Frontiers in Microbiology

The Clostridium estertheticum complex (CEC) consists of closely related bacterial species that are mostly isolated from meat processing environment. Genome mining studies have recently established CEC as a source of class I bacteriocins. However, up until now, class II bacteriocins have not been reported from CEC. In the present study, we determined the presence of class II bacteriocin biosynthetic clusters in 33 CEC genomes through genome mining followed by bacteriocin production through heterologous expression in Escherichia coli . Six biosynthetic gene clusters belonging to class IIa ( n  = 1), IIb ( n  = 2), IId ( n  = 2) and an undefined class containing three precursor peptides were identified in six different CEC strains. Using molecular biology, we developed dedicated expression vectors for the class IIa bacteriocin, clesteriocin A. Its precursor peptide, CleA, and protease, CleB150, were initially expressed in insoluble form in E. coli . Through a systematic analysis of suitable solubility enhancing protein tags, both CleA and CleB150 were expressed in significant amounts of soluble fractions using tandem tags derived from Small Ubiquitin-like Modifier, superfolder Green fluorescent protein, Maltose binding protein and N-utilization substance. Mature clesteriocin A was obtained following in vitro maturation reactions between the tagged CleA and CleB150. The novel bacteriocin displayed antimicrobial activity against Listeria monocytogenes , which was mediated by the mptC gene. By combining genome mining and molecular biology, we have shown CEC is a source of novel class II bacteriocins that have potential for application as food biopreservatives.

“ Candidatus Liberibacter asiaticus” (CLas) is an uncultivable α -proteobacterium causing the most destructive and currently incurable citrus disease, Huanglongbing (HLB). The transcription factors (TFs) of CLas are involved in various biological processes. However, the functions of most TFs remain unverified. BolA is reported to be an important transcriptional regulator related to bacterial growth and virulence. Here, the role of BolA in CLas was investigated using gene deletion and complementation assays in the heterologous host Sinorhizobium meliloti ( Sme ). The results showed that BolA CLas and BolA Sme are similar in sequence and transcriptional regulation. BolA positively regulates biofilm formation—evidenced by the significant downregulation of a key gene ( cyaA ) in the mutant (Δ BolA Sme ), without affecting bacterial growth. The upregulation of 16 differentially expressed genes (DEGs) related to flagellar assembly indicated that BolA negatively regulates CLas motility. BolA deletion also led to the downregulation of ABC transporters (15 DEGs) and lipid metabolism genes (13 DEGs), correlating with reduced stress tolerance. Furthermore, BolA CLas is involved in modulating heme metabolism, as well as protein export, folding, sorting, and degradation. Finally, in vivo screening identified two compounds as BolA inhibitors, which significantly reduced CLas titer in infected periwinkle leaves. Taken together, this study constitutes a relevant step toward the understanding of CLas virulence by demonstrating that BolA is a key TF involved in biofilm formation, stress response, motility, and bacterial physiology, thereby presenting a potential target for disease control.

Polyphosphate-accumulating organisms (PAOs) play a crucial role in enhanced biological phosphorus removal (EBPR) processes. In addition to biosynthesis, they rely on phosphate for energy generation. However, Candidatus Accumulibacter, a model PAO, has been shown to adapt to low phosphate conditions by switching to a glycogen-accumulating metabolism (GAM), with variable success across genus members and experiments. This study aimed to explore the metabolic shift of several Accumulibacter species subjected to low-phosphate concentration in different operating conditions using metatranscriptomics analysis. Furthermore, the study enabled a comparison of the transcriptomic profiles of Accumulibacter with those of Propionivibrio , a glycogen-accumulating organism typically found in EBPR plants. Two sequencing batch reactors were operated with different carbon sources to enrich for different populations of Accumulibacter . After decreasing the influent phosphate concentration, carbon removal performance was maintained while anaerobic phosphate release dropped dramatically, suggesting a shift from a phosphate-accumulating to a glycogen-accumulating metabolism. Analysis of metatranscriptomics data indicated that Accumulibacter regalis (type I) and Propionivibrio aalborgensis remained the most abundant species after the phosphate decrease in the reactor with acetate-propionate and allylthiourea, while Accumulibacter delftensis (type I) and Accumulibacter phosphatis (type II) remained active in the reactor with acetate-glucose and no allylthiourea. Transcription of the genes from the ethylmalonyl-CoA pathway involved in the production of propionyl-CoA and regulation of the anaerobic redox balance was enhanced under low-phosphate conditions, especially for type I Accumulibacter . Conversely, the transcription of the methylmalonyl-CoA pathway was enhanced under low-phosphate conditions in Propionivibrio and type II Accumulibacter .

IntroductionEndosymbiotic bacteria show diverse strategies to manipulate host reproduction for their survival in nature. The diversity of symbionts infecting hematophagous insects and their impact on host ecology could be crucial for developing effective control measures of disease-transmitting vectors. Sand flies are a group of small insects, with some species serving as vectors for leishmaniasis, bartonellosis, and arboviral diseases. Sergentomyia squamirostris is the only known species of sand flies found on the main islands of Japan. Although no medical implications exist for S. squamirostris, we conducted whole-genome sequencing to explore its potential relevance.MethodsWe conducted whole-genome sequencing and de novo assembly of S. squamirostris using genomic DNA isolated from a single field-collected female insect sample. During this attempt, we incidentally obtained closed genomes of two new bacteria, wSSQ and RiSSQ, belonging to Wolbachia and Candidatus Tisiphia, respectively. We then investigated infection rates of both bacteria in two natural populations of S. squamirostris in Japan.ResultsPhylogenetic analysis indicated that wSSQ and RiSSQ belonged to Wolbachia and Ca. Tisiphia, respectively. Ca. Tisiphia is also known as “Torix Rickettsia,” which is considered a large group of endosymbionts of invertebrates with no known pathogenicity to humans and animals. NGS read depths for both wSSQ and RiSSQ genomes were found to be high coverages, indicating that these bacteria are S. squamirostris endosymbionts. The infection rates of wSSQ and RiSSQ in the wild population of S. squamirostris varied in the two different localities in Japan, and co-infection with both bacteria was commonly seen. wSSQ was detected in both sexes of S. squamirostris, whereas RiSSQ was detected only in female sand flies.ConclusionCa. Tisiphia has recently been recognized as an underexplored endosymbiont with a widespread presence in invertebrates, including disease vectors. RiSSQ represents the first complete genomic information resource of Ca. Tisiphia infecting sand flies. Further research is needed to understand potential interactions between its host and other endosymbionts, as well as to explore the potential implications of disease control in the future.

Marine heterotrophic bacteria in coastal waters respond to the influx of carbon from natural and anthropogenic sources. We identified two nearly identical, (99.9% average nucleotide identity; 100% amino acid identity; same DNA G + C content of 52.3 mol%) high-quality (≥99% CheckM completeness and ≤ 1.3% contamination) draft metagenome-assembled genomes (MAGs; SJ0813 and SJ0972) from seawater microbiomes of a southern island of Singapore that is in a protected marine park. The MAGs were only assigned to the Cellvibrionaceae family according to Genome Taxonomy Database. Overall genome related indices to Pseudomaricurvus alkylphenolicus KU41GT as the closest phylogenetic relative revealed no more than 70.45% average nucleotide identity (ANIcutoff < 95%), below the 50% percentage of conserved proteins (POCPcutoff = 43.54%) for genera cutoff and low digital DNA–DNA hybridization values (DDH = 20.6 and 20.8%). The major respiratory quinone is predicted to be ubiquinone-9 from the annotation of 3-demethylubiquinone-9 3-methyltransferase (ubiG, K00568) involved in the last step of the ubiquinone biosynthesis pathway (M00117), which differed from the ubiquinone-8 utilized by known members of Cellvibrionaceae. Both MAGs contained a complete pathway for dissimilatory nitrate reduction to ammonia, which increases bioavailability of nitrogen in seawater. An identical choline dehydrogenase found in both MAGs have a low amino-acid identity (≤64.47%) compared to existing GMC family oxidoreductases, expanding on the diversity of this family of enzymes. The MAGs meet nearly all the minimum requirements but lack a 16S rRNA gene of sufficient length required for the proposed novel genus and species under SeqCode. Nevertheless, phylogenetic trees based on core-genome and RpoB as an alternative phylogenetic marker are congruent with the taxon standing as a monophyletic clade to other taxa of the order Cellvibrionales. Taken together, the MAGs (SJ0813 and SJ0972) represent an uncultured, undescribed genus and species in which we tentatively propose the name Candidatus Pelagadaptatus aseana gen. nov., sp. nov. and strain SJ0813TS (=BAABNI000000000.1TS) as type sequence. Phylogenetic inference from core-genome and RpoB phylogenetic trees placed Umboniibacter marinipuniceus KMM 3891T outside Cellvibrionaceae. We, therefore, propose the transfer of the genus Umboniibacter from the family Cellvibrionaceae to a new family Umboniibacteraceae according to the International Code of Nomenclature of Prokaryotes.

The anammox bacteria “Candidatus Kuenenia stuttgartiensis” (Ca. Kuenenia) are able to gain energy by combining ammonium and nitrite to produce nitrogen gas, which is an ecologically and technically significant activity process. In this reaction, nitric oxide serves as a recognized intermediate in the reduction of nitrite, which is subsequently combined with ammonium to produce hydrazine. However, the enzyme that converts nitrite to nitric oxide remains elusive. In this study, we investigated the nitrite-reducing activity in “Ca. Kuenenia stuttgartiensis” strain CSTR1 to identify candidates for such an enzyme. An optimized in vitro assay was established to measure nitrite-reducing activities, with which we followed the activity in protein fractions obtained from various fractionation methods. Separation of the cell extract of strain CSTR1 with size exclusion chromatography yielded active fractions corresponding to a molecular size range of 150–200 kDa. Several proteins coeluted with the nitrite-reducing activity, including the hydroxylamine dehydrogenase HOX, an NADP-dependent isopropanol dehydrogenase (Adh), an electron-transfer 4Fe-4S subunit protein (Fcp), and a nitric oxide detoxifying flavorubredoxin (NorVW). However, further separation of the cell extract with anion exchange chromatography, resulted in much lower activity yields, and activities were distributed among several fractions. In addition, fractionation of cell extracts using ultracentrifugation and ultrafiltration linked the activity to HOX, but could not exclude the involvement of other proteins in the activity. Overall, our results suggest that the molecular mechanism for nitrite reduction in “Ca. Kuenenia” strains is more complex than that currently described in the literature. Nitrite reduction appears to be strongly associated with HOX but may additionally require the participation of other proteins.

The class Limnochordia harbors a single cultivated member, the mesophilic Limnochorda pilosa, which was isolated from a meromictic lake. Despite numerous molecular signatures reported in various ecosystems, the ecophysiological versatility of this deeply branched lineage of Firmicutes (Bacillota) remains poorly understood. The objective of this study was to use targeted cultivation, based on metagenome-assembled genomes from a deep terrestrial aquifer in Western Siberia, to isolate two new thermophilic members of the class. These isolates, described as Geochorda subterranea gen. nov. sp. nov. and Carboxydochorda subterranea gen. nov. sp. nov. within the Geochordaceae fam. nov., were capable of both anaerobic and aerobic respiration using fumarate and O2, respectively, with simple sugars as electron donors. The cultivated Geochordaceae have demonstrated fermentative growth and degradation of various polymers, including starch, maltose, maltodextrin, xylan, and chitin. The carboxydotrophic C. subterranea sp. nov. exhibited autotrophic growth via the Calvin–Benson–Bassham cycle, using CO, H2, and formate as electron donors and O2 as an electron acceptor, adding metabolic flexibility to the bacterium in the nutrient-depleted “deep biosphere” and supporting the possibility of aerobic metabolism in the deep subsurface. The broad physiological potential deciphered from physiological experiments and comparative genomic data explains the widespread distribution of uncultivated members of the class Limnochordia in various ecosystems, where they can oxidize complex organic substrates through both aerobic and anaerobic respiration, as well as pursue a chemolithotrophic lifestyle through the oxidation of H2 or CO.

Hadal zones account for the deepest 45% of the oceanic depth range and play an important role in ocean biogeochemical cycles. As the least-explored aquatic habitat on earth, hadal ecosystems contain a vast diversity of so far uncultured microorganisms that cannot be grown on conventional laboratory culture media. Therefore, it has been difficult to gain a true understanding of the detailed metabolic characteristics and ecological functions of those difficult-to-culture microorganisms in hadal environments. In this study, a novel anaerobic bacterial strain, MT110T, was isolated from a hadal sediment–water interface sample of the Mariana Trench at 10,890 m. The level of 16S rRNA gene sequence similarity and percentage of conserved proteins between strain MT110T and the closest relatives, Anaerovorax odorimutans DSM 5092T (94.9 and 46.6%) and Aminipila butyrica DSM 103574T (94.4 and 46.7%), indicated that strain MT110T exhibits sufficient molecular differences for genus-level delineation. Phylogenetic analyses based on both 16S rRNA gene and genome sequences showed that strain MT110T formed an independent monophyletic branch within the family Anaerovoracaceae. The combined evidence showed that strain MT110T represents a novel species of a novel genus, proposed as Anoxybacterium hadale gen. nov. sp. nov. (type strain MT110T = KCTC 15922T = MCCC 1K04061T), which represents a previously uncultured lineage of the class Clostridia. Physiologically, no tested organic matter could be used as sole carbon source by strain MT110T. Genomic analysis showed that MT110T had the potential capacity of utilizing various carbon sources, but the pathways of sulfur reduction were largely incomplete. Our experiments further revealed that cysteine is one of the essential nutrients for the survival of strain MT110T, and cannot be replaced by sulfite, leucine, or taurine. This result suggests that organic sulfur compounds might play an important role in metabolism and growth of the family Anaerovoracaceae and could be one of the key factors affecting the cultivation of the uncultured microbes. Our study brings a new perspective to the role of dissolved organic sulfur in hadal ecosystems and also provides valuable information for optimizing the conditions of isolating related microbial taxa from the hadal environment.

This study delves into the evolutionary history of Anaerolineaceae, a diverse bacterial family within the Chloroflexota phylum. Employing a multi-faceted approach, including phylogenetic analyses, genomic comparisons, and exploration of adaptive features, the research unveils novel insights into the family’s taxonomy and evolutionary dynamics. The investigation employs metagenome-assembled genomes (MAGs), emphasizing their prevalence in anaerobic environments. Notably, a novel mesophilic lineage, tentatively named Mesolinea, emerges within Anaerolineaceae, showcasing a distinctive genomic profile and apparent adaptation to a mesophilic lifestyle. The comprehensive genomic analyses shed light on the family’s complex evolutionary patterns, including the conservation of key operons in thermophiles, providing a foundation for understanding the diverse ecological roles and adaptive strategies of Anaerolineaceae members.