Publications

4374

ABSTRACTThe orderRickettsiales(Alphaproteobacteria) is a well-known group containing obligate endocellular prokaryotes. The order encompasses three families (Rickettsiaceae,Anaplasmataceae, andHolosporaceae) and a fourth, family-level cluster, which includes only one candidate species, “CandidatusMidichloria mitochondrii,” as well as several unnamed bacterial symbionts. The broad host range exhibited by the members of the “CandidatusMidichloria” clade suggests their eventual relevance for a better understanding of the evolution of symbiosis and host specificity ofRickettsiales. In this paper, two new bacteria belonging to the “CandidatusMidichloria” clade, hosted by two different strains of the ciliate protistEuplotes harpa, are described on the basis of ultrastructural observations, comparative 16S rRNA gene sequence analysis, and an estimation of the percentage of infection. Ultrastructure of these bacteria shows some unusual features: one has an electron-dense cytoplasm, and the other one lacks a symbiosomal membrane. The latter was up to now considered an exclusive feature of bacteria belonging to the familyRickettsiaceae. 16S rRNA gene phylogenetic analysis unambiguously places the new bacteria in the “CandidatusMidichloria” clade, although their phylogenetic relationships with other members of the clade are not clearly resolved. This is the first report of a ciliate-borne bacterium belonging to the “CandidatusMidichloria” clade. On the basis of the data obtained, the two bacteria are proposed as two new candidate genera and species, “CandidatusAnadelfobacter veles” and “CandidatusCyrtobacter comes.”

ABSTRACT The complete genome sequence of “ Candidatus Puniceispirillum marinum” IMCC1322, the first cultured representative of the SAR116 clade in the Alphaproteobacteria , is reported here. The genome contains genes for proteorhodopsin, aerobic-type carbon monoxide dehydrogenase, dimethylsulfoniopropionate demethylase, and C 1 compound metabolism. The genome information proposes the SAR116 group to be metabolic generalists in ocean nutrient cycling.

A moderately thermophilic chemoheterotrophic bacterium, strain Mat9-16T, was isolated from microbial mats developed in hot spring water streams from Yumata, Nagano, Japan. Cells of strain Mat9-16Twere strictly anaerobic, Gram-stain-negative, non-sporulating, non-motile and short to long rods (2.0–15.5 μm in length). Strain Mat9-16Tgrew fermentatively with optimum growth at 45 °C, pH 7.0–7.5 and 1 % NaCl (w/v). Phylogenetic analysis based on the 16S rRNA gene revealed that strain Mat9-16Twas affiliated with an uncultivated lineage, and the nearest cultivated neighbours were green sulfur bacteria belonging to the classChlorobeawith 77–83 % sequence similarity. However, strain Mat9-16Tcould not grow phototrophically and did not possess light-harvesting structures, morphologically and genetically, such as the chlorosomes of green sulfur bacteria. On the basis of phenotypic features and phylogenetic position, a novel genus and species are proposed for strain Mat9-16T, to be namedIgnavibacterium albumgen. nov., sp. nov. (=NBRC 101810T=DSM 19864T). We also propose to place the cultivated bacterial lineage accommodating the sole representative Mat9-16Tin a novel class,Ignavibacteriaclassis nov. In addition, we present a formal description of the phylum-level taxon‘Chlorobi’asChlorobiphyl. nov.

The citrus industries of North and South America are endangered by Huanglongbing (HLB), also known as citrus greening, a devastating disease associated with ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’, two species of fastidious phloem-limited bacteria spread by the Asian citrus psyllid (ACP), Diaphorina citri, Kuwayama. The first reports of HLB from the Americas were from Brazil in 2004 followed by Florida in 2005 (3). The ACP was found in Belize in 2005 (S. Williams, personal communication) and is now present throughout Central America. On the basis of the report that the HLB-associated bacteria can be easily detected in the ACP vector (4), an initial sampling of ACP from 67 locations was collected in February 2009 from trees in the Belize, Corozal, Orange Walk, Stann Creek, and Toledo Districts of Belize, and shipped in 95% ethanol to Riverside, CA for analysis. DNA was extracted from lots containing three to five psyllids from each of the 67 samples with Fast DNA kits (MP Biomedicals, Solon, OH) and analyzed by multiplex qPCR for ‘Ca. L. asiaticus’ and ‘Ca. L. americanus’ with a Stratagene MX3005P thermocycler with primers and Taqman probes to detect the 16sRNA gene of ‘Ca. L. asiaticus’ or ‘Ca. L. americanus’ and a psyllid gene, wingless, as an internal control target (4). Nine of the sixty-seven psyllid extractions were clearly positive for ‘Ca. L. asiaticus’ with cycle threshold values of 24 to 29. ‘Ca. L. americanus’ was not detected in any of the samples. From the districts previously sampled for ACP, leaves and fruit peduncles were collected from Citrus sinensis and C. aurantifolia plants showing HLB symptoms of asymmetrical leaf mottle and lopsided fruit with aborted seeds. DNA extracted from 10 of the 12 plant samples with a Qiagen Plant DNeasy kit (Qiagen Inc., Valencia, CA) was positive for ‘Ca. L. asiaticus’ with the qPCR procedure of Li et al (3). The presence of ‘Ca. L. asiaticus’ in the positive plant and ACP samples was corroborated by amplification, cloning, and sequencing of a 1,168-bp region of the 16S rRNA gene (2) with SpeedSTAR HS DNA polymerase (TaKaRa Bio Inc., Shiga, Japan). Consensus sequences obtained from three clones each from psyllids (Accession No. GQ502291) and plants (Accession No. GU061003) showed >99% identity to corresponding regions of ‘Ca. L. asiaticus’ in GenBank. The presence of ‘Ca. L. asiaticus’ was further indicated by amplification of a 227-bp fragment from the same 10 positive plant samples using primers for the ‘Ca. L. asiaticus’ preprotein translocase subunit SecE gene (nucleotides 31418 to 31644 of the genomic DNA) (1). Presence of trees with HLB symptoms and the detection of the associated ‘Ca. L. asiaticus’ confirm the disease in the Cayo, Corozal, Stann Creek, and Toledo districts in Belize. Analyses of psyllids from limited surveys conducted from 2006 to 2008 had not detected ‘Ca. L. asiaticus’ or ‘Ca. L. americanus’. Confirmation of HLB in Belize has significant implications to the citrus industries in Central America. References: (1) T. H. Hung et al. J. Phytopathol. 147:599, 1999. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006. (4) K. L. Manjunath et al. Phytopathology 98:387, 2008.

Metagenomics has provided access to genomes of as yet uncultivated microorganisms in natural environments, yet there are gaps in our knowledge—particularly for Archaea—that occur at relatively low abundance and in extreme environments. Ultrasmall cells (<500 nm in diameter) from lineages without cultivated representatives that branch near the crenarchaeal/euryarchaeal divide have been detected in a variety of acidic ecosystems. We reconstructed composite, near-complete ~1-Mb genomes for three lineages, referred to as ARMAN (archaeal Richmond Mine acidophilic nanoorganisms), from environmental samples and a biofilm filtrate. Genes of two lineages are among the smallest yet described, enabling a 10% higher coding density than found genomes of the same size, and there are noncontiguous genes. No biological function could be inferred for up to 45% of genes and no more than 63% of the predicted proteins could be assigned to a revised set of archaeal clusters of orthologous groups. Some core metabolic genes are more common in Crenarchaeota than Euryarchaeota , up to 21% of genes have the highest sequence identity to bacterial genes, and 12 belong to clusters of orthologous groups that were previously exclusive to bacteria. A small subset of 3D cryo-electron tomographic reconstructions clearly show penetration of the ARMAN cell wall and cytoplasmic membranes by protuberances extended from cells of the archaeal order Thermoplasmatales . Interspecies interactions, the presence of a unique internal tubular organelle [Comolli, et al. (2009) ISME J 3:159–167], and many genes previously only affiliated with Crenarchaea or Bacteria indicate extensive unique physiology in organisms that branched close to the time that Cren - and Euryarchaeotal lineages diverged.

A novel halophilic actinomycete, designated strain YIM 93223T, was isolated from a salt lake in Xinjiang Province, north-west China, and was subjected to a polyphasic taxonomic study. The isolate grew at 25–45 °C, at pH 6–8 and in the presence of 1–15 % (w/v) NaCl; no growth was observed in the absence of NaCl. Strain YIM 93223T contained meso-diaminopimelic acid, glutamic acid and alanine as cell-wall amino acids, and glucose and galactose as major whole-cell-wall sugars. Major fatty acids were iso-C16 : 0, C16 : 0 and C16 : 1 ω7c/iso-C15 : 0 2-OH. MK-8(H4) was the predominant menaquinone. The genomic DNA G+C content was 66.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 93223T belongs to the genus Amycolatopsis. It shared highest 16S rRNA gene sequence similarity with Amycolatopsis palatopharyngis 1BDZT (96.6 %) and Amycolatopsis marina Ms392AT (96.4 %), but lower values (94.5–96.2 %) with the type strains of other recognized species of the genus Amycolatopsis. On the basis of the data from this polyphasic study, strain YIM 93223T is considered to represent a novel species of the genus Amycolatopsis, for which the name Amycolatopsis halophila sp. nov. is proposed. The type strain is YIM 93223T (=DSM 45216T =KCTC 19403T).

Huanglongbing (HLB), considered to be the most serious insect-vectored bacterial disease of citrus, is transmitted in nature by the Asian citrus psyllid Diaphorina citri and the African citrus psyllid Trioza erytreae. D. citri was discovered in southern Florida in 1998 and the HLB disease in 2005. Both have become established throughout citrus-producing areas of Florida. Murraya species are widely grown in southern Florida as ornamental hedges and are readily colonized by D. citri vectors. Colonies of D. citri, isolates of ‘Candidatus Liberibacter asiaticus’ from Taiwan and Florida, and the Murraya species were established in the BSL-3 biosecurity facility at Fort Detrick. In controlled inoculation experiments, D. citri transmitted ‘Ca. L. asiaticus’ into M. paniculata (34/36 plants) and M. exotica (22/23 plants), but not into Bergera (Murraya) koenigii. Disease symptoms rarely developed in Murraya plants; however, positive infections were determined by conventional and real-time polymerase chain reaction (PCR). Back-inoculations of ‘Ca. L. asiaticus’ from M. paniculata to Madam Vinous sweet orange resulted in disease development in 25% of the inoculated plants. Considerable variability was observed in infection rates, titer, and persistence of ‘Ca. L. asiaticus’ in infected Murraya.