Publications

4731

Abstract Chemoheterotrophic marine bacteria of the SAR11 clade are Earth’s most abundant organisms. Following the first cultivation of a SAR11 bacterium, ‘Candidatus Pelagibacter ubique’ strain HTCC1062 (Ca. P. ubique) in 2002, unusual nutritional requirements were identified for reduced sulfur compounds and glycine or serine. These requirements were linked to genome streamlining resulting from selection for efficient resource utilization in nutrient-limited ocean habitats. Here we report the first successful cultivation of Ca. P. ubique on a defined artificial seawater medium (AMS1), and an additional requirement for pyruvate or pyruvate precursors. Optimal growth was observed with the collective addition of inorganic macro- and micronutrients, vitamins, methionine, glycine and pyruvate. Methionine served as the sole sulfur source but methionine and glycine were not sufficient to support growth. Optimal cell yields were obtained when the stoichiometry between glycine and pyruvate was 1:4, and incomplete cell division was observed in cultures starved for pyruvate. Glucose and oxaloacetate could fully replace pyruvate, but not acetate, taurine or a variety of tricarboxylic acid cycle intermediates. Moreover, both glycine betaine and serine could substitute for glycine. Interestingly, glycolate partially restored growth in the absence of glycine. We propose that this is the result of the use of glycolate, a product of phytoplankton metabolism, as both a carbon source for respiration and as a precursor to glycine. These findings are important because they provide support for the hypothesis that some micro-organisms are challenging to cultivate because of unusual nutrient requirements caused by streamlining selection and gene loss. Our findings also illustrate unusual metabolic rearrangements that adapt these cells to extreme oligotrophy, and underscore the challenge of reconstructing metabolism from genome sequences in organisms that have non-canonical metabolic pathways.

ABSTRACTThree cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined forChlamydia-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 orderChlamydiales. 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 “CandidatusPiscichlamydia salmonis” (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35ChlamydiaandChlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the orderChlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as “CandidatusParilichlamydia carangidicola” and that the new family be known as “CandidatusParilichlamydiaceae.”

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.

Summary Knowledge concerning microbial infectious diseases in the current amphibian crisis is rudimentary and largely limited to ranavirosis and chytridiomycosis. The family Chlamydiaceae is gaining attention as a common cause of disease in amphibians and may harbour new and emerging amphibian pathogens. We identified a novel species of Chlamydiales (Candidatus Amphibiichlamydia ranarum ) with a prevalence of 71% in exotic invasive bullfrog tadpoles ( Lithobates catesbeianus ) from an introduced population in the Netherlands. The sequence of a 1474 bp 16S rRNA gene fragment showed that the novel taxon forms a well‐defined clade with ‘ Candidatus Amphibiichlamydia salamandrae’ within the Chlamydiaceae family. Although none of the tadpoles examined showed signs of clinical disease, urgent evaluation of its pathogenic potential for native amphibian species is required.

Abstract Bacteria from the uncultured SUP05/Arctic96BD-19 clade of gamma proteobacterial sulfur oxidizers (GSOs) have the genetic potential to oxidize reduced sulfur and fix carbon in the tissues of clams and mussels, in oxygen minimum zones and throughout the deep ocean (>200 m). Here, we report isolation of the first cultured representative from this GSO clade. Closely related cultures were obtained from surface waters in Puget Sound and from the deep chlorophyll maximum in the North Pacific gyre. Pure cultures grow aerobically on natural seawater media, oxidize sulfur, and reach higher final cell densities when glucose and thiosulfate are added to the media. This suggests that aerobic sulfur oxidation enhances organic carbon utilization in the oceans. The first isolate from the SUP05/Arctic96BD-19 clade was given the provisional taxonomic assignment ‘Candidatus: Thioglobus singularis’, alluding to the clade’s known role in sulfur oxidation and the isolate’s planktonic lifestyle.

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive citrus diseases worldwide and is seen as a major threat to the multimillion dollar citrus industry in California. The vector of the two bacterial species associated with this disease, Candidatus Liberibacter asiaticus and Ca. L. americanus, is the Asian citrus psyllid (ACP), Diaphorina citri (4). ACP was detected in California in August of 2008 and has since been detected in nine counties in southern California. As part of a long term survey and testing program for the ACP carrying the HLB associated bacteria, groups of ACP nymphs and adults were submitted to the Jerry Dimitman Citrus Research Board/Citrus Pest and Disease Prevention Program Laboratory in Riverside, CA. In March 2012, DNA extracted using the Qiagen MagAttract 96 DNA plant kit (QIAGEN Inc., 27220 Turnberry Lane, Suite 200, Valencia, CA 91355) from a group of three ACP adults tested positive for Ca. L. asiaticus with the real-time PCR assay developed by Li et al. (4). ACP adults were collected from a residential citrus tree located in the Hacienda Heights area of Los Angeles County, California. The approximately 1.8 meter tall lemon tree had 23 graft unions, primarily of lemon (Citrus × meyeri) and pomelo (Citrus maxima) varieties. The tree was unthrifty, with yellow shoots and chlorotic leaves. Symptoms on the lemon and pomelo leaves included asymmetrical blotchy mottling, yellowing, and corking of the leaf veins, with the blotchy mottle more prominent in the pomelo leaves. Pomelo leaves appeared crinkled along the thickened veins. Lemon leaves had yellow veins and a few had islands of green tissue completely surrounded by yellow tissue. The entire tree was removed, cut into sections, bagged, and transported to the CDFA Plant Pest Diagnostics Lab for analysis. Two hundred milligrams of petiole and midrib tissue from leaves apical to each graft union was collected, and DNA from each sample was extracted using the Qiagen DNeasy plant mini kit. DNA extracted from both lemon and pomelo leaves tested positive for Ca. L. asiaticus using real-time PCR (4). A 1,160-bp fragment of the 16S ribosomal RNA gene was amplified from the insect and plant DNA extracts using conventional PCR with primers Ol1 and OI2c (2). A 703-bp fragment of the β-operon gene was amplified from the insect and plant extracts with primers A2 and J5 (1). The 16S rDNA fragments from the insect and plant respectively (GenBank Accession Nos. JX430434 and JX455745) and the β-operon fragments (JX430435 and JX455746) showed 100% identity with the corresponding regions of Ca. L. asiaticus (CP001677) strain psy 62. Our 16S rDNA sequence showed 98% identity with Ca. L. africanus (EU921620), 97% identity with Ca. L. solanacearum (HM246509), and 96% with Ca. L. americanus (FJ036892). In response to the detection of HLB, a 241 km2 quarantine area around the detection site was established. Surveys for ACP and symptomatic host plants within the HLB quarantine area are ongoing. To date, there have been no additional positive detections. In the United States, HLB was first detected in Florida in 2005 (4) and in Texas in January of 2012 (3). To our knowledge, this is the first confirmed report of Ca. L. asiaticus associated with HLB in California. References: (1) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) M. Kunta et al. Phytopathology 102:S4.66, 2012. (4) W. Li et al. J. Microbiol. Methods 66:104, 2006.