Publications

4467

Abstract The DPANN superphylum is a deep-branching radiation of Archaea with small cell and genome sizes. Most DPANN lineages are predicted or validated to be host-dependent. However, certain lineages have substantial biosynthetic capacities and are potentially less dependent on hosts or even free-living. Here, we reconstructed 163 Micrarchaeota genomes, comprising 48 assigned to previously undescribed orders and 115 affiliated with known orders. Investigation of their genetic repertoire revealed substantial metabolic capacity in Norongarragalinales, Anstonellales, and the newly proposed Wunengiarchaeales-associated lineages, including complete or near-complete glycolysis and de novo biosynthetic pathways for nucleotides, amino acids, co-factors, and cell envelopes. We classified these genes related to central metabolism, but which are uncommon in DPANN archaea as putative free-living associated genes (pFLAGs). The extensive presence of pFLAGs in Norongarragalinales suggests a potential host-independent lifestyle. Reconstruction of evolutionary history revealed that these pFLAGs were not ancestral within the DPANN superphylum. Instead, we suggest that less host-dependent organisms evolved from symbionts through the gradual acquisition of pFLAGs through horizontal gene transfer, whereas other Micrarchaeota lineages with streamlined genomes experienced reductive evolution due to thermal adaptation. Our analyses demonstrate that host dependency is not always an evolutionary dead end, but can be reversed through acquisition of new metabolic capabilities by horizontal transfer.

Phloem feeders, such as the psyllid Diaphorina citri ( D. citri ), feed on plants by inserting their stylet bundle followed by probing the apoplast before reaching the phloem. The psyllids secrete watery saliva containing various proteins into the phloem, which can act as effectors to facilitate their feeding or modulate host defense responses. Concomitantly, feeding is the main mode of transmitting the Candidatus Liberibacter asiaticus ( C Las) bacteria to the phloem. C Las produces several effectors that have been hypothesized to contribute to Huanglongbing (HLB) virulence. Here, we aimed to identify putative effector proteins from both D. citri and C Las. To achieve this, we used different omics techniques on different tissues and organs from both plants and insects. More specifically, we performed transcriptomics on the heads of healthy and C Las-infected D. citri and proteomics of artificial diet and of phloem of four different plant species fed on by healthy and C Las-infected D. citri. Subsequently, we used various criteria and bioinformatics tools to predict putative effectors. This resulted in the identification of four proteins from D. citri [ferritin, prisilkin, CG31997-PA, and pterin-4-alpha-carbinolamine dehydratase-like protein (PCBD)] and two Sec-dependent effectors from C Las, CLIBASIA_04560 and CLIBASIA_05320, that were used for further functional studies. The expression of these six proteins in Nicotiana benthamiana modified the ROS burst triggered by flagellin, indicating that they can indeed function as effector proteins in planta . In addition, expression of the psyllid effectors in planta significantly reduced the growth of Pseudomonas syringae pv. tabaci ( Pta ).

Abstract Background Hemotropic Mycoplasma spp. (hemoplasmas) parasitize erythrocytes and cause hemolytic anemia in several mammalian species, including cats. Mycoplasma haemofelis ( Mhf ), “ Candidatus Mycoplasma haemominutum” ( C Mhm) and “ Candidatus Mycoplasma turicensis” ( C Mt) are the three main feline hemoplasma species. A species closely related to C Mhm was recently proposed as a putative novel species based on the 23S ribosomal RNA (rRNA) gene. Methods In this study, 16S and 23S rRNA genes were used to investigate hemoplasma diversity in cats. Blood samples from 388 cats were obtained and screened for hemoplasma infection based on a PCR assay targeting the 16S rRNA gene. Positive samples were sequenced based on the 16S and 23S rRNA genes. All obtained sequences were analyzed by the nucleotide Basic Local Alignment Search Tool (BLASTn), the DnaSP6 computer program, phylogenetic construction, genetic network and pairwise identity matrix. Results The 388 blood samples collected from the cats were screened for hemoplasma infection. The tests showed that 68 cats (17.5%, 95% confidence interval [CI] 13.9–21.7%) were positive for hemoplasmas. Of these 68 positive samples, 49 were successfully sequenced for both the 16S and 23S rRNA genes and the sequences subsequently assigned to 11 nucleotide sequence types (ntSTs). The 16S rDNA analysis revealed one Mhf group, at least three groups within C Mhm and at least two groups within C Mt. Notably, we identified C Mhm as well as two putative species closely related to C Mhm from 23S rDNA analysis, including one that has been previously identified. In contrast, the identity of the C Mt-derived 23S rDNA sequence ntST#11 remains unclear due to the lack of C Mt reference sequences, highlighting the need for more comprehensive C Mt data in public databases. Conclusions Our data suggested the presence of two putative species related to C Mhm identified in domestic cats in Thailand. Integrating analyses of independent genetic markers, such as 16S and 23S rRNA genes, would enhance hemoplasma species identification and novel species discovery. Graphical abstract

ABSTRACT Huanglongbing (HLB) is a bacterial disease that affects citrus trees and is considered the most severe citrus disease in the world. The three HLB-associated “ Ca . Liberibacter” species harbor prophage regions which have been described to play critical roles in their evolution and biology. In this study, using infected insect vectors, we assembled and characterized the accessory genome of the first circular de novo “ Candidatus Liberibacter asiaticus” (CLas) assembly (V1R1) from Réunion, one of the sparse areas worldwide hosting CLas and “ Ca . Liberibacter africanus” (CLaf). This 1.272 Mb-long whole-genome harbored 1,129 coding sequences and two complete prophages, including a 37,934 bp-long Type 1 prophage, frequently present in CLas genomes, and a 40,501 bp-long undescribed CLas prophage designated as P-V1R1-5. Comparative genomic approaches suggested that P-V1R1-5 has all the genetic elements to produce new viral particles and revealed that it likely belongs to a new CLas Type 5 prophage. While P-V1R1-5-like prophages had been identified previously in CLaf genomes, our study demonstrates the first lysogenic conversion of these prophages in CLas, suggesting a positive selection of prophage-like sequences among HLB-associated “ Ca . Liberibacter” species. Using PCR amplifications targeting P-V1R1-5, we also showed that 85.7% of the tested CLas strains from Réunion and all tested CLaf strains from Madagascar and Réunion harbored a Type 5-like prophage. The identification of CLas Type 5-like prophages not only expanded our knowledge of CLas genomic diversity in Réunion but also provided new insights into the role of horizontally transferred elements in the evolution of the sympatric HLB-associated bacteria. IMPORTANCE Huanglongbing (HLB) is the most severe citrus disease worldwide. The disease is associated with three “ Candidatus Liberibacter” species harboring prophage regions that have been described to play critical roles in their biology. In this study, using infected insect vectors, we assembled and characterized the accessory genome of the first circular de novo “ Ca . Liberibacter asiaticus” (CLas) assembly from Réunion, one of the very few areas in the world where both of CLas and “ Ca . Liberibacter africanus” (CLaf) coexist. Comparative genomic approaches demonstrated that this genome harbored two complete prophages, including a new CLas Type 5 prophage that was previously identified in CLaf but was reported for the first time in a CLas strain. This first evidence of shared prophage-like sequences among HLB-associated species expands our knowledge of CLas genomic diversity but also provides new insights into the role of the accessory genome in the evolution of these bacteria.

‘ Candidatus Ferrigenium straubiae’ strain KS (KCTC 25982, DSM 118991) is a neutrophilic, Fe(II)-oxidizing bacterium representing up to 98% of the community in culture KS, the most extensively studied mixed culture known for autotrophic nitrate-reducing Fe(II) oxidation. The phylogeny and genome of this bacterium were previously analysed and identified as ‘ Candidatus Ferrigenium straubiae’. In this study, we report the first-time successful isolation of ‘ Candidatus Ferrigenium straubiae’ strain KS and its experimental physiological characterization. The bacterium was identified as a non-stalk-forming, rod-shaped and non-halophilic strain with a Gram-negative classification. We characterized its physiology when grown in agarose-stabilized Fe(II)-O 2 gradient tubes where Fe(II) stemming from FeS functions as the electron donor and O 2 as the electron acceptor. It showed growth at temperatures of 20–30 °C (optimal at 25°C) and at pH levels of 6.0–7.5 (optimal at pH 6.5–7.0). The doubling time at 20 °C and pH 6.5 was 16 h. We further optimized the gradient tubes for sustainable culture maintenance using modified Wolfe’s mineral medium (MWMM; 1 g l −1 NH 4 Cl) supplemented with 7-vitamin solution, SL-10 trace elements, selenite-tungstate solution and selenite-molybdate-nickel-copper-arsenic-vanadium solution (final concentrations of 10 µM Se, 10 µM Mo, 0.1 µM Ni, 0.1 µM Cu, 0.1 µM As and 5 nM V). We also evaluated several Fe(II) sources (with O 2 as electron acceptor), as well as both inorganic and organic substrates for their influence on growth. Although a known member of the denitrifying community in culture KS, the isolated strain ‘ Candidatus Ferrigenium straubiae’ KS exhibited exclusively microaerobic and autotrophic growth in agarose-stabilized Fe(II)-O 2 gradients, utilizing Fe(II) from FeS as the electron donor.