Publications

4537

Summary Here we report the first metatranscriptomic analysis of gene expression and regulation of ‘ Candidatus Accumulibacter’‐enriched lab‐scale sludge during enhanced biological phosphorus removal (EBPR). Medium density oligonucleotide microarrays were generated with probes targeting most predicted genes hypothesized to be important for the EBPR phenotype. RNA samples were collected at the early stage of anaerobic and aerobic phases (15 min after acetate addition and switching to aeration respectively). We detected the expression of a number of genes involved in the carbon and phosphate metabolisms, as proposed by EBPR models (e.g. polyhydroxyalkanoate synthesis, a split TCA cycle through methylmalonyl‐CoA pathway, and polyphosphate formation), as well as novel genes discovered through metagenomic analysis. The comparison between the early stage anaerobic and aerobic gene expression profiles showed that expression levels of most genes were not significantly different between the two stages. The majority of upregulated genes in the aerobic sample are predicted to encode functions such as transcription, translation and protein translocation, reflecting the rapid growth phase of Accumulibacter shortly after being switched to aerobic conditions. Components of the TCA cycle and machinery involved in ATP synthesis were also upregulated during the early aerobic phase. These findings support the predictions of EBPR metabolic models that the oxidation of intracellularly stored carbon polymers through the TCA cycle provides ATP for cell growth when oxygen becomes available. Nitrous oxide reductase was among the very few Accumulibacter genes upregulated in the anaerobic sample, suggesting that its expression is likely induced by the deprivation of oxygen.

Pear decline (PD) is a serious disease of pear (Pyrus communis L.) caused by ‘Candidatus Phytoplasma pyri’, which belongs to the subgroup 16SrX-C of the apple proliferation (AP) group of phytoplasmas (3). Pear seedlings from the Agriculture and Agri-Food Canada (AAFC) pear breeding program, which have been selected for advanced test and grower trials, are routinely submitted to the Canadian Food Inspection Agency (CFIA) Sidney Laboratory (formerly, CFIA Centre for Plant Health, Saanichton, BC) for virus testing at the same time that propagation is initiated to produce trees for further evaluations. In early 2007, the CFIA reported that samples of two seedling selections submitted in 2005 tested positive for phytoplasmas by a nested PCR assay with phytoplasma universal primers P1/P7 (1), followed by phytoplasma universal primers fU5/rU3 (2) and real time PCR with universal phytoplasma primers developed by the CFIA-Sidney (personal communication). Phytoplasmas present in both selections were subsequently identified as ‘Ca. P. pyri' strains by nested PCR with the P1/P7 primers followed by PD/peach yellow leaf roll (PYLR)-specific primers fPD/rPDS (2,4). These were the first PD-positive results from many samples submitted over the years for testing. Following PD-positive diagnoses for the seedling trees, others propagated from these seedling trees were removed from the nursery. When tested by PD-specific nested PCR (P1/P7 then fPD/rPDS), one selection had 39 of 79 nursery trees (49%) that were PD positive, while the other selection had 27 of 96 trees (28%) testing as PD positive. PCR amplification of DNA isolated from leaves of six of the propagated trees, with primer pair fPD/rPDS, yielded an ~1,400-bp product that was sequenced. A consensus sequence of 1,313 bp (GenBank Accession No. GU565959) was subjected to a nucleotide BLAST search of the NCBI database and showed 100% nt identity with sequences of phytoplasmas PD1 (AJ542543) and PYLR (Y16394). Subsequently, the PD-positive results from leaf, dormant shoot, and root tissues from the original seedling trees were confirmed by PD-specific nested PCR. On the original seedling trees, visible symptoms typical of PD, especially premature leaf coloration, were observed in late summer 2008 and samples taken of green and red leaves were subjected to PD-specific PCR. Red leaves were PD-positive, while green leaves were mostly PD-negative. Pear leaves, dormant shoots, and roots collected from research and commercial orchards in southern Ontario in 2007 and 2008 were subjected to PD-specific nested PCR (P1/P7 then fPD/rPDS), AP-specific nested PCR (P1/P7 then fO1/rO1) (2), as well as the universal phytoplasma nested PCR (P1/P7 then fU5/rU3), resulting in the identification of PD-positive trees of several cultivars. The sequence of the 1,057-bp amplicon from accession PYR0190 (selection HW615), with AP-specific primers fO1/rO1, was deposited in GenBank (GU475131). Although there have been no previous reports of PD in Ontario, Canada, it would appear that PD has been present for some time based on the number and distribution (both geographic and cultivar) of positive samples. References: (1) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (2) K.-H. Lorenz et al. Phytopathology 85:771, 1995. (3) E. Seemüller et al. J. Plant Pathol. 80:3, 1998. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

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.

Huanglongbing (HLB) is a serious disease of citrus worldwide. Three different ‘Candidatus Liberibacter’ species are associated with HLB: ‘Ca. Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’ (1). ‘Ca. L. africanus’ and its vector, Trioza erytreae, are both heat sensitive, and when present, occur in citrus when temperatures remain below 30 to 32°C. In Africa, ‘Ca. L. africanus’ and T. erytreae have been reported in South Africa, Zimbabwe, Malawi, Burundi, Kenya, Somalia, Ethiopia, Cameroon, and Madagascar (1). Inspection of citrus trees in orchards and budwood sources in nurseries located in the warmer citrus-growing areas of Tigray and North Wollo in northern Ethiopia revealed nearly 100 trees with symptoms of leaf yellowing with a blotchy mottle pattern, dead branches, and decreased fruit quality and yield. Two symptomatic sweet orange budwood trees and three symptomatic orchard plants were sampled in April 2009, along with three healthy-looking sweet orange plants. DNA was extracted from 200 mg of desiccated leaf midribs using the CTAB method (4) and subjected to conventional PCR using the primer pairs A2/J5 (2) and OI2/23S1 (3) that amplify the ribosomal protein gene in the rplKAJL-rpoBC operon and the 16S/23S ribosomal intergenic regions, respectively, of ‘Ca. L. africanus’ and ‘Ca. L. asiaticus’. Positive PCR reactions were obtained for all five symptomatic samples with both primer pairs. PCR amplicons of 703 bp (A2/J5) and 892 bp (OI2/23S) recovered from two of these samples were purified, cloned, and sequenced. BLAST analysis revealed that the nucleotide sequences we obtained for the ribosomal protein (GenBank Accessions Nos. GQ890155 and GQ890156) shared 100% identity with each other and 99% identity with sequences of ‘Ca. L. asiaticus’ from Brazil (DQ471904), Indonesia (AB480161), China (DQ157277), and Florida (CP001677). Similarly, the 16S/23S ribosomal intergenic sequences (GU296538 and GU296539) shared 100% identity with each other and 99% identity with homologous ‘Ca. L. asiaticus’ sequences from Brazil (DQ471903), Indonesia (AB480102), China (DQ778016), and Florida (CP001677) and contained two tRNA genes as occurs in ‘Ca. L. asiaticus’ but not in ‘Ca. L. africanus’ (3). To our knowledge, this is the first report of ‘Ca. L. asiaticus’ in Africa. The presence of ‘Ca. L. asiaticus’ is a threat for warmer citrus-growing areas of Africa that are less favorable for ‘Ca. L. africanus’ and T. erytreae. In areas where ‘Ca. L. asiaticus’ was confirmed, symptomatic trees must be promptly eradicated and surveys to determine spread of the disease and its vectors are necessary. References: (1) J. M. Bove. J. Plant Pathol. 88:7, 2006. (2) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (3) S. Jagoueix et al. Int. J. Syst. Bacteriol. 47:224, 1997. (4) M. G. Murray and W. F Thompson. Nucleic Acids Res. 8:4321, 1980.

Abstract Analyses of archaeal membrane lipids are increasingly being included in ecological studies as a comparatively unbiased complement to gene-based microbiological approaches. For example, crenarchaeol, a glycerol dialkyl glycerol tetraether (GDGT) with a unique cyclohexane moiety, has been postulated as biomarker for ammonia-oxidizing Archaea (AOA). Crenarchaeol has been detected in Nitrosopumilus maritimus and ‘Candidatus Nitrosocaldus yellowstonii’ representing two of the three lineages within the Crenarchaeota containing described AOA. In this paper we present the membrane GDGT composition of ‘Candidatus Nitrososphaera gargensis’, a moderately thermophilic AOA, and the only cultivated Group I.1b Crenarchaeon. At a cultivation temperature of 46 °C, GDGTs of this organism consisted primarily of crenarchaeol, its regioisomer, and a novel GDGT. Intriguingly, ‘Ca. N. gargensis’ is the first cultivated archaeon to synthesize substantial amounts of the crenarchaeol regioisomer, a compound found in large relative abundances in tropical ocean water and some soils, and an important component of the TEX86 paleothermometer. Intact polar lipid (IPL) analysis revealed that ‘Ca. N. gargensis’ synthesizes IPLs similar to those reported for the Goup I.1a AOA, Nitrosopumilus maritimus SCMI, in addition to IPLs containing uncharacterized headgroups. Overall, the unique GDGT composition of ‘Ca. N. gargensis’ extends the known taxonomic distribution of crenarchaeol synthesis to the Group I.1b Crenarchaeota, implicating this clade as a potentially important source of crenarchaeol in soils and moderately high temperature environments. Moreover, this work supports the hypothesis that crenarchaeol is specific to all AOA and highlights specific lipids, which may prove useful as biomarkers for ‘Ca. N. gargensis’-like AOA.