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
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.
ABSTRACT
The primary endosymbiotic bacteria from three species of parasitic primate lice were characterized molecularly. We have confirmed the characterization of the primary endosymbiont (P-endosymbiont) of the human head/body louse
Pediculus humanus
and provide new characterizations of the P-endosymbionts from
Pediculus schaeffi
from chimpanzees and
Pthirus pubis
, the pubic louse of humans. The endosymbionts show an average percent sequence divergence of 11 to 15% from the most closely related known bacterium “
Candidatus
Arsenophonus insecticola.” We propose that two additional species be added to the genus “
Candidatus
Riesia.” The new species proposed within “
Candidatus
Riesia” have sequence divergences of 3.4% and 10 to 12% based on uncorrected pairwise differences. Our Bayesian analysis shows that the branching pattern for the primary endosymbionts was the same as that for their louse hosts, suggesting a long coevolutionary history between primate lice and their primary endosymbionts. We used a calibration of 5.6 million years to date the divergence between endosymbionts from human and chimpanzee lice and estimated an evolutionary rate of nucleotide substitution of 0.67% per million years, which is 15 to 30 times faster than previous estimates calculated for
Buchnera
, the primary endosymbiont in aphids. Given the evidence for cospeciation with primate lice and the evidence for fast evolutionary rates, this lineage of endosymbiotic bacteria can be evaluated as a fast-evolving marker of both louse and primate evolutionary histories.