Publications

4490

Citrus Huanglongbing, caused by ‘ Candidatus Liberibacter asiaticus’ (CLas), is the most devastating citrus disease worldwide. The CLas genome is much smaller than those of its relatives, such as Sinorhizobium meliloti, due to its reductive evolution. Because CLas has not been cultured in artificial media, despite some progress in co-cultivating, and because genetic manipulation of CLas remains impossible, the understanding of CLas biology is very limited. Usually, 10% of total genes in bacteria are regulatory genes, but only 2% of CLas genes encode transcriptional factors. Here, 20 transcriptional regulators were predicted, including nine genes ( lsrB, ldtR, rem, visR, visN, ctrA, mucR, pelD, and atoC) directly or indirectly involved in regulating motility, and five genes ( rpoH, prbP, phrR, rirA, and lsrB) involved in oxidative stress response. We demonstrated that rem, lsrB, and visNR of CLas can complement the corresponding mutants of S. meliloti in their reduced motility. We further investigated traits controlled by Rem in S. meliloti and CLas using RNA sequencing analyses of rem mutant versus complementation strains with remSmc or remLas. Transcriptomic analysis showed that RemLas significantly regulates the expression of genes in S. meliloti, which was used to infer its regulation of CLas genes by identification of homologous genes. We found that Rem is involved in regulating motility, chemotaxis, transporters, and oxidative phosphorylation in S. meliloti and regulating flagellar and transporter genes in CLas. Among the 39 putative RemLas-regulated genes in CLas, 16 contain the Rem-binding motif, including 10 genes involved in flagellar assembly. Taken together, this study offers valuable insights regarding CLas regulatory genes, with many of them involved in regulating motility and oxidative stress response. The regulation of flagellar genes by Rem in CLas unravels critical information regarding motility in CLas infection of hosts.

Diaphorina citri (Hemiptera: Liviidae) is the main vector for the bacterium ‘ Candidatus Liberibacter asiaticus’, which is associated with citrus greening, also known as Huanglongbing. D. citri transmits ‘ Ca. L. asiaticus’ during its feeding on citrus phloem sap. Transmission occurs in a circulative, propagative, and persistent manner. ‘ Ca. L. asiaticus’ has a small genome (1.2 Mb). Therefore, it acquires most of its nutrients and energetic nucleotides from its hosts. The objective of this study was to assess the effect of ‘ Ca. L. asiaticus’ infection on the level of the free fatty acids in its vector. The fatty acids were extracted from adult D. citri using ethyl acetate, derivatized with boron trifluoride-methanol, and analyzed using gas chromatography-mass spectrometry. Nine fatty acids were identified in the D. citri extracts. Oleic acid was the most predominant fatty acid, followed by stearic and palmitic acid, whereas the rest of the fatty acids were present in low amounts. In general, the levels of the detected fatty acids in ‘ Ca. L. asiaticus’-infected D. citri were lower than those found in healthy psyllids. Our findings showed that the reduction of fatty acids in ‘ Ca. L. asiaticus’-infected psyllids resulted from the higher activity of β-oxidation to generate acetyl-coenzyme A, which causes more production of ATP. Our results indicated that ‘ Ca. L. asiaticus’ may enhance the β-oxidation of fatty acids in its vector insect to fulfill its nutrient and energetic nucleotide requirements.

Objectives This study examined feline haemoplasmas ( Mycoplasma haemofelis , ‘ Candidatus Mycoplasma haemominutum’ [ C Mhm] and ‘ Candidatus Mycoplasma turicensis’) infecting Thai domestic cats, using the 16S and 23S rRNA genes as genetic markers. Methods Blood samples from 20 cats were obtained from a diagnostic laboratory and nucleic acids were extracted from each sample using a commercial kit. PCR targeting the 16S rRNA gene was used to screen haemoplasmas in the samples. Positive PCR samples were further sequenced using the 16S and 23S rRNA genes. The sequences from each genetic marker were analysed using Nucleotide BLAST, phylogeny and genetic network analyses. Results Among the 20 samples, five were infected with haemoplasmas. In the 16S rRNA gene sequencing, four sequences were assigned to C Mhm and the remaining sequence was likely to be a closely related species of C Mhm. In the 23S rRNA gene sequencing, four sequences from the same samples used for 16S rRNA gene sequencing were identified as C Mhm and one sequence could be a putative novel haemoplasma species closely related to C Mhm. Conclusions and relevance Only C Mhm and its closely related species were identified in this study. Although C Mhm has been recognised as a low-virulence parasite, cases of severe anaemia in cats infected with C Mhm have been found. Thus, such cases could be confirmed via the analysis of 16S and 23S rRNA genes. Furthermore, molecular detection and genetic analyses of feline haemoplasmas in additional cat blood samples should be conducted using PCR assay and DNA sequencing based on universal primers of 16S rRNA and 23S rRNA genes to enable more specific identification.

A novel, strictly anaerobic, slightly alkaliphilic, halotolerant, peptide- and amino acid-utilizing bacterial strain, SD1T, was isolated from a hypersaline lake in Western Australia. The strain stained Gram-negative and was a motile, spore-forming rod. The strain grew between 15 and 50 °C (optimum 40 °C), 1–15% w/v sodium chloride (optimum 5%) and pH 6.0–10.0 (optimum 9.0). Major fatty acids included anteiso-C15 : 0 (24.9%), C14 : 0 dimethyl acetyl (13.2%), anteiso-C15 : 0 dimethyl acetyl (11.5%) and iso-C15 : 0 (10.4%). The DNA G+C content was 30.3 mol%. The isolate did not grow using any tested sugars but grew well on arginine and glycine. It is capable of using elemental sulfur and thiosulfate as alternate electron acceptors, but not sulfide, sulfate, nitrate or nitrite. 16S rRNA gene similarity indicates that the isolate is related to Sporosalibacterium tautonense MRo-4T (94.33% identity). SD1T showed 76.18%–76.31% average nucleotide identity with other strains within the family Thermohalobacteraceae. Phylogenetics, based on the 16S rRNA gene and whole-genome sequence, as well as phenotypic analysis, differentiates the isolate from close neighbors. We propose that SD1T represents a novel species in a new genus, which we have named Dethiothermospora halolimnae gen. nov., sp. nov., type strain SD1T (DSM 117405T = TSD-443T). From this work, we also propose repositioning of the genus Anaeromonas to the family Thermohalobacteraceae.

ABSTRACT The Coxiellaceae bacterial family, within the order Legionellales, is defined by a collection of poorly characterized obligate intracellular bacteria. The zoonotic pathogen and causative agent of human Q fever, Coxiella burnetii , represents the best-characterized member of this family. Coxiellaceae establish replicative niches within diverse host cells and rely on their host for survival, making them challenging to isolate and cultivate within a laboratory setting. Here, we describe a new genus within the Coxiellaceae family that has been previously shown to infect economically significant freshwater crayfish. Using culture-independent long-read metagenomics, we reconstructed the complete genome of this novel organism and demonstrate that the species previously referred to as Candidatus Coxiella cheraxi represents a novel genus within this family, herein denoted Candidatus Paracoxiella cheracis . Interestingly, we demonstrate that Candidatus P. cheracis encodes a complete, putatively functional Dot/Icm type 4 secretion system that likely mediates the intracellular success of this pathogen. In silico analysis defined a unique repertoire of Dot/Icm effector proteins and highlighted homologs of several important C. burnetii effectors, including a homolog of CpeB that was demonstrated to be a Dot/Icm substrate in C. burnetii . IMPORTANCE Using long-read sequencing technology, we have uncovered the full genome sequence of Candidatus Paracoxiella cheracis , a pathogen of economic importance in aquaculture. Analysis of this sequence has revealed new insights into this novel member of the Coxiellaceae family, demonstrating that it represents a new genus within this poorly characterized family of intracellular organisms. Importantly, the genome sequence reveals invaluable information that will support diagnostics and potentially both preventative and treatment strategies within crayfish breeding facilities. Candidatus P. cheracis also represents a new member of Dot/Icm pathogens that rely on this system to establish an intracellular niche. Candidatus P. cheracis possesses a unique cohort of putative Dot/Icm substrates that constitute a collection of new eukaryotic cell biology-manipulating effector proteins.

Abstract A new distribution map is provided for Candidatus Liberibacter solanacearum (Liefting et al.) Alphaproteobacteria: Rhizobiales: Rhizobiaceae Hosts: Solanaceae, including potato (Solanum tuberosum), tomato (S. lycopersicum), pepper (Capsicum annuum), tobacco (Nicotiana tabacum), Solanum melongena (eggplant), also Apiaceae, including carrot (Daucus carota), celery (Apium graveolens) and parsnip (Pastinaca saliva).