Publications

3805

ABSTRACT Filamentous Chloroflexota are abundant in activated sludge wastewater treatment plants (WWTPs) worldwide and are occasionally associated with poor solid-liquid separation or foaming, but most of the abundant lineages remain undescribed. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide, using high-quality metagenome-assembled genomes (MAGs) and 16S rRNA amplicon data from 740 Danish and global WWTPs. Many novel taxa were described, encompassing 4 families, 13 genera, and 29 novel species. These were widely distributed across most continents, influenced by factors such as climate zone and WWTP process design. Visualization by fluorescence in situ hybridization (FISH) confirmed their high abundances in many WWTPs based on the amplicon data and showed a filamentous morphology for nearly all species. Most formed thin and short trichomes integrated into the floc structure, unlikely to form the typical inter-floc bridging that hinders activated sludge floc settling. Metabolic reconstruction of 53 high-quality MAGs, representing most of the novel genera, offered further insights into their versatile metabolisms and suggested a primary role in carbon removal and involvement in nitrogen cycling. The presence of glycogen reserves, detected by FISH-Raman microspectroscopy, seemed widespread across the phylum, demonstrating that these bacteria likely utilize glycogen as energy storage to survive periods with limited resources. This study gives a broad overview of the Chloroflexota community in global activated sludge WWTPs and improves our understanding of their roles in these engineered ecosystems. IMPORTANCE Chloroflexota are often abundant members of the biomass in wastewater treatment plants (WWTPs) worldwide, typically with a filamentous morphology, forming the backbones of the activated sludge floc. However, their overgrowth can often cause operational issues connected to poor settling or foaming, impairing effluent quality and increasing operational costs. Despite their importance, few Chloroflexota genera have been characterized so far. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide and an in-depth characterization of their morphology, phylogeny, and ecophysiology, obtaining a broad understanding of their ecological role in activated sludge.

This study presents the assembly and comparative genomic analysis of luminous Photobacterium strains isolated from the light organs of 12 fish species using Oxford Nanopore Technologies (ONT) sequencing. The majority of assemblies achieved chromosome-level continuity, consisting of one large (>3 Mbp) and one small (~1.5 Mbp) contig, with near complete BUSCO scores along with varying plasmid sequences. Leveraging this dataset, this study significantly expanded the available genomes for P. leiognathi and its subspecies P. ‘mandapamensis’, enabling a comparative genomic analysis between the two lineages. An analysis of the large and small chromosomes unveiled distinct patterns of core and accessory genes, with a larger fraction of the core genes residing on the large chromosome, supporting the hypothesis of secondary chromosome evolution from megaplasmids in Vibrionaceae. In addition, we discovered a proposed new species, Photobacterium acropomis sp. nov., isolated from an acropomatid host, with an average nucleotide identify (ANI) of 93 % compared to the P. leiognathi and P. ‘mandapamensis’ strains. A comparison of the P. leiognathi and P. ‘mandapamensis’ lineages revealed minimal differences in gene content, yet highlighted the former’s larger genome size and potential for horizontal gene transfer. An investigation of the lux-rib operon, responsible for light production, indicated congruence between the presence of luxF and host family, challenging its role in differentiating P. ‘mandapamensis’ from P. leiognathi . Further insights were derived from the identification of metabolic differences, such as the presence of the NADH:quinone oxidoreductase respiratory complex I in P. leiognathi as well as variations in the type II secretion system (T2S) genes between the lineages, potentially impacting protein secretion and symbiosis. In summary, this study advances our understanding of Photobacterium genome evolution, highlighting subtle differences between closely related lineages, specifically P. leiognathi and P. ‘mandapamensis’. These findings highlight the benefit of long read sequencing for bacterial genome assembly and pangenome analysis and provide a foundation for exploring early bacterial speciation processes of these facultative light organ symbionts.

The taxonomic relationships of 10 strains isolated from seaweeds collected from two beaches in Republic of Korea were studied by sequencing and analyses of 16S rRNA genes and whole genomes. For the construction of a more reliable and robust 16S rRNA gene phylogeny, the authentic and nearly complete 16S rRNA gene sequences of all the Microbacterium type strains were selected through pairwise comparison of the sequences contained in several public databases including the List of Prokaryotic names with Standing in Nomenclature (LPSN). The clustering of the ten study strains into five distinct groups was apparent in this single gene-based phylogenetic tree. In addition, the 16S rRNA gene sequences of a few type strains were shown to be incorrectly listed in LPSN. An overall phylogenomic clustering of the genus Microbacterium was performed with a total of 113 genomes by core genome analysis. As a result, nine major (≥ three type strains) and eight minor (two type strains) clusters were defined mostly at gene support index of 92 and mean intra-cluster OrthoANIu of >80.00%. All of the study strains were assigned to a Microbacterium liquefaciens clade and distributed further into four subclusters in the core genome-based phylogenetic tree. In vitro phenotypic assays for physiological, biochemical, and chemotaxonomic characteristics were also carried out with the ten study strains and seven closely related type strains. Comparison of the overall genomic relatedness indices (OGRI) including OrthoANIu and digital DNA–DNA hybridization supported that the study strains constituted four new species of the genus Microbacterium. In addition, some Microbacterium type strains were reclassified as members of preexisting species. Moreover, some of them were embedded in a new genus of the family Microbacteriaceae based on their distinct separation in the core genome-based phylogenetic tree and amino acid identity matrices. Based on the results here, four new species, namely, Microbacterium aurugineum sp. nov., Microbacterium croceum sp. nov., Microbacterium galbinum sp. nov., and Microbacterium sufflavum sp. nov., are described, along with the proposal of Paramicrobacterium gen. nov. containing five reclassified Microbacterium species from the “Microbacterium agarici clade”, with Paramicrobacterium agarici gen. nov., comb. nov. as the type species.

‘Candidatus Liberibacter solanacearum’ (Lso) is a plant pathogenic bacterium transmitted by psyllids that causes significant agricultural damage. Several Lso haplotypes have been reported. Among them, LsoA and LsoB are transmitted by the potato psyllid Bactericera cockerelli and infect solanaceous crops, and LsoD is transmitted by the carrot psyllid B. trigonica and infects apiaceous crops. Several studies evaluated the transmission of these haplotypes by adult psyllids. However, fewer data are available on the transmission of different Lso haplotypes by psyllid nymphs. In this study, we investigated the transmission of these three haplotypes by psyllid nymphs to expand our basic understanding of Lso transmission. Specifically, the objective was to determine if the haplotypes differed in their transmission rates by nymphs and if LsoA and LsoB accumulated at different rates in the guts of nymphs as it occurs in adults. First, we quantified LsoA and LsoB titers in the guts of third- and fifth-instar potato psyllid nymphs. We found similar LsoA titers in the two nymphal stages, while LsoB titer was lower in the gut of the third-instar nymphs compared to fifth-instar nymphs. Second, we assessed the transmission efficiency of LsoA and LsoB by third-instar nymphs to tomato plants, revealing that LsoA was transmitted earlier and with higher efficiency than LsoB. Finally, we examined the transmission of LsoD by carrot psyllid nymphs to celery plants and demonstrated an age-related difference in the transmission rate. These findings provide valuable insights into the transmission dynamics of different Lso haplotypes by nymphal vectors, shedding light on their epidemiology and interactions with their psyllid vectors.

This study aimed to evaluate the efficacy of biotic inducers for the management of phyllody disease in sesame, as well as in analyzing the defense enzyme activity. The field trial experiments were conducted with the biotic inducers [salicylic acid (SA), methyl jasmonate (MeJA), and beta amino butyric acid (BABA)] in individually (50 ppm, 100 ppm and 150 ppm) and in combination. Concerning biotic inducers, SA 50 / 100 ppm pre-seed treatment and exogenous application at 30th, 45th and 60th DAS resulted in no/less phyllody disease incidence and added significant value by increasing defense enzyme activity (PO, PPO, PAL and SOD).

The bacterial strain WB46 was isolated from the rhizosphere of willow plants (Salix purpurea L.) growing in soil contaminated with petroleum hydrocarbons. The strain was subjected to whole-genome shotgun sequencing using Illumina HiSeq. Its draft genome is 7.15 Mb, with a 69.55% GC content, containing 6387 protein-coding genes and 51 tRNA and 15 rRNA sequences. The quality and reliability of the genome were assessed using CheckM, attaining an estimated genome completeness of 98.75% and an estimated contamination of 1.68%. These results indicate a high-quality genome (>95%) and low contamination (<5%). Many of these genes are responsible for petroleum hydrocarbon degradation, such as alkane 1-monooxygenase (alkB) and naphthalene dioxygenase (ndo). 16S rRNA gene analysis, including in silico DNA–DNA hybridization (DDH) and average nucleotide identity (ANI), showed that strain WB46 belongs to the genus Nocardia, and the most closely related species is Nocardia asteroides. The strain WB46 showed a distance of 63.4% and sequence identity of 88.63%, respectively. These values fall below the threshold levels of 70% and 95%, respectively, suggesting that the strain WB46 is a new species. We propose the name of Nocardia canadensis sp. nov. for this new species. Interestingly, the sequence divergence of the 16S rRNA gene showed that the divergence only occurred in the V2 region. Therefore, the conventional V3–V4, V5–V7, or V8–V9 targeting metabarcoding, among others, would not be able to assess the diversity related to this new species.