Thermostability optimization of the aspartate/alanine exchange transporter from Tetragenococcus halophilus.

Aspartate/alanine exchange transporter (AspT) is a secondary transporter isolated from the lactic acid bacterium Tetragenococcus halophilus D10 strain. This transporter cooperates with aspartate decarboxylase to produce proton-motive force through decarboxylative phosphorylation. A method that successfully analyzes the AspT mechanism could serve as a prototype for elucidating the substrate transport mechanism of other exchange transporters; therefore, the purpose of this study was to search for conditions that improve the thermal stability of AspT for 3D structure analysis. We used the fluorescence size-exclusion chromatography-based thermostability assay to evaluate conditions that contribute to AspT stability. We found that the AspT thermostability was enhanced at pH 5.0 to 6.0 and in the presence of Na+ and Li+. Pyridoxal phosphate, a coenzyme of aspartate decarboxylase, also had a thermostabilizing effect on AspT. Under the conditions obtained from these results, it was possible to increase the temperature at which 50% of dimer AspT remained by 14°C. We expect these conditions to provide useful information for future structural analysis of AspT.

Real-time monitoring of mycelial growth in liquid culture using hyphal dispersion mutant of Aspergillus fumigatus.

Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.

Thermostability optimization of the aspartate/alanine exchange transporter from Tetragenococcus halophilus.

Aspartate/alanine exchange transporter (AspT) is a secondary transporter isolated from the lactic acid bacterium Tetragenococcus halophilus D10 strain. This transporter cooperates with aspartate decarboxylase to produce proton-motive force through decarboxylative phosphorylation. A method that successfully analyzes the AspT mechanism could serve as a prototype for elucidating the substrate transport mechanism of other exchange transporters; therefore, the purpose of this study was to search for conditions that improve the thermal stability of AspT for 3D structure analysis. We used the fluorescence size-exclusion chromatography-based thermostability assay to evaluate conditions that contribute to AspT stability. We found that the AspT thermostability was enhanced at pH 5.0 to 6.0 and in the presence of Na+ and Li+. Pyridoxal phosphate, a coenzyme of aspartate decarboxylase, also had a thermostabilizing effect on AspT. Under the conditions obtained from these results, it was possible to increase the temperature at which 50% of dimer AspT remained by 14°C. We expect these conditions to provide useful information for future structural analysis of AspT.

Involvement of ionic interactions in self-assembly and resultant rodlet formation of class I hydrophobin RolA from Aspergillus oryzae.

Hydrophobins are small amphiphilic proteins that are conserved in filamentous fungi. They localized on the conidial surface to make it hydrophobic, which contributes to conidial dispersal in the air, and helps fungi to infect plants and mammals and degrade polymers. Hydrophobins self-assemble and undergo structural transition from the amorphous state to the rodlet (rod-like multimeric structure) state. However, it remains unclear whether the amorphous or rodlet state is biologically functional and what external factors regulate state transition. In this study, we analyzed the self-assembly of hydrophobin RolA of Aspergillus oryzae in detail and identified factors regulating this process. Using atomic force microscopy, we observed RolA rodlet formation over time, and determined "rodlet elongation rate" and "rodlet formation frequency." Changes in these kinetic parameters in response to pH and salt concentration suggest that RolA rodlet formation is regulated by the strength of ionic interactions between RolA molecules.

Novel transporter screening technology for chemical production by microbial fermentation.

In the fermentative production of compounds by using microorganisms, control of the transporter activity responsible for substrate uptake and product efflux, in addition to intracellular metabolic modification, is important from a productivity perspective. However, there has been little progress in analyses of the functions of microbial membrane transporters, and because of the difficulty in finding transporters that transport target compounds, only a few transporters have been put to practical use. Here, we constructed a Corynebacterium glutamicum-derived transporter expression library (CgTP-Express library) with the fusion partner gene mstX and used a peptide-feeding method with the dipeptide L-Ala-L-Ala to search for alanine exporters in the library. Among 39 genes in the library, five candidate alanine exporters (NCgl2533, NCgl2683, NCgl0986, NCgl0453, and NCgl0929) were found; expression of NCgl2533 increased the alanine concentration in cell culture. The CgTP-Express library was thus effective for finding a new transporter candidate.

Conformational transition induced in the aspartate:alanine antiporter by L-Ala binding.

An aspartate:alanine antiporter (AspT) from the lactic acid bacterium Tetragenococcus halophilus catalyzes the electrogenic aspartate1-:alanine0 exchange reaction. Our previous kinetic analyses of transport reactions mediated by AspT in reconstituted liposomes suggested that, although the substrate transport reactions are physiologically coupled, the putative binding sites of L-aspartate (-Asp) and L-alanine (-Ala) are independently located on AspT. By using the fluorescent probe Oregon Green maleimide (OGM), which reacts specifically with cysteine, we also found that the presence of L-Asp changes the conformation of AspT. In this study, we conducted an OGM labeling assay in the presence of L-Ala. The labeling efficiency of single cysteine mutants (G62C and P79C) in transmembrane helix 3 of the AspT showed novel patterns depending on the presence of L-Ala or analogs. A concentration-dependent shift of AspT from the conformation in the presence of one substrate to that specific to the substrate added subsequently (L-Ala or L-Asp) was observed. Moreover, size-exclusion-chromatography-based thermostability assays indicated that the thermal stability of AspT in the presence of L-Ala differed from that in the presence of L-Asp. From these results, we concluded that L-Ala binding yields a conformation different from the apo or L-Asp binding conformations.

Aspergillus Hydrophobins: Physicochemical Properties, Biochemical Properties, and Functions in Solid Polymer Degradation.

Hydrophobins are small amphipathic proteins conserved in filamentous fungi. In this review, the properties and functions of Aspergillus hydrophobins are comprehensively discussed on the basis of recent findings. Multiple Aspergillus hydrophobins have been identified and categorized in conventional class I and two non-conventional classes. Some Aspergillus hydrophobins can be purified in a water phase without organic solvents. Class I hydrophobins of Aspergilli self-assemble to form amphipathic membranes. At the air-liquid interface, RolA of Aspergillus oryzae self-assembles via four stages, and its self-assembled films consist of two layers, a rodlet membrane facing air and rod-like structures facing liquid. The self-assembly depends mainly on hydrophobin conformation and solution pH. Cys4-Cys5 and Cys7-Cys8 loops, disulfide bonds, and conserved Cys residues of RodA-like hydrophobins are necessary for self-assembly at the interface and for adsorption to solid surfaces. AfRodA helps Aspergillus fumigatus to evade recognition by the host immune system. RodA-like hydrophobins recruit cutinases to promote the hydrolysis of aliphatic polyesters. This mechanism appears to be conserved in Aspergillus and other filamentous fungi, and may be beneficial for their growth. Aspergilli produce various small secreted proteins (SSPs) including hydrophobins, hydrophobic surface-binding proteins, and effector proteins. Aspergilli may use a wide variety of SSPs to decompose solid polymers.

Oligomeric state of the aspartate:alanine transporter from Tetragenococcus halophilus.

The aspartate:alanine exchanger family of membrane transporters includes industrially important transporters such as succinate exporter and glutamate exporter. No high-resolution structure is available from this family so far, and the transport mechanism of these transporters also remains unclear. In the present study, we focus on the oligomeric status of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus, which is the prototype of this family. To investigate the oligomeric structure of AspT, we established a system that produces high yields of highly purified AspT and determined the oligomeric structure of AspT by analysis with size exclusion chromatography coupled with multi-angle light scattering and blue native PAGE and by comparison of the wild-type AspT with a single-cysteine mutant that forms spontaneous inter-molecular thiol crosslinking. All the results consistently support the notion that AspT is a homodimer in solutions and in membranes.

Cell Wall Integrity and Its Industrial Applications in Filamentous Fungi.

Signal transduction pathways regulating cell wall integrity (CWI) in filamentous fungi have been studied taking into account findings in budding yeast, and much knowledge has been accumulated in recent years. Given that the cell wall is essential for viability in fungi, its architecture has been analyzed in relation to virulence, especially in filamentous fungal pathogens of plants and humans. Although research on CWI signaling in individual fungal species has progressed, an integrated understanding of CWI signaling in diverse fungi has not yet been achieved. For example, the variety of sensor proteins and their functional differences among different fungal species have been described, but the understanding of their general and species-specific biological functions is limited. Our long-term research interest is CWI signaling in filamentous fungi. Here, we outline CWI signaling in these fungi, from sensor proteins required for the recognition of environmental changes to the regulation of cell wall polysaccharide synthesis genes. We discuss the similarities and differences between the functions of CWI signaling factors in filamentous fungi and in budding yeast. We also describe the latest findings on industrial applications, including those derived from studies on CWI signaling: the development of antifungal agents and the development of highly productive strains of filamentous fungi with modified cell surface characteristics by controlling cell wall biogenesis.

Adsorption Kinetics and Self-Assembled Structures of Aspergillus oryzae Hydrophobin RolA on Hydrophobic and Charged Solid Surfaces.

Hydrophobins are small secreted amphipathic proteins ubiquitous among filamentous fungi. Hydrophobin RolA produced by Aspergillus oryzae attaches to solid surfaces, recruits polyesterase CutL1, and thus promotes hydrolysis of polyesters. Because the N-terminal region of RolA is involved in the interaction with CutL1, the orientation of RolA on the solid surface is important. However, the kinetic properties of RolA adsorption to solid surfaces with various chemical properties remain unclear, and RolA structures assembled after the attachment to surfaces are unknown. Using a quartz crystal microbalance (QCM), we analyzed the kinetic properties of RolA adsorption to the surfaces of QCM electrodes that had been chemically modified to become hydrophobic or charged. We also observed the assembled RolA structures on the surfaces by atomic force microscopy and performed molecular dynamics (MD) simulations of RolA adsorption to self-assembled monolayer (SAM)-modified surfaces. The RolA-surface interaction was considerably affected by the zeta potential of RolA, which was affected by pH. The interactions of RolA with the surface seemed to be involved in the self-assembly of RolA. Three types of self-assembled structures of RolA were observed: spherical, rod-like, and mesh-like. The kinetics of RolA adsorption and the structures formed depended on the amount of RolA adsorbed, chemical properties of the electrode surface, and the pH of the buffer. Adsorption of RolA to solid surfaces seemed to depend mainly on its hydrophobic interaction with the surfaces; this was supported by MD simulations, which suggested that hydrophobic Cys-Cys loops of RolA attached to all SAM-modified surfaces at all pH values. IMPORTANCE The adsorption kinetics of hydrophobins to solid surfaces and self-assembled structures formed by hydrophobin molecules have been studied mostly independently. In this report, we combined the kinetic analysis of hydrophobin RolA adsorption onto solid surfaces and observation of RolA self-assembly on these surfaces. Since RolA, whose isoelectric point is close to pH 4.0, showed higher affinity to the solid surfaces at pH 4.0 than at pH 7.0 or 10.0, the affinity of RolA to these surfaces depends mainly on hydrophobic interactions. Our combined analyses suggest that not only the adsorbed amount of RolA but also the chemical properties of the solid surfaces and the zeta potential of RolA affect the self-assembled RolA structures formed on these surfaces.

Quantitative Monitoring of Mycelial Growth of Aspergillus fumigatus in Liquid Culture by Optical Density.

Filamentous fungi form multicellular hyphae, which generally form pellets in liquid shake cultures, during the vegetative growth stage. Because of these characteristics, growth-monitoring methods commonly used in bacteria and yeast have not been applied to filamentous fungi. We have recently revealed that the cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) contribute to hyphal aggregation in Aspergillus oryzae. Here, we tested whether Aspergillus fumigatus shows dispersed growth in liquid media that can be quantitatively monitored, similar to that of yeasts. We constructed a double disruptant mutant of both the primary α-1,3-glucan synthase gene ags1 and the putative GAG synthase gene gtb3 in A. fumigatus AfS35 and found that the hyphae of this mutant were fully dispersed. Although the mutant lost α-1,3-glucan and GAG, its growth and susceptibility to antifungal agents were not different from those of the parental strain. Mycelial weight of the mutant in shake-flask cultures was proportional to optical density for at least 18 h. We were also able to quantify the dose response of hyphal growth to antifungal agents by measuring optical density. Overall, we established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly used for screening for novel antifungals against Aspergillus species. IMPORTANCE Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi so that we may apply the methods used for unicellular organisms such as yeast and bacteria. In the present study, by using the fungal pathogen Aspergillus fumigatus strain with modified hyphal surface polysaccharides, we succeeded in monitoring the hyphal growth quantitatively by optical density. The principle of this easy measurement by optical density could lead to a novel standard of hyphal quantification such as those that have been used for yeasts and bacteria. Dose response of hyphal growth by antifungal agents could also be monitored. This method could be useful for screening for novel antifungal reagents against Aspergillus species.

Cleavage of α-1,4-glycosidic linkages by the glycosylphosphatidylinositol-anchored α-amylase AgtA decreases the molecular weight of cell wall α-1,3-glucan in Aspergillus oryzae.

Aspergillus fungi contain α-1,3-glucan with a low proportion of α-1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored α-amylases are conserved in Aspergillus fungi. The GPI-anchored α-amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall α-1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall α-1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall α-1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial α-1,3-glucan. Furthermore, rAoAgtA cleaved 3-α-maltotetraosylglucose with a structure similar to the predicted boundary structure between the α-1,3-glucan main chain and a short spacer composed of α-1,4-linked glucose residues in cell wall α-1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the α-1,4-glycosidic bonds of 3-α-maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall α-1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight of cell wall α-1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored α-amylases can degrade the spacer α-1,4-glycosidic linkages in cell wall α-1,3-glucan before its insolubilization, and this spacer cleavage decreases the molecular weight of cell wall α-1,3-glucan in vivo.

Corrigendum: A Glycosylphosphatidylinositol-Anchored α-Amylase Encoded by amyD Contributes to a Decrease in the Molecular Mass of Cell Wall α-1,3-Glucan in Aspergillus nidulans.

[This corrects the article DOI: 10.3389/ffunb.2021.821946.].

Discovery of a gene cluster for the biosynthesis of novel cyclic peptide compound, KK-1, in Curvularia clavata.

KK-1, a cyclic depsipeptide with 10 residues produced by a filamentous fungus Curvularia clavata BAUA-2787, is a promising pesticide active compound with high activity against many plant pathogens, especially Botrytis cinerea. As a first step toward the future mass production of KK-1 through synthetic biological approaches, we aimed to identify the genes responsible for the KK-1 biosynthesis. To achieve this, we conducted whole genome sequencing and transcriptome analysis of C. clavata BAUA-2787 to predict the KK-1 biosynthetic gene cluster. We then generated the overexpression and deletion mutants for each cluster gene using our originally developed transformation system for this fungus, and analyzed the KK-1 production and the cluster gene expression levels to confirm their involvement in KK-1 biosynthesis. As a result of these, a region of approximately 71 kb was found, containing 10 open reading frames, which were co-induced during KK-1 production, as a biosynthetic gene cluster. These include kk1B, which encodes nonribosomal peptide synthetase with a domain structure that is consistent with the structural features of KK-1, and kk1F, which encodes a transcription factor. The overexpression of kk1F increased the expression of the entire cluster genes and, consequently, improved KK-1 production, whereas its deletion decreased the expression of the entire cluster genes and almost eliminated KK-1 production, demonstrating that the protein encoded by kk1F regulates the expressions of the other nine cluster genes cooperatively as the pathway-specific transcription factor. Furthermore, the deletion of each cluster gene caused a reduction in KK-1 productivity, indicating that each gene is involved in KK-1 production. The genes kk1A, kk1D, kk1H, and kk1I, which showed a significant decrease in KK-1 productivity due to deletion, were presumed to be directly involved in KK-1 structure formation, including the biosynthesis of the constituent residues. kk1C, kk1E, kk1G, and kk1J, which maintained a certain level of KK-1 productivity despite deletion, were possibly involved in promoting or assisting KK-1 production, such as extracellular transportation and the removal of aberrant units incorporated into the peptide chain.

Vulcanimicrobium alpinus gen. nov. sp. nov., the first cultivated representative of the candidate phylum "Eremiobacterota", is a metabolically versatile aerobic anoxygenic phototroph.

The previously uncultured phylum "Candidatus Eremiobacterota" is globally distributed and often abundant in oligotrophic environments. Although it includes lineages with the genetic potential for photosynthesis, one of the most important metabolic pathways on Earth, the absence of pure cultures has limited further insights into its ecological and physiological traits. We report the first successful isolation of a "Ca. Eremiobacterota" strain from a fumarolic ice cave on Mt. Erebus volcano (Antarctica). Polyphasic analysis revealed that this organism is an aerobic anoxygenic photoheterotrophic bacterium with a unique lifestyle, including bacteriochlorophyll a production, CO2 fixation, a high CO2 requirement, and phototactic motility using type IV-pili, all of which are highly adapted to polar and fumarolic environments. The cells are rods or filaments with a vesicular type intracytoplasmic membrane system. The genome encodes novel anoxygenic Type II photochemical reaction centers and bacteriochlorophyll synthesis proteins, forming a deeply branched monophyletic clade distinct from known phototrophs. The first cultured strain of the eighth phototrophic bacterial phylum which we name Vulcanimicrobium alpinus gen. nov., sp. nov. advances our understanding of ecology and evolution of photosynthesis.

Improved recombinant protein production in Aspergillus oryzae lacking both α-1,3-glucan and galactosaminogalactan in batch culture with a lab-scale bioreactor.

Filamentous fungi are used as production hosts for various commercially valuable enzymes and chemicals including organic acids and secondary metabolites. We previously revealed that α-1,3-glucan and galactosaminogalactan (GAG) contribute to hyphal aggregation in the industrial fungus Aspergillus oryzae, and that production of recombinant protein in shake-flask culture is higher in a mutant lacking both α-1,3-glucan and GAG (AGΔ-GAGΔ) than in the parental strain. Here, we compared the productivity of the wild type, AGΔ-GAGΔ, and mutants lacking α-1,3-glucan (AGΔ) or GAG (GAGΔ) in batch culture with intermittent addition of glucose in a 5-L lab-scale bioreactor. The hyphae of the wild type and all mutants were dispersed by agitation, although the wild type and AGΔ formed small amounts of aggregates. Although mycelial weight was similar among the strains, the concentration of a secreted recombinant protein (CutL1) was the highest in AGΔ-GAGΔ. Evaluation of fluid properties revealed that the apparent viscosities of mycelial cultures of the wild type and AGΔ-GAGΔ decreased as the agitation speed was increased. The apparent viscosity of the AGΔ-GAGΔ culture tended to be lower than that of the wild-type strain at each agitation speed, and was significantly lower at 600 rpm. Overall, the lack of α-1,3-glucan and GAG in the hyphae improved culture rheology, resulting in an increase in recombinant protein production in AGΔ-GAGΔ. This is the first report of flow behavior improvement by a cell-surface component defect in a filamentous fungus.

Oryzapsins, the orthologs of yeast yapsin in Aspergillus oryzae, affect ergosterol synthesis.

The oryzapsin genes opsA and opsB in Aspergillus oryzae encoding glycosylphosphatidylinositol (GPI)-anchored aspartic endopeptidase are homologs of Saccharomyces cerevisiae yapsins. We recently found another homolog, opsC, in the A. oryzae genome database, which was suggested to be a pseudogene. However, the profiles and roles of the proteins encoded by these genes have not yet been clarified. Toward this end, we first produced opsA- and opsB-overexpression strains and performed enzymatic analyses, revealing that OpsA and OpsB can attack sites other than the carboxyl-terminal peptide bonds of basic amino acids. Moreover, OpsA and OpsB were confirmed to bind to the cell membrane with a GPI anchor. Second, opsA and opsB single-deletion and double-deletion strains (ΔopsA, ΔopsB, and ΔopsAΔopsB) were constructed to explore the expected roles of oryzapsins in cell wall synthesis, similar to the role of yapsins. The transcription level of mpkA in the cell wall integrity pathway was increased in ΔopsB and ΔopsAΔopsB strains, suggesting that OpsB might be involved in processing cell wall synthesis-related proteins. Treatment with an ergosterol biosynthesis inhibitor reduced the growth of the ΔopsAΔopsB strain. Moreover, the mRNA levels of Aoerg1, Aoerg3-1, Aoerg3-2, Aoerg7b, Aoerg11, and Aohmg1,2 showed a decreasing tendency in the ΔopsAΔopsB strain, and the ergosterol content in the membrane was reduced in the ΔopsAΔopsB strain. These results suggest that oryzapsins exist in the cell membrane and play roles in the formation of cell membranes. This is the first report of the involvement of GPI-anchored aspartic endopeptidases in ergosterol biosynthesis.Key points• The oryzapsins have wider substrate specificity than yaspins in S. cerevisiae.• Unlike the yapsins, the oryzapsins might not be involved in the main structure synthesis of the cell wall.• The oryzapsins would be involved in ergosterol biosynthesis.

Hyperosmotic medium partially restores the growth defect and the impaired production of sterigmatocystin of an Aspergillus nidulans ΔpmtC mutant in a HogA-independent manner.

The protein O-mannosyltransferase catalyzes O-mannosylation in the endoplasmic reticulum by transferring mannose to the seryl or threonyl residues of substrate proteins. We previously reported a deletion mutant of O-mannosyltransferase C (ΔpmtC) in Aspergillus nidulans with impaired vegetative growth and sterigmatocystin (ST) production. In this study, we investigated whether osmotic conditions contribute to the developmental processes and ST biosynthesis of the ΔpmtC deletion mutant. We found that hyphal growth and ST production partially improved in the presence of NaCl, KCl or sorbitol as osmotic stabilizers. Conidiation of the ΔpmtC deletion mutant was not restored under osmotic stress conditions when the hogA gene was deleted. The hogA gene encodes a protein required for the cellular response to osmotic pressure. However, the yield of ST and the vegetative growth of the ΔhogA ΔpmtC double deletant was restored by high osmolarity in a HogA-independent manner.

Reticulibacter mediterranei gen. nov., sp. nov., within the new family Reticulibacteraceae fam. nov., and Ktedonospora formicarum gen. nov., sp. nov., Ktedonobacter robiniae sp. nov., Dictyobacter formicarum sp. nov. and Dictyobacter arantiisoli sp. nov., belonging to the class Ktedonobacteria.

The aerobic, Gram-positive, mesophilic Ktedonobacteria strains, Uno17T, SOSP1-1T, 1-9T, 1-30T and 150040T, formed mycelia of irregularly branched filaments, produced spores or sporangia, and numerous secondary metabolite biosynthetic gene clusters. The five strains grew at 15-40 °C (optimally at 30 °C) and pH 4.0-8.0 (optimally at pH 6.0-7.0), and had 7.21-12.67 Mb genomes with 49.7-53.7 mol% G+C content. They shared MK9(H2) as the major menaquinone and C16 : 1-2OH and iso-C17 : 0 as the major cellular fatty acids. Phylogenetic and phylogenomic analyses showed that Uno17T and SOSP1-9T were most closely related to members of the genus Dictyobacter, with 94.43-96.21 % 16S rRNA gene similarities and 72.16-81.56% genomic average nucleotide identity. The strain most closely related to SOSP1-1T and SOSP1-30T was Ktedonobacter racemifer SOSP1-21T, with 91.33 and 98.84 % 16S rRNA similarities, and 75.13 and 92.35% average nucleotide identities, respectively. Strain 150040T formed a distinct clade within the order Ktedonobacterales, showing <90.47 % 16S rRNA gene similarity to known species in this order. Based on these results, we propose: strain 150040T as Reticulibacter mediterranei gen. nov., sp. nov. (type strain 150 040T=CGMCC 1.17052T=BCRC 81202T) within the family Reticulibacteraceae fam. nov. in the order Ktedonobacterales; strain SOSP1-1T as Ktedonospora formicarum gen. nov., sp. nov. (type strain SOSP1-1T=CGMCC 1.17205T=BCRC 81203T) and strain SOSP1-30T as Ktedonobacter robiniae sp. nov. (type strain SOSP1-30T=CGMCC 1.17733T=BCRC 81205T) within the family Ktedonobacteraceae; strain Uno17T as Dictyobacter arantiisoli sp. nov. (type strain Uno17T=NBRC 113155T=BCRC 81116T); and strain SOSP1-9T as Dictyobacter formicarum sp. nov. (type strain SOSP1-9T=CGMCC 1.17206T=BCRC 81204T) within the family Dictyobacteraceae.