Applied and Environmental Microbiology

ABSTRACT Three cohorts of farmed yellowtail kingfish ( Seriola lalandi ) from South Australia were examined for Chlamydia -like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales . A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported “ Candidatus Piscichlamydia salmonis” (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia -like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales . Based on these observations, we propose this bacterium of yellowtail kingfish be known as “ Candidatus Parilichlamydia carangidicola” and that the new family be known as “ Candidatus Parilichlamydiaceae.”

ABSTRACT The bacteria that cause necrotizing hepatopancreatitis in Penaeus vannamei adversely affect penaeid shrimp cultured in the western hemisphere. 16S rRNA and gyrase B gene analyses determined the taxonomic position of these bacteria. The name “ Candidatus Hepatobacter penaei” is proposed for these pathogenic bacteria, which are members of the Rickettsiales order.

ABSTRACT “ Candidatus Neoehrlichia mikurensis” was detected by PCR in 4.0% (34/841) of the rodents tested in this study. The 34 rodents represented nine species from seven regions of China. Phylogenetic analyses based on the partial groEL and nearly entire 16S rRNA gene sequences of the agent revealed genetic diversity, which was correlated with its geographic origins.

ABSTRACTTo characterize the denitrifying phosphorus (P) uptake properties of “CandidatusAccumulibacter phosphatis,” a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of “Ca. Accumulibacter” and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescencein situhybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized “Ca. Accumulibacter” subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria (Dechloromonas[from 1.2% to 19.2%] and “CandidatusCompetibacter phosphatis” [from 16.4% to 20.0%]), while the overall “Ca. Accumulibacter” abundance decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses was performed to characterize the denitrifying P uptake properties of the “Ca. Accumulibacter” clades. In FISH/MAR experiments using slightly diluted sludge (∼0.5 g/liter), all “Ca. Accumulibacter” clades successfully took up phosphorus in the presence of nitrate. However, the “Ca. Accumulibacter” clades showed no P uptake in the presence of nitrate when the sludge was highly diluted (∼0.005 g/liter); under these conditions, reduction of nitrate to nitrite did not occur, whereas P uptake by “Ca. Accumulibacter” clades occurred when nitrite was added. These results suggest that the “Ca. Accumulibacter” cells lack nitrate reduction capabilities and that P uptake by “Ca. Accumulibacter” is dependent upon nitrite generated by associated nitrate-reducing bacteria such asDechloromonasand “Ca. Competibacter.”

ABSTRACT“CandidatusPortiera aleyrodidarum” is the primary endosymbiont of whiteflies. We report two complete genome sequences of this bacterium from the worldwide invasive B and Q biotypes of the whiteflyBemisia tabaci. Differences in the two genome sequences may add insights into the complex differences in the biology of both biotypes.

ABSTRACT The recently described bacterium “ Candidatus Methylomirabilis oxyfera” couples the oxidation of the important greenhouse gas methane to the reduction of nitrite. The ecological significance of “ Ca . Methylomirabilis oxyfera” is still underexplored, as our ability to identify the presence of this bacterium is thus far limited to DNA-based techniques. Here, we investigated the lipid composition of “ Ca . Methylomirabilis oxyfera” to identify new, gene-independent biomarkers for the environmental detection of this bacterium. Multiple “ Ca . Methylomirabilis oxyfera” enrichment cultures were investigated. In all cultures, the lipid profile was dominated up to 46% by the fatty acid (FA) 10-methylhexadecanoic acid (10MeC 16:0 ). Furthermore, a unique FA was identified that has not been reported elsewhere: the monounsaturated 10-methylhexadecenoic acid with a double bond at the Δ7 position (10MeC 16:1Δ7 ), which comprised up to 10% of the total FA profile. We propose that the typical branched fatty acids 10MeC 16:0 and 10MeC 16:1Δ7 are key and characteristic components of the lipid profile of “ Ca . Methylomirabilis oxyfera.” The successful detection of these fatty acids in a peatland from which one of the enrichment cultures originated supports the potential of these unique lipids as biomarkers for the process of nitrite-dependent methane oxidation in the environment.

ABSTRACT Here we present comparative data on the localization and identity of intracellular symbionts among the superfamily Lygaeoidea (Insecta: Hemiptera: Heteroptera: Pentatomomorpha). Five different lygaeoid species from the families Blissidae and Lygaeidae (sensu stricto; including the subfamilies Lygaeinae and Orsillinae) were analyzed. Fluorescence in situ hybridization (FISH) revealed that all the bugs studied possess paired bacteriomes that are differently shaped in the abdomen and harbor specific endosymbionts therein. The endosymbionts were also detected in female gonads and at the anterior poles of developing eggs, indicating vertical transmission of the endosymbionts via ovarial passage, in contrast to the posthatch symbiont transmission commonly found among pentatomoid bugs (Pentatomomorpha: Pentatomoidea). Phylogenetic analysis based on 16S rRNA and groEL genes showed that the endosymbionts of Ischnodemus sabuleti , Arocatus longiceps , Belonochilus numenius , Orsillus depressus , and Ortholomus punctipennis constitute at least four distinct clades in the Gammaproteobacteria . The endosymbiont phylogeny did not agree with the host phylogeny based on the mitochondrial cytochrome oxidase I ( COI ) gene, but there was a local cospeciating pattern within the subfamily Orsillinae. Meanwhile, the endosymbiont of Belonochilus numenius (Lygaeidae: Orsillinae), although harbored in paired bacteriomes as in other lygaeoid bugs of the related genera Nysius , Ortholomus , and Orsillus , was phylogenetically close to “ Candidatus Rohrkolberia cinguli,” the endosymbiont of Chilacis typhae (Lygaeoidea: Artheneidae), suggesting an endosymbiont replacement in this lineage. The diverse endosymbionts and the differently shaped bacteriomes may reflect independent evolutionary origins of the endosymbiotic systems among lygaeoid bugs.

ABSTRACT “ Candidatus Liberibacter asiaticus” is a psyllid-transmitted, phloem-limited alphaproteobacterium and the most prevalent species of “ Ca . Liberibacter” associated with a devastating worldwide citrus disease known as huanglongbing (HLB). Two related and hypervariable genes ( hyv I and hyv II ) were identified in the prophage regions of the Psy62 “ Ca. Liberibacter asiaticus” genome. Sequence analyses of the hyv I and hyv II genes in 35 “ Ca. Liberibacter asiaticus” DNA isolates collected globally revealed that the hyv I gene contains up to 12 nearly identical tandem repeats (NITRs, 132 bp) and 4 partial repeats, while hyv II contains up to 2 NITRs and 4 partial repeats and shares homology with hyv I . Frequent deletions or insertions of these repeats within the hyv I and hyv II genes were observed, none of which disrupted the open reading frames. Sequence conservation within the individual repeats but an extensive variation in repeat numbers, rearrangement, and the sequences flanking the repeat region indicate the diversity and plasticity of “ Ca. Liberibacter asiaticus” bacterial populations in the world. These differences were found not only in samples of distinct geographical origins but also in samples from a single origin and even from a single “ Ca. Liberibacter asiaticus”-infected sample. This is the first evidence of different “ Ca. Liberibacter asiaticus” populations coexisting in a single HLB-affected sample. The Florida “ Ca. Liberibacter asiaticus” isolates contain both hyv I and hyv II , while all other global “ Ca. Liberibacter asiaticus” isolates contain either one or the other. Interclade assignments of the putative Hyv I and Hyv II proteins from Florida isolates with other global isolates in phylogenetic trees imply multiple “ Ca. Liberibacter asiaticus” populations in the world and a multisource introduction of the “ Ca. Liberibacter asiaticus” bacterium into Florida.

ABSTRACT “ Candidatus Accumulibacter” and total bacterial community dynamics were studied in two lab-scale enhanced biological phosphorus removal (EBPR) reactors by using a community fingerprint technique, automated ribosomal intergenic spacer analysis (ARISA). We first evaluated the quantitative capability of ARISA compared to quantitative real-time PCR (qPCR). ARISA and qPCR provided comparable relative quantification of the two dominant “ Ca . Accumulibacter” clades (IA and IIA) detected in our reactors. The quantification of total “ Ca . Accumulibacter” 16S rRNA genes relative to that from the total bacterial community was highly correlated, with ARISA systematically underestimating “ Ca . Accumulibacter” abundance, probably due to the different normalization techniques applied. During 6 months of normal (undisturbed) operation, the distribution of the two clades within the total “ Ca . Accumulibacter” population was quite stable in one reactor while comparatively dynamic in the other reactor. However, the variance in the clade distribution did not appear to affect reactor performance. Instead, good EBPR activity was positively associated with the abundance of total “ Ca . Accumulibacter.” Therefore, we concluded that the different clades in the system provided functional redundancy. We disturbed the reactor operation by adding nitrate together with acetate feeding in the anaerobic phase to reach initial reactor concentrations of 10 mg/liter NO 3 -N for 35 days. The reactor performance deteriorated with a concomitant decrease in the total “ Ca . Accumulibacter” population, suggesting that a population shift was the cause of performance upset after a long exposure to nitrate in the anaerobic phase.

ABSTRACT To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “ Candidatus Accumulibacter phosphatis” (henceforth referred to as “ Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “ Ca . Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “ Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “ Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “ Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “ Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 ( ppk1 ) gene homologs were amplified by using a ppk1 -specific PCR primer set for “ Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that “ Ca. Accumulibacter” strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.