Publications

4466

Background: Huanglongbing (HLB) is currently one of the most devasting diseases in citrus plants worldwide. Resistance against its causal agent, Candidatus Liberibacter asiaticus (CLas), in commercial Citrus species remains a challenge, even though they show differences in CLas multiplication. Methods: A total of 14 citrandarins and their parents (Sunki mandarin and Poncirus trifoliata cv. Rubidoux) were top-grafted onto the canopy of potted ‘Valencia’ sweet orange plants with high CLas titers. The grafted genotypes were tested for CLas infection and physiological effects of the disease (starch accumulation and callose deposition) for 12 months. Results: All tested genotypes were infected by CLas during the time frame of the experiment. However, a decrease in the infection rate ranging from 50% to 80% for the hybrids H109, H126, H157, and H222 was observed 360 days from the top-grafting. CLas was undetected by real-time PCR in H106 at the end of the experiment, which had low levels of starch and callose deposition. Conclusions: CLas infected all of the tested citrandarins, but a decrease in the infection rate over time was detected for some specific genotypes, which led to less starch accumulation and callose deposition.

Huanglongbing (HLB), the current major threat for Citrus species, is caused by intracellular alphaproteobacteria of the genus Candidatus Liberibacter (CaL), with CaL asiaticus (CLas) being the most prevalent species. This bacterium inhabits phloem cells and is transmitted by the psyllid Diaphorina citri. A gene encoding a putative serralysin-like metalloprotease (CLIBASIA_01345) was identified in the CLas genome. The expression levels of this gene were found to be higher in citrus leaves than in psyllids, suggesting a function for this protease in adaptation to the plant environment. Here, we study the putative role of CLas-serralysin (Las1345) as virulence factor. We first assayed whether Las1345 could be secreted by two different surrogate bacteria, Rhizobium leguminosarum bv. viciae A34 (A34) and Serratia marcescens. The protein was detected only in the cellular fraction of A34 and S. marcescens expressing Las1345, and increased protease activity of those bacteria by 2.55 and 4.25-fold, respectively. In contrast, Las1345 expressed in Nicotiana benthamiana leaves did not show protease activity nor alterations in the cell membrane, suggesting that Las1345 do not function as a protease in the plant cell. Las1345 expression negatively regulated cell motility, exopolysaccharide production, and biofilm formation in Xanthomonas campestris pv. campestris (Xcc). This bacterial phenotype was correlated with reduced growth and survival on leaf surfaces as well as reduced disease symptoms in N. benthamiana and Arabidopsis. These results support a model where Las1345 could modify extracellular components to adapt bacterial shape and appendages to the phloem environment, thus contributing to virulence.

A working hypothesis is that less common species of Campylobacter (other than C. jejuni and C. coli) play a role in enteric disease among children in low resource settings and explain the gap between the detection of Campylobacter using culture and culture independent methods. “Candidatus Campylobacter infans” (C. infans), was recently detected in stool samples from children and hypothesized to play a role in Campylobacter epidemiology in low- and middle-income countries (LMIC). This study determined the prevalence of C. infans in symptomatic and asymptomatic stool samples from children living in Iquitos, Peru. Stool samples from 215 children with diarrhea and 50 stool samples from children without diarrhea under the age of two were evaluated using a multiplex qPCR assay to detect Campylobacter spp. (16S rRNA), Campylobacter jejuni / Campylobacter coli (cadF gene), C. infans (lpxA), and Shigella spp. (ipaH). C. infans was detected in 7.9% (17/215) symptomatic samples and 4.0% (2/50) asymptomatic samples. The association between diarrhea and the presence of these targets was evaluated using univariate logistic regressions. C. infans was not associated with diarrhea. Fifty-one percent (75/146) of Campylobacter positive fecal samples were negative for C. jejuni, C. coli, and C. infans via qPCR. Shotgun metagenomics confirmed the presence of C. infans among 13 out of 14 positive C. infans positive stool samples. C infans explained only 20.7% of the diagnostic gap in stools from children with diarrhea and 16.7% of the gap in children without diarrhea. We posit that poor cadF primer performance better explains the observed gap than the prevalence of atypical non-C. jejuni/coli species.

AbstractCandidatusAccumulibacter is a key genus of polyphosphate-accumulating organisms (PAOs) found in laboratory- and full-scale wastewater treatment systems, mediating enhanced biological phosphorus removal (EBPR). However, little is known about their ability to resist phage infection. We conducted a systematic analysis of the occurrence and characteristics of clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) systems and prophages in diverseCa.Accumulibacter taxa (43 in total, including 10 newly recovered genomes). Fourty complete CRISPR loci were identified in 28 genomes, falling into 6 subtypes. The occurrence and distribution of CRISPR-Cas systems did not follow a vertical evolutionary relationship. Phylogenetic analyses of thecasgenes and direct repeats (DRs) suggested that the CRISPR-Cas systems were likely acquired via horizontal gene transfer, with acquisition rates higher than those of speciation, rendering differentCa.Accumulibacter distinct adaptivity to phage predations. 2448 spacers were identified, 67 of them matched to known phages. Major differences were observed among the numbers of spacers for differentCa.Accumulibacter, showing unique phages that could be resisted by different members. A comparison of the spacers in genomes having the samecasgene but from distinct geographical locations indicated that habitat isolation may have resulted in the acquisition of different spacers by differentCa. Accumulibacter. Metagenomic analysis allowed the identification of 26 viral contigs (18 are Caudovirales members) in 6 metagenomic datasets from three lab-scale enrichment reactors, matching to 73 spacers in 10Ca.Accumulibacter genomes in these reactors, showing the specific immunity of theseCa.Accumulibacter. Metatranscriptomic analyses showed the activity of the CRISPR-Cas system under both anaerobic and aerobic conditions. Extra efforts were made to identify prophages inCa.Accumulibacter genomes. In total, 133 prophage regions were identified. Twenty-seven of them were classified as potentially active prophages. Three prophages (all are Caudovirales members, in DS2011, SCELSE-7IIH and SCELSE-5IIH, respectively) are readily activable. Differences in the occurrence of CRISPR-Cas systems and prophages inCa.Accumulibacter will lead to their distinct responses under phage predation. This study represents the first systematic analysis of CRISPR-Cas systems and prophages with combined experimental and bioinformatic methods in theCa.Accumulibacter lineage, providing new perspectives on the potential impacts of phages onCa.Accumulibacter and EBPR systems.

AbstractAmmonia-oxidizing archaea (AOA) are key players in the nitrogen cycle of polar soils. Here, we analyzed metagenomic data from tundra soils in Rásttigáisá, Norway, and recovered four metagenome-assembled genomes (MAGs) assigned to the genus ‘UBA10452’, an uncultured lineage of putative AOA in the order Nitrososphaerales (‘terrestrial group I.1b’), phylum Thaumarchaeota. Analysis of other eight previously reported MAGs and publicly available amplicon sequencing data revealed that the UBA10452 lineage is predominantly found in acidic polar and alpine soils. In particular, UBA10452 MAGs were more abundant in highly oligotrophic environments such as mineral permafrost than in more nutrient-rich, vegetated tundra soils. UBA10452 MAGs harbour multiple copies of genes related to cold tolerance, particularly genes involved in DNA replication and repair. Based on the phylogenetic, biogeographic, and ecological characteristics of 12 UBA10452 MAGs, which include a high-quality MAG (90.8% complete, 3.9% redundant) with a nearly complete 16S rRNA gene, we propose a novel Candidatus genus, Ca. Nitrosopolaris, with four species representing clear biogeographic/habitat clusters.

AbstractSeveral species of luminous bacteria in the genusPhotobacteriumare the light organ symbionts of teleost fishes.Photobacterium leiognathiand its subspecies,P. mandapamensis, in particular, commonly form bioluminescent symbioses with fish hosts in the Leiognathidae and Acropomatidae families as well as with cardinalfish in the genusSiphamia(Apogonidae). These two closely related lineages ofPhotobacteriumare right at the cutoff average nucleotide identity used to delimit bacterial species (95-96%) and show overlapping ecological niches, including their host fish range. However, there are only a few whole genome assemblies available for these bacterial species, particularly for symbiotic strains isolated from fish light organs, that can be used to explore genome evolution of these two lineages. Here we used Oxford Nanopore Technologies sequencing to produce long reads for assembling highly contiguous genomes ofPhotobacteriumstrains isolated from fish light organs, including severalP. kishitaniistrains isolated from deep water fishes. We were able to assemble 31 high-quality genomes with near complete BUSCO scores, many at the chromosome-level, and compare their gene contents, including plasmid genes. In doing so, we discovered a new candidate species ofPhotobacterium, CandidatusPhotobacterium acropomis, which originated from the light organ of the acropomid fish,Acropoma japonicum. We also describe a lack of congruency between the presence of theluxFgene, which is involved in light production, and the phylogenetic relationships between closely relatedP. leiognathiandP. mandapamensisstrains. In contrast, there was strong congruency betweenluxFand the host fish family of origin, suggesting this gene might be essential to initiate bioluminescent symbioses with certain hosts, includingSiphamiaandAcropomaspecies. Our study shows the benefit of using long reads in the assembly of bacterial genomes and outlines an assembly pipeline that results in highly contiguous genomes, even from low-coverage ONT reads.

In Saudi Arabia (SA), the citrus greening disease is caused by ‘Candidatus Liberibacter asiaticus’ (CLas) transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. The origin and route(s) of the ACP-CLas pathosystem invasion in SA have not been studied. Adult ACP were collected from citrus trees in SA and differentiated by analysis of the mitochondrial cytochrome oxidase I (mtCOI) and nuclear copper transporting protein (atox1) genes. A phylogenetic analysis of the Wolbachia spp. surface protein (wsp) gene was used to identify the ACP-associated Wolbachia spp. A phylogenetic analysis of the atox1 and mtCOI gene sequences revealed one predominant ACP haplotype most closely related to the Indian subcontinent founder populations. The detection and identification of CLas in citrus trees were carried out by polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA gene. The CLas-integrated prophage genomes were sequenced, annotated, and used to differentiate CLas populations. The ML and ASTRAL trees reconstructed with prophages type 1 and 2 genome sequences, separately and concatenated, resolved two major lineages, CLas-1 and -2. The CLas-1 clade, reported here for the first time, consisted of isolates from SA isolates and Pakistan. The CLas-2 sequences formed two groups, CLas-2-1 and -2-2, previously the ‘Asiatic’ and ‘Floridian’ strains, respectively. Members of CLas-2-1 originated from Southeast Asia, the USA, and other worldwide locations, while CLas-2-2 was identified only in Florida. This study provides the first snapshot into the status of the ACP-CLas pathosystem in SA. In addition, the results provide new insights into the pathosystem coevolution and global invasion histories of two ACP-CLas lineages with a predicted center of origin in South and Southeast Asia, respectively.

AbstractMicrobes perform critical functions in corals yet most knowledge is derived from the photic zone. Here, we discovered two mollicutes that dominate the microbiome of the deep-sea octocoral,Callogorgia delta,and reside in the mesoglea. These symbionts were abundant across the host’s range, absent in the water, and rare in sediments. The symbionts lack all known fermentative capabilities including glycolysis and can only generate energy from arginine provided by the coral host. Their genomes feature extensive mechanisms to interact with foreign DNA which may be indicative of their role in symbiosis. We erect the novel family Oceanoplasmataceae which includes these symbionts and others associated with four marine invertebrate phyla. Its exceptionally broad host range suggests that the diversity of this enigmatic family remains largely undiscovered. Oceanoplasmataceae genomes are the most highly reduced among mollicutes providing new insight into their reductive evolution and the roles of coral symbionts.