Publications

3825

We investigated the bacterial community compositions and phosphorus removal performance under sludge bulking and non-bulking conditions in two biological wastewater treatment systems (conventional A2/O (anaerobic/anoxic/aerobic) and inverted A2/O (anoxic/anaerobic/aerobic) processes) receiving the same raw wastewater. Sludge bulking resulted in significant shift in bacterial compositions from Proteobacteria dominance to Actinobacteria dominance, characterized by the significant presence of filamentous ‘Candidatus Microthrix parvicella’. Quantitative real-time polymerase chain reaction (PCR) analysis revealed that the relative abundance of ‘Candidatus Accumulibacter phosphatis’, a key polyphosphate-accumulating organism responsible for phosphorus removal, with respect to 16s rRNA genes of total bacteria was 0.8 and 0.7%, respectively, for the conventional and inverted A2/O systems when sludge bulking occurred, which increased to 8.2 and 12.3% during the non-bulking period. However, the total phosphorus removal performance during the bulking period (2-week average: 97 ± 1 and 96 ± 1%, respectively) was not adversely affected comparable to that during the non-bulking period (2-week average: 96 ± 1 and 96 ± 1%, respectively). Neisser staining revealed the presence of large polyphosphate granules in ‘Candidatus Microthrix parvicella’, suggesting that this microbial group might have been responsible for phosphorus removal during the sludge bulking period when ‘Candidatus Accumulibacter phosphatis’ was excluded from the systems.

ABSTRACT “ Candidatus Synechococcus spongiarum” is a cyanobacterial symbiont widely distributed in sponges, but its functions at the genome level remain unknown. Here, we obtained the draft genome (1.66 Mbp, 90% estimated genome recovery) of “ Ca. Synechococcus spongiarum” strain SH4 inhabiting the Red Sea sponge Carteriospongia foliascens . Phylogenomic analysis revealed a high dissimilarity between SH4 and free-living cyanobacterial strains. Essential functions, such as photosynthesis, the citric acid cycle, and DNA replication, were detected in SH4. Eukaryoticlike domains that play important roles in sponge-symbiont interactions were identified exclusively in the symbiont. However, SH4 could not biosynthesize methionine and polyamines and had lost partial genes encoding low-molecular-weight peptides of the photosynthesis complex, antioxidant enzymes, DNA repair enzymes, and proteins involved in resistance to environmental toxins and in biosynthesis of capsular and extracellular polysaccharides. These genetic modifications imply that “ Ca. Synechococcus spongiarum” SH4 represents a low-light-adapted cyanobacterial symbiont and has undergone genome streamlining to adapt to the sponge’s mild intercellular environment. IMPORTANCE Although the diversity of sponge-associated microbes has been widely studied, genome-level research on sponge symbionts and their symbiotic mechanisms is rare because they are unculturable. “ Candidatus Synechococcus spongiarum” is a widely distributed uncultivated cyanobacterial sponge symbiont. The genome of this symbiont will help to characterize its evolutionary relationship and functional dissimilarity to closely related free-living cyanobacterial strains. Knowledge of its adaptive mechanism to the sponge host also depends on the genome-level research. The data presented here provided an alternative strategy to obtain the draft genome of “ Ca. Synechococcus spongiarum” strain SH4 and provide insight into its evolutionary and functional features.

Huanglongbing is an unculturable vascular citrus pathogen transmitted from infected to healthy plants through grafting or by citrus psyllids, Diaphorina citri mainly in Asia and America and Trioza erytreae in Africa. This phloem limited gram-negative bacterium causes dramatic yield losses and is classified into three species based on 16S rDNA sequence analysis (2): (i) ‘Candidatus Liberibacter asiaticus’ (Las), the most epidemiologically active, widespread and heat tolerant species; (ii) ‘Ca. L. africanus’ (Laf), only found in Africa; and (iii) the newly described ‘Ca. L. americanus’ (Lam), which appeared in 2005 in Brazil (5). Considered as a quarantine organism in America and Europe, Las is actively affecting North America and Asia, and research is leading toward psyllid management and resistance breeding. Despite the fact that Reunion Island has successfully controlled Las by introducing a psyllid parasitoid, Tamarixia radiata (1), this strategy was less effective or reproducible within other territories. D. citri was first detected in Guadeloupe in 1998, where the control of the the psyllid population has been effective with T. radiata (3); and was first detected in Martinique in 2012. Following the outbreak in the United States and the Caribbean, and also supported by reports of symptoms in citrus orchards, local National Plant Protection Organizations (NPPO) organized a detection survey across both islands to verify the occurrence of Huanglongbing. Since 2012, 450 sites were prospected each year in Martinique and Guadeloupe, where 20 leaves from 10 to 30 trees were analyzed. DNA extraction was performed (DNeasy Plant Mini Kit, Qiagen) on fresh or dried leaf midribs, along with negative control midribs (Citrus paradisi ‘Star Rubis’) and PCR amplification was done with the species-specific primers A2/J5 (4) and GB1/GB3 (5). Only Las-specific 703-bp amplicons were obtained (n = 43) and 20 were sequenced (Beckman Coulter Genomics, United Kingdom; sequences available through GenBank Accession Nos. KF699074 to KF699093) and blasted against the National Center for Biotechnology Information non-redondant database (NCBI-nr). BLAST analysis revealed 100% identity with the 50S ribosomal protein subunit L1 (rplA) and L10 (rplJ) of ‘Ca. L. asiaticus’ (all strains), and no significant homology to other organisms. Additionally, sequence assembly on a reference genome (NC_012985) showed 100% homology. Huanglongbing was detected in Guadeloupe on March 2012 at Le Moule (East coast) in a Tahiti lime orchard (C. latifolia) and crossed the island in 6 months. Las was detected in Martinique on May 2013 on Tahiti lime (C. latifolia) at Bellefontaine (Northwest) in a private garden and at Le Lorrain (Northeast) in an orchard. Other species from the Rutaceae family were affected by HLB (C. reticulat and C. sinensis) on both islands; however, few of the positive samples showed HLB symptoms (blotchy mottle patterns and green islands on leaves), but presented symptoms similar to nutrient deficiencies. Despite the former presence of T. radiata in Guadeloupe and its detection in Martinique a few weeks after the detection of D. citri, where it had a mean parasitism rate of 70%, an outbreak of HLB spread across both islands. These analyses confirm the presence of HLB in Martinique and Guadeloupe and to our knowledge represent the first report of Las in the French West Indies. Introduction events remain unclear, but this report raises the importance of plant certification, psyllid population control, and surveillance of territories close to the French West Indies, with regards to the risk that HLB presents to citrus production worldwide. References: (1) B. Aubert et al. Fruits. 38, 1983. (2) J. M. Bové. J. Plant Pathol. 88:1, 2006. (3) J. Etienne et al. Fruits. 56:05, 2001. (4) A. Hocquellet et al. Mol. Cell. Probes 13:5, 1999. (5) D. C. Teixeira et al. Mol. Cell. Probes 19:3, 2005.

ABSTRACT Methane is an important greenhouse gas and the most abundant hydrocarbon in the Earth's atmosphere. Methanotrophic microorganisms can use methane as their sole energy source and play a crucial role in the mitigation of methane emissions in the environment. “ Candidatus Methylomirabilis oxyfera” is a recently described intra-aerobic methanotroph that is assumed to use nitric oxide to generate internal oxygen to oxidize methane via the conventional aerobic pathway, including the monooxygenase reaction. Previous genome analysis has suggested that, like the verrucomicrobial methanotrophs, “ Ca. Methylomirabilis oxyfera” encodes and transcribes genes for the Calvin-Benson-Bassham (CBB) cycle for carbon assimilation. Here we provide multiple independent lines of evidence for autotrophic carbon dioxide fixation by “ Ca. Methylomirabilis oxyfera” via the CBB cycle. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), a key enzyme of the CBB cycle, in cell extracts from an “ Ca. Methylomirabilis oxyfera” enrichment culture was shown to account for up to 10% of the total methane oxidation activity. Labeling studies with whole cells in batch incubations supplied with either 13 CH 4 or [ 13 C]bicarbonate revealed that “ Ca. Methylomirabilis oxyfera” biomass and lipids became significantly more enriched in 13 C after incubation with 13 C-labeled bicarbonate (and unlabeled methane) than after incubation with 13 C-labeled methane (and unlabeled bicarbonate), providing evidence for autotrophic carbon dioxide fixation. Besides this experimental approach, detailed genomic and transcriptomic analysis demonstrated an operational CBB cycle in “ Ca. Methylomirabilis oxyfera.” Altogether, these results show that the CBB cycle is active and plays a major role in carbon assimilation by “ Ca. Methylomirabilis oxyfera” bacteria. Our results suggest that autotrophy might be more widespread among methanotrophs than was previously assumed and implies that a methanotrophic community in the environment is not necessarily revealed by 13 C-depleted lipids.