Publications

4495

ABSTRACT Although the high-altitude limit for microbial survival in the Earth–atmosphere system has remained a scientific curiosity and topic of study, the ecological significance of long-distance microbial dispersal in the atmosphere has been perceived to have marginal relevance. Here, we report the characterization of novel plant pathogenic species of Curtobacterium that were isolated from samples collected at altitudes ranging from 1.5 to 29 km above sea level. Whole genome-based phylogenies of three strains, paired with plant challenge assays, indicate that each is a previously unrecognized species and causes disease on beans comparable to Curtobacterium flaccumfaciens . Isolates from the stratosphere (strain L6-1) and agricultural millet (G77) were identified to be the same species and designated as Curtobacterium aetherium sp. nov. C. aetherium displays high levels of tolerance to desiccation and UV radiation, which are stresses that increase in intensity with altitude. Back trajectory air mass analysis implied that the phytopathogens may have had an intercontinental source, but regional origins in the continental US cannot be excluded. The environmentally robust phytopathogens we have documented in the upper atmosphere provide new perspective on the role that high-altitude transport may play in microbial dispersal, gene flow, and the epidemiology of aerially dispersed plant disease. IMPORTANCE Enormous quantities and varieties of microorganisms are continually aerosolized and transported in the atmosphere, yet there is a limited understanding of the consequences to downwind ecosystems. While studying bacteria that survive extreme conditions at altitudes up to 29 km in the atmosphere, we discovered new species that have the capacity to cause disease in agriculturally relevant bean varieties. The hardiest isolate we characterized from the stratosphere is a member of the same species as an isolate from an agricultural source, which we have designated Curtobacterium aetherium . C. aetherium is a phytopathogen capable of enduring high-altitude and long-distance atmospheric transport while also possessing the potential to opportunistically infect crops in deposition locations.

Abstract Defining the minimal genetic requirements for cellular life remains a fundamental question in biology. Genomic exploration continually reveals novel microbial lineages, often exhibiting extreme genome reduction, particularly within symbiotic relationships. Here, we report the discovery ofCandidatusSukunaarchaeum mirabile, a novel archaeon with an unprecedentedly small genome of only 238 kbp —less than half the size of the smallest previously known archaeal genome— from a dinoflagellate-associated microbial community. Phylogenetic analyses place Sukunaarchaeum as a deeply branching lineage within the tree of Archaea, representing a novel major branch distinct from established phyla. Environmental sequence data indicate that sequences closely related to Sukunaarchaeum form a diverse and previously overlooked clade in microbial surveys. Its genome is profoundly stripped-down, lacking virtually all recognizable metabolic pathways, and primarily encoding the machinery for its replicative core: DNA replication, transcription, and translation. This suggests an unprecedented level of metabolic dependence on a host, a condition that challenges the functional distinctions between minimal cellular life and viruses. The discovery of Sukunaarchaeum pushes the conventional boundaries of cellular life and highlights the vast unexplored biological novelty within microbial interactions, suggesting that further exploration of symbiotic systems may reveal even more extraordinary life forms, reshaping our understanding of cellular evolution. Graphical Abstract

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.

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.

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.