Applied and Environmental Microbiology

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.

ABSTRACT Methanol, the simplest alcohol, has long been known to be a key energy and carbon source for soil and plant-associated bacteria and fungi and is increasingly recognized as an important substrate for marine bacteria. Lanthanide-dependent methanol dehydrogenases (encoded by the gene xoxF ) have been shown to be key catalysts for methylotrophy in many environments, yet the identity of the most transcriptionally active methylotrophs in open ocean waters (“Clade X”) has remained elusive. Here, we show that “Clade X” methylotrophs belong to the deep-branching alphaproteobacterial order TMED127, which we propose be renamed “Methylaequorales”: “methyl” for “methylotrophic metabolism” and “aequor” for “ocean surface,” as these bacteria are most transcriptionally active near the sea surface. TMED127/Methylaequorales are present in surface waters of tropical and subtropical oceans throughout the global ocean. They have small, streamlined genomes (~1.5 Mb) and appear to be obligate methylotrophs that use the serine cycle for carbon assimilation. They display a diel pattern of xoxF5 and glucose dehydrogenase ( gdh ) transcription, peaking in the late afternoon, in oligotrophic surface water of the Sargasso Sea. Several other highly transcribed genes of unknown function had no homologs outside of TMED127/Methylaequorales genomes. Our findings illuminate an overlooked marine methylotrophic bacterium and predict a diel cycle of methanol production in surface seawater by an unknown pathway. IMPORTANCE Methanol metabolism is increasingly recognized as an important process in the marine carbon cycle, yet the identity and metabolism of the microorganisms mediating methylotrophy in the open ocean have remained unknown. This study reveals that bacteria in the TMED127 order of Alphaproteobacteria, renamed here as “Methylaequorales,” abundantly transcribe the key gene for lanthanide-dependent methylotrophy in oligotrophic surface waters of the world’s oceans. TMED127/Methylaequorales likely require methanol as a carbon and energy source and display a diel pattern of transcription of key genes for methylotrophy that peaks in the late afternoon. These findings motivate future studies on the mechanisms of methanol production in surface seawater.

ABSTRACT Huanglongbing (HLB) is a devastating citrus disease associated with the gram-negative, phloem-limited, and unculturable bacterium “ Candidatus Liberibacter asiaticus ( C Las),” which is transmitted by the Asian citrus psyllid Diaphorina citri . Despite extensive research, effective, long-term, and sustainable solutions for managing HLB remain elusive. Oxytetracycline (OTC) is currently used as an emergency measure, but there is an urgent need for alternative compounds to complement or replace OTC. In this study, we identified amicoumacins, a class of antimicrobial compounds produced by the bacterium Bacillus safensis CB729 isolated from the citrus microbiome, and demonstrated their ability to suppress C Las. Genome mining of B. safensis CB729, combined with metabolomic analysis and bioassay-guided fractionation, revealed the presence of amicoumacins and related derivatives in fractions inhibitory to Liberibacter crescens , a culturable surrogate for C Las. We tested commercially available synthetic amicoumacins A and B, along with a B. safensis -derived amicoumacin mixture, against L. crescens and C Las. We determined the MICs of amicoumacin A (1.25 µg/mL) and amicoumacin B (10 µg/mL) against L. crescens . Furthermore, amicoumacin B and the amicoumacin mixture significantly reduced C Las populations in ex vivo citrus hairy root assays. This study highlights the potential of amicoumacins as a promising group of natural products for the management of HLB, offering valuable insights for the development of novel and sustainable disease control strategies. IMPORTANCE For two decades, the citrus industry has been severely impacted by Huanglongbing (HLB), a devastating disease caused by “ Candidatus Liberibacter asiaticus ( C Las)” and transmitted by the Asian citrus psyllid ( Diaphorina citri ). Despite extensive research, effective, long-term, and sustainable solutions remain unavailable for growers. Currently, medically relevant antibiotics, such as oxytetracycline (OTC), are used as an emergency response to combat HLB in Florida, the most affected citrus-producing state in the U.S. This underscores the urgent need for alternative treatments that can be used in rotation or as replacements for OTC. Here, we present amicoumacins, a group of bioactive secondary metabolites with antibiotic properties. We identified amicoumacin B and its derivatives from the culture broth of a Bacillus safensis isolate, native to citrus, and demonstrated their ability to inhibit Liberibacter spp. and reduce C Las populations in citrus tissue. This study highlights how microbial discovery can lead to the identification of antimicrobial compounds with potential applications in plant disease management.

ABSTRACT Species belonging to the genus Leptothrix are widely distributed in the environment and in activated sludge (AS) wastewater treatment plants (WWTPs). They are commonly found in iron-rich environments and reported to cause filamentous bulking in WWTPs. In this study, the diversity, distribution, and metabolic potential of the most prevalent Leptothrix spp. found in AS worldwide were studied. Our 16S rRNA amplicon survey showed that Leptothrix belongs to the general core community of AS worldwide, comprising 32 species with four species being most commonly found. Their taxonomic classification was re-evaluated based on both 16S rRNA gene and genome-based phylogenetic analysis showing that three of the most abundant “ Leptothrix” species represented species in three other genera, Rubrivivax , Ideonella , and the novel genus, Ca . Intricatilinea. New fluorescence in situ hybridization (FISH) probes revealed rod-shaped morphology for the novel Ca . Rubrivivax defluviihabitans and Ca . Ideonella esbjergensis , while filamentous morphology was found only for Ca . Intricatilinea gracilis. Analysis of high-quality metagenome-assembled genomes revealed metabolic potential for aerobic growth, fermentation, storage of intracellular polymers, partial denitrification, photosynthesis, and iron reduction. FISH in combination with Raman microspectroscopy confirmed the in situ presence of chlorophyll and carotenoids in Ca . Rubrivivax defluviihabitans and Ca . Intricatilinea gracilis. This study resolves the taxonomy of abundant but poorly classified “ Leptothrix” species, providing important insights into their diversity, morphology, and function in global AS wastewater treatment systems. IMPORTANCE The genus Leptothrix has been extensively studied and described since the 1880s, with six species currently described but with the majority uncultured and undescribed. Some species are assumed to have a filamentous morphology and can cause settling problems in wastewater treatment plants (WWTPs). Here, we revised the classification of the most abundant Leptothrix spp. present in WWTPs across the world, showing that most belong to other genera, such as Rubrivivax and Ideonella . Furthermore, most do not have a filamentous morphology and are not problematic in WWTPs as previously believed. Metabolic reconstruction, including some traits validated in situ by the application of new fluorescence in situ hybridization probes and Raman microspectroscopy, provided additional insights into their metabolism. The study has contributed to a better understanding of the diversity, morphology, and function of “ Leptothrix ,” which belong to the abundant core community across global activated sludge WWTPs.

ABSTRACT The archaeal mevalonate pathway is a recently discovered modified version of the eukaryotic mevalonate pathway. This pathway is widely conserved in archaea, except for some archaeal lineages possessing the eukaryotic or other modified mevalonate pathways. Although the pathway seems almost exclusive to the domain Archaea, the whole set of homologous genes of the pathway is found in the metagenome-assembled genome sequence of an uncultivated bacterium, Candidatus Promineifilum breve, of the phylum Chloroflexota. To prove the existence of the archaea-specific pathway in the domain Bacteria, we confirmed the activities of the enzymes specific to the pathway, phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, because only these two enzymes are absent in closely related Chloroflexota bacteria that possess a different type of modified mevalonate pathway. The activity of anhydromevalonate phosphate decarboxylase was evaluated by carotenoid production via the archaeal mevalonate pathway reconstituted in Escherichia coli cells, whereas that of phosphomevalonate dehydratase was confirmed by an in vitro assay using the recombinant enzyme after purification and iron-sulfur cluster reconstruction. Phylogenetic analyses of some mevalonate pathway-related enzymes suggest an evolutionary route for the archaeal mevalonate pathway in Candidatus P. breve, which probably involves horizontal gene transfer events. IMPORTANCE The recent discovery of various modified mevalonate pathways in microorganisms, such as archaea and Chloroflexota bacteria, has shed light on the complexity of the evolution of metabolic pathways, including those involved in primary metabolism. The fact that the archaeal mevalonate pathway, which is almost exclusive to the domain Archaea, exists in a Chloroflexota bacterium provides valuable insights into the molecular evolution of the mevalonate pathways and associated enzymes. Putative genes probably involved in the archaeal mevalonate pathway have also been found in the metagenome-assembled genomes of Chloroflexota bacteria. Such genes can contribute to metabolic engineering for the bioproduction of valuable isoprenoids because the archaeal mevalonate pathway is known to be an energy-saving metabolic pathway that consumes less ATP than other mevalonate pathways do.

Chlorinated ethenes are risk drivers at many contaminated sites, and current bioremediation efforts focus on organohalide-respiring Dehalococcoides mccartyi strains to achieve detoxification. We isolated and characterized the first non- Dehalococcoides bacterium, “ Candidatus Dehalogenimonas etheniformans” strain GP, capable of metabolic reductive dechlorination of TCE, all DCE isomers, and VC to environmentally benign ethene.