Publications

4358

ABSTRACT We report the draft genomes of five Clostridium isolates from soil and agricultural by-products, four of which are proposed as Candidatus species. Members of the genus Clostridium are of significant industrial interest, and the availability of their genome sequences facilitates the understanding and exploration of their functional potential.

Bacterial strain SL12-8T was characterized and isolated from the skin microbiota of Pelophylax perezi, the Perez’s frog. Strain SL12-8T stained Gram-negative and formed rod-shaped cells that grew optimally at 25 °C and pH 7.0–7.5. The G+C content of the DNA was 66.2 mol%. Ubiquinone 8 was the respiratory quinone identified in the studied strain and the most closely related taxon. The major fatty acids were summed in feature 3 and summed in feature 8 and C16:0, representing 84% of the total fatty acids. Phylogenetic analyses based on the 16S rRNA gene sequence placed strain SL12-8T within the order Burkholderiales in a distinct lineage. The 16S rRNA gene sequence similarities of strain SL12-8T to that of Rubrivivax albus, Scleromatobacter humisilvae, Piscinibacter aquaticus, Azohydromonas caseinilytica and Aquincola agrisoli were 94.41, 94.08, 93.72, 93.72 and 93.64%, respectively. The draft genome sequence of strain SL12-8T comprises 3,115,197 bases with a 313-fold mapped genome coverage. The assembled genome consists of 53 large contigs with more than 500 bp, and the genome encodes 2,814 putative coding sequences. The analysis of the available genomes from the closest genera showed 124 core genes that reveal a novel genus-level clade including the strain SL12-8T. Analysis of the SL12-8T genome revealed the presence of the beta-lactone and terpene biosynthetic gene clusters. The phylogenomic, phylogenetic, phenotypic and chemotaxonomic data showed that strain SL12-8T (=UCCCB 131T=CECT 30762T) represents the type of a novel species and genus, for which we propose the name Amphibiibacter pelophylacis gen. nov., sp. nov.

Candidatus Liberibacter asiaticus (CLas) is a host-dependent obligate bacteria that causes citrus greening disease in citrus plants. The prolonged association of CLas with the host led to the loss of essential metabolic genes, including transaldolase in the pentose phosphate pathway, hindering the development of axenic culture in the host-free environment. In this study, in silico functional annotation of pyrophosphate-fructose 6-phosphate 1-phosphotransferase (PFP) protein was performed by utilizing sequence analysis and molecular modeling, revealing its potential role in bypassing the loss of transaldolase. The findings suggest how PFP and fructose bisphosphate aldolase (FBPA) can reconstruct the disrupted central carbon metabolism and help CLas in ATP production. The molecular modeling of PFP showed binding affinity towards fructose 6-phosphate and sedoheptulose 7-phosphate, showing its dual function. The sequence-based analysis and metabolic modeling study also highlight the presence of other alternate enzymes or transport proteins in CLas, elucidating the broader metabolic resilience. This is the first report to provide the structural insights of PFP in compensating for the lost metabolic function and survival strategies adopted by CLas despite the restrained metabolic capability.

Lethal wilt (LW), also known as “Marchitez Letal (ML)” in Colombia, is an endemic disease affecting oil palms (Elaeis guineensis Jacq.) and is a leading cause of crop loss. The disease is characterized by the drying of leaflets from the tip to the base, primarily impacting the lower third of the plant and progressively moving upward. This progression leads to physiological disturbances, including necrosis at the tips of immature inflorescence bracts (spines) and the detachment of bunch fruits, ultimately causing wilting. As a phytosanitary measure, infected palms are eradicated to prevent further spread of the disease. The primary goal of this research was to identify the bacteria associated with LW and to validate a molecular detection method. A 16S amplicon-based analysis was employed to identify and compare the microbial diversity in LW-affected palm tissues with those of healthy plants. Among the 16 OTUs corresponding to different bacterial genera found in all LW samples, taxonomic classification and symptomatology suggested that the bacteria closely associated with LW belong to the genus Candidatus Liberibacter. Further phylogenetic analysis indicated that these bacteria are part of the Rhizobiaceae family, grouping closely with other species of the genus Candidatus. Liberibacter. The concentration of the pathogen in different oil palm tissues was determined using droplet digital PCR (ddPCR) and quantitative PCR (qPCR), expressed in copies/µL in the LW samples. This study represents the first report of ‘Candidatus Liberibacter sp’. being associated with lethal wilt in oil palms of the Arecaceae family in Colombia. The findings from this research have the potential to contribute significantly to the development of effective management strategies to prevent crop losses.

The strain TOYAMA8T is a deep-sea alkaliphilic cellulolytic bacterium isolated from a slurry-adhered epiphytic site of Megalodicopia hians. Cells of this strain are Gram-negative, aerobic, curved rods or spirilla, motile with monopolar flagella, and grow on cellulose as the sole carbon source. Compared to other closely related species, this bacterium is characterized by a large number of cellulase genes. Strain TOYAMA8T showed alkaliphilic growth within the pH range 7.5–9.0. The major cellular fatty acids were C18 : 1 ω7, C14 : 0, C16 : 0 and C16 : 1 ω7. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, unidentified phospholipids and aminolipids. A major respiratory lipoquinone was Q-9. Phylogenomic analysis using the 16S rRNA gene and whole-genome sequence data showed that the strain is related to the families Gynuellaceae, Saccharospirillaceae and Natronospirillaceae. The values of 16S rRNA gene sequence similarity, amino acid identity and percentage of conserved proteins between the strain TOYAMA8T and related species were low, with maximum values of 90.6, 48.1 and 34.6%, respectively. These results, together with differences in phenotypic and biochemical characteristics, indicate that the new isolate TOYAMA8T represents a novel genus and species, for which the name Marinicellulosiphila megalodicopiae gen. nov., sp. nov., is proposed. The type strain is TOYAMA8T (JCM 31119T=DSM 114864T).

The Asian citrus psyllid (ACP) is the vector of ‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus greening or Huanglongbing (HLB), one of the most devastating citrus diseases worldwide. The citrus industry in Georgia (U.S.A.) is in the process of a rapid expansion, and based on experiences with HLB in Florida, there is great concern about the potential impacts of HLB on this emerging industry. Prior to 2023, ACP had been identified in residential citrus trees in isolated Georgia counties, but little to no testing of psyllids for CLas had occurred. However, in 2023, one individual psyllid collected from Chatham County was confirmed positive for CLas by PCR and sequencing. Furthermore, during 2023, ACP adults and nymphs were identified for the first time in a Georgia commercial citrus grove. The finding of ACP in a commercial planting represents a significant risk for CLas dissemination and thereby has the potential to stall the rapid expansion of Georgia’s citrus industry. In the coming years, surveillance and testing of ACP from commercial groves will be essential for the early detection and management of HLB and its vector to reduce HLB spread within Georgia’s commercial groves.