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4391

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.

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.

Murraya paniculata (orange jasmine) is a popular ornamental rutaceaous plant and is known to be a preferred host for the Asian citrus psyllid, Diaphorina citri (Kuwayana), the primary vector of ‘Candidatus Liberibacter spp.’ that causes citrus Huanglongbing (HLB). HLB is a highly destructive citrus disease worldwide. However, the presence of ‘Ca. Liberibacter spp.’ in M. paniculata remains uncertain (2). Clarification of M. paniculata as a host of ‘Ca. Liberibacter spp.’ has direct impact on HLB control programs. During June of 2006, we identified three M. paniculata trees near a mandarin orchard affected by HLB in Luoding City and two trees from Guangzhou City, Guangdong Province, People's Republic of China. All trees had leaves showing yellowing and mottling symptoms characteristic of HLB. Both symptomatic and asymptomatic leaves were collected. DNA was extracted using the CTAB (cetyltrimethylammoniumbromide) method and assayed by nested-PCR. The general bacterial 16S rDNA primer set fDl/rD1 (3) was used for the first round of amplification. Amplification was conducted as previously described (1), and 2 μl of PCR reaction product were used for a second round of amplification using the same procedure but with 35 PCR cycles with primer set OI1/OI2c (3,4). After agarose gel electrophoresis and staining with ethidium bromide, a 1.1-kb DNA band was unambiguously associated with symptomatic but not asymptomatic leaf samples. Nonnested-PCR using primer set OI1/OI2c alone did not yield a target DNA band or yielded a very weak DNA band. XbaI digestion of the nested-PCR DNA product yielded two fragments, 520 and 640 bp long, characteristic of ‘Ca. L. asiaticus’. PCR amplicons were sequenced and were 1,095 bp long. This sequence shared >98% similarity to sequences of ‘Ca. L. asiaticus’ in the GenBank database. We observed that nested-PCR is necessary for consistent amplification of DNA from ‘Ca. L. asiaticus’ from M. paniculata. We excluded the possible nonspecific amplification associated with nested-PCR by XbaI restriction enzyme digestion and by nucleotide sequence analysis. Our data indicate that M. paniculata is a host of ‘Ca. L. asiaticus’ but the bacterial titer might be low. References: (1) X. Deng et al. Online publication. doi:10.1094/PHP-2007-0419-01-BR. Plant Health Progress, 2007. (2) M. Garnier and J. Bove. Huanglongbing (Greening). Page 46 in: Compendium of Citrus Diseases. 2nd ed. L. W. Timmer et al., eds. The American Phytopathological Society, St. Paul, MN, 2000. (3) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (4) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996.