proteobacteria , and candidate phylum TM7, with the exception of cluster 1, which did not colonize TM7 filaments. The three epiphytic clusters were all widespread in domestic and industrial wastewater treatment plants with or without biological phosphorus removal, constituting, in total, up to 9% of the bacterial biovolume. A new genus, “ Candidatus Epiflobacter,” is proposed for this epiphytic group in activated-sludge treatment plants, where it presumably plays an important role in protein degradation.

ABSTRACT Microscopic examination of the hemolymph from diseased daphniids in 17 lakes in southwestern Michigan and five rock pools in southern Finland revealed the presence of tightly coiled bacteria that bore striking similarities to the drawings of a morphologically unique pathogen, “ Spirobacillus cienkowskii ,” first described by Elya Metchnikoff more than 100 years ago. The uncultivated microbe was identified as a deeply branching member of the Deltaproteobacteria through phylogenetic analyses of two conserved genes: the 16S rRNA-encoding gene ( rrs ) and the β-subunit of topoisomerase ( gyrB ). Fluorescence in situ hybridization confirmed that the rRNA gene sequence originated from bacteria with the tightly coiled morphology. Microscopy and PCR amplification with pathogen-specific primers confirmed infections by this bacterium in four species of Daphnia : Daphnia dentifera, D. magna, D. pulicaria , and D. retrocurva . Extensive field surveys reveal that this bacterium is widespread geographically and able to infect many different cladoceran species. In a survey of populations of D. dentifera in lakes in Michigan, we found the bacterium in 17 of 18 populations studied. In these populations, 0 to 12% of the individuals were infected, with an average of 3% during mid-summer and early autumn. Infections were less common in rock pool populations of D. magna in southern Finland, where the pathogen was found in 5 of 137 populations. The broad geographic distribution, wide host range, and high virulence of S. cienkowskii suggest it plays an important role in the ecology and evolution of daphniids.

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.

ABSTRACT Pointed, rod-shaped bacteria colonizing the cuticular surface of the hindgut of the terrestrial isopod crustacean Porcellio 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 class Mollicutes , within which they represent a novel and deeply branched lineage, sharing less than 82.6% sequence similarity to known Mollicutes . The lineage has been positioned as a sister group to the clade comprising the Spiroplasma group, the Mycoplasma pneumoniae group, and the Mycoplasma hominis group. 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 of P. scaber and 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 “ Candidatus Bacilloplasma” has been proposed for this new lineage of bacteria specifically associated with the gut surface of P. 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.