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Abstract The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.

Bacteria of the family Flavobacteriaceae (flavobacteria) primarily comprise nonpathogenic bacteria that inhabit soil and water (both marine and freshwater). However, some bacterial species in the family, including Flavobacterium psychrophilum and Flavobacterium columnare, are known to be pathogenic to fish. Flavobacteria, including the abovementioned pathogenic bacteria, belong to the phylum Bacteroidota and possess two phylum-specific features, gliding motility and a protein secretion system, which are energized by a common motor complex. Herein, we focused on Flavobacterium collinsii (GiFuPREF103) isolated from a diseased fish (Plecoglossus altivelis). Genomic analysis of F. collinsii GiFuPREF103 revealed the presence of a type IX secretion system and additional genes associated with gliding motility and spreading. Using transposon mutagenesis, we isolated two mutants with altered colony morphology and colony spreading ability; these mutants had transposon insertions in pep25 and lbp26. The glycosylation material profiles revealed that these mutants lacked the high-molecular-weight glycosylated materials present in the wild-type strain. In addition, the wild-type strains exhibited fast cell population movement at the edge of the spreading colony, whereas reduced cell population behavior was observed in the pep25- and lbp26-mutant strains. In the aqueous environment, the surface layers of these mutant strains were more hydrophobic, and they formed biofilms with enhanced microcolony growth compared to those with the wild-type. In Flavobacterium johnsoniae, the Fjoh_0352 and Fjoh_0353 mutant strains were generated, which were based on the ortholog genes of pep25 and lbp26. In these F. johnsoniae mutants, as in F. collinsii GiFuPREF103, colonies with diminished spreading capacity were formed. Furthermore, cell population migration was observed at the edge of the colony in wild-type F. johnsoniae, whereas individual cells, and not cell populations, migrated in these mutant strains. The findings of the present study indicate that pep25 and lbp26 contribute to the colony spreading of F. collinsii.

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins. [ABSTRACT FROM AUTHOR]

Biofilms are three-dimensional structures containing one or more bacterial species embedded in extracellular polymeric substances. Although most biofilms are stationary, Flavobacterium johnsoniae forms a motile spherical biofilm called a zorb, which is propelled by its base cells and contains a polysaccharide core. Here, we report formation of spatially organized, motile, multispecies biofilms, designated "co-zorbs," that are distinguished by a core-shell structure. F. johnsoniae forms zorbs whose cells collect other bacterial species and transport them to the zorb core, forming a co-zorb. Live imaging revealed that co-zorbs also form in zebrafish, thereby demonstrating a new type of bacterial movement in vivo. This discovery opens new avenues for understanding community behaviors, the role of biofilms in bulk bacterial transport, and collective strategies for microbial success in various environments.

ABSTRACT Bacterial alternative complex III (ACIII) catalyzes menaquinol (MKH2) oxidation, presumably fulfilling the role of cytochromes bc1/b6f in organisms that lack these enzymes. The molecular mechanism of ACIII is unknown and so far the complex has remained inaccessible for genetic modifications. The recently solved cryo-electron microscopy (cryo-EM) structures of ACIII from Flavobacterium johnsoniae, Rhodothermus marinus, and Roseiflexus castenholzii revealed no structural similarity to cytochrome bc1/b6f and there were variations in the heme-containing subunits ActA and ActE. These data implicated intriguing alternative electron transfer paths connecting ACIII with its redox partner, and left the contributions of ActE and the terminal domain of ActA to the catalytic mechanism unclear. Here, we report genetic deletion and complementation of F. johnsoniae actA and actE and the functional implications of such modifications. Deletion of actA led to the loss of activity of cytochrome aa3 (a redox partner of ACIII in this bacterium), which confirmed that ACIII is the sole source of electrons for this complex. Deletion of actE did not impair the activity of cytochrome aa3, revealing that ActE is not required for electron transfer between ACIII and cytochrome aa3. Nevertheless, absence of ActE negatively impacted the cell growth rate, pointing toward another, yet unidentified, function of this subunit. Possible explanations for these observations, including a proposal of a split in electron paths at the ActA/ActE interface, are discussed. The described system for genetic manipulations in F. johnsoniae ACIII offers new tools for studying the molecular mechanism of operation of this enzyme. IMPORTANCE Energy conversion is a fundamental process of all organisms, realized by specialized protein complexes, one of which is alternative complex III (ACIII). ACIII is a functional analogue of well-known mitochondrial complex III, but operates according to a different, still unknown mechanism. To understand how ACIII interacts functionally with its protein partners, we developed a genetic system to mutate the Flavobacterium johnsoniae genes encoding ACIII subunits. Deletion and complementation of heme-containing subunits revealed that ACIII is the sole source of electrons for cytochrome aa3 and that one of the redox-active subunits (ActE) is dispensable for electron transfer between these complexes. This study sheds light on the operation of the supercomplex of ACIII and cytochrome aa3 and suggests a division in the electron path within ACIII. It also shows a way to manipulate protein expression levels for application in other members of the Bacteroidetes phylum.

GldL is an inner‐membrane protein that is essential for the function of the type IX secretion system (T9SS) in Flavobacterium johnsoniae. The complex that it forms with GldM is supposed to act as a new rotary motor involved in the gliding motility of the bacterium. In the context of structural studies of GldL to gain information on the assembly and function of the T9SS, two camelid nanobodies were selected, produced and purified. Their interaction with the cytoplasmic domain of GldL was characterized and their crystal structures were solved. These nanobodies will be used as crystallization chaperones to help in the crystallization of the cytoplasmic domain of GldL and could also help to solve the structure of the complex using molecular replacement. [ABSTRACT FROM AUTHOR]

In a previous study, it was shown that Riemerella anatipestifer, a member of Flavobacteriaceae, is naturally competent. However, whether natural competence is universal in Flavobacteriaceae remains unknown. In this study, it was shown for the first time that Riemerella columbina was naturally competent in the laboratory condition; however, Flavobacterium johnsoniae was not naturally competent under the same conditions. The competence of R. columbina was maintained throughout the growth phases, and the transformation frequency was highest during the logarithmic phase. A competition assay revealed that R. columbina preferentially took up its own genomic DNA over heterologous DNA. The natural transformation frequency of R. columbina was significantly increased in GCB medium without peptone or phosphate. Furthermore, natural transformation of R. columbina was inhibited by 0.5 mM EDTA, but could be restored by the addition of CaCl2, MgCl2, ZnCl2, and MnCl2, suggesting that these divalent cations promote the natural transformation of R. columbina. Overall, this study revealed that natural competence is not universal in Flavobacteriaceae members and triggering of competence differs from species to species.

In a previous study, it was shown that Riemerella anatipestifer , a member of Flavobacteriaceae , is naturally competent. However, whether natural competence is universal in Flavobacteriaceae remains unknown. In this study, it was shown for the first time that Riemerella columbina was naturally competent in the laboratory condition; however, Flavobacterium johnsoniae was not naturally competent under the same conditions. The competence of R. columbina was maintained throughout the growth phases, and the transformation frequency was highest during the logarithmic phase. A competition assay revealed that R. columbina preferentially took up its own genomic DNA over heterologous DNA. The natural transformation frequency of R. columbina was significantly increased in GCB medium without peptone or phosphate. Furthermore, natural transformation of R. columbina was inhibited by 0.5 mM EDTA, but could be restored by the addition of CaCl2, MgCl2, ZnCl2, and MnCl2, suggesting that these divalent cations promote the natural transformation of R. columbina. Overall, this study revealed that natural competence is not universal in Flavobacteriaceae members and triggering of competence differs from species to species. [ABSTRACT FROM AUTHOR]

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins.

Glycoside hydrolase family (GH) 31 contains a large variety of enzymes, but the major members are enzymes that act on relatively small oligosaccharides such as α‐glucosidase. Here, we determined the crystal structure of Flavobacterium johnsoniae dextranase (FjDex31A), an enzyme from F. johnsoniae that hydrolyzes a polysaccharide, dextran. FjDex31A is composed of four domains: an N‐terminal domain, a catalytic domain, a proximal C‐terminal domain, and a distal C‐terminal domain, as observed in typical GH31 enzymes. However, the architecture of active site residues in FjDex31A, other than subsite −1, is markedly different from that of other GH31 enzymes. The FjDex31A structure in complex with isomaltotriose shows that Gly273 and Tyr524, both of which interact with an α‐glucose residue at subsite −2, as well as Trp376 and Leu308‐cisGln309, are especially unique to FjDex31A. Site‐directed mutagenesis of Gly273 and Tyr524 resulted in a decrease in the hydrolysis of polysaccharides dextran and pullulan, as well as that of the disaccharide isomaltose. These results suggest that, regardless of the length of sugar chains of the substrates, binding of FjDex31A to the substrates at subsite −2 is likely to be important for its activity. Database: Structural data are available in the Protein Data Bank under the accession numbers 6JR6, 6JR7, and 6JR8. [ABSTRACT FROM AUTHOR]

ABSTRACT The quest to manipulate microbiomes has intensified, but many microbial communities have proven to be recalcitrant to sustained change. Developing model communities amenable to genetic dissection will underpin successful strategies for shaping microbiomes by advancing an understanding of community interactions. We developed a model community with representatives from three dominant rhizosphere taxa, the Firmicutes, Proteobacteria, and Bacteroidetes. We chose Bacillus cereus as a model rhizosphere firmicute and characterized 20 other candidates, including “hitchhikers” that coisolated with B. cereus from the rhizosphere. Pairwise analysis produced a hierarchical interstrain-competition network. We chose two hitchhikers, Pseudomonas koreensis from the top tier of the competition network and Flavobacterium johnsoniae from the bottom of the network, to represent the Proteobacteria and Bacteroidetes, respectively. The model community has several emergent properties, induction of dendritic expansion of B. cereus colonies by either of the other members, and production of more robust biofilms by the three members together than individually. Moreover, P. koreensis produces a novel family of alkaloid antibiotics that inhibit growth of F. johnsoniae, and production is inhibited by B. cereus. We designate this community THOR, because the members are the hitchhikers of the rhizosphere. The genetic, genomic, and biochemical tools available for dissection of THOR provide the means to achieve a new level of understanding of microbial community behavior. IMPORTANCE The manipulation and engineering of microbiomes could lead to improved human health, environmental sustainability, and agricultural productivity. However, microbiomes have proven difficult to alter in predictable ways, and their emergent properties are poorly understood. The history of biology has demonstrated the power of model systems to understand complex problems such as gene expression or development. Therefore, a defined and genetically tractable model community would be useful to dissect microbiome assembly, maintenance, and processes. We have developed a tractable model rhizosphere microbiome, designated THOR, containing Pseudomonas koreensis, Flavobacterium johnsoniae, and Bacillus cereus, which represent three dominant phyla in the rhizosphere, as well as in soil and the mammalian gut. The model community demonstrates emergent properties, and the members are amenable to genetic dissection. We propose that THOR will be a useful model for investigations of community-level interactions.

Plants expend significant resources to select and maintain rhizosphere communities that benefit their growth and protect them from pathogens. A better understanding of assembly and function of rhizosphere microbial communities will provide new avenues for improving crop production. Secretion of antibiotics is one means by which bacteria interact with neighboring microbes and sometimes change community composition. In our analysis of a taxonomically diverse consortium from the soybean rhizosphere, we found that Pseudomonas koreensis selectively inhibits growth of Flavobacterium johnsoniae and other members of the Bacteroidetes grown in soybean root exudate. A genetic screen in P. koreensis identified a previously uncharacterized biosynthetic gene cluster responsible for the inhibitory activity. Metabolites were isolated based on biological activity and were characterized using tandem mass spectrometry, multidimensional nuclear magnetic resonance, and Mosher ester analysis, leading to the discovery of a new family of bacterial tetrahydropyridine alkaloids, koreenceine A to D (metabolites 1 to 4). Three of these metabolites are analogs of the plant alkaloid γ-coniceine. Comparative analysis of the koreenceine cluster with the γ-coniceine pathway revealed distinct polyketide synthase routes to the defining tetrahydropyridine scaffold, suggesting convergent evolution. Koreenceine-type pathways are widely distributed among Pseudomonas species, and koreenceine C was detected in another Pseudomonas species from a distantly related cluster. This work suggests that Pseudomonas and plants convergently evolved the ability to produce similar alkaloid metabolites that can mediate interbacterial competition in the rhizosphere.IMPORTANCE The microbiomes of plants are critical to host physiology and development. Microbes are attracted to the rhizosphere due to massive secretion of plant photosynthates from roots. Microorganisms that successfully join the rhizosphere community from bulk soil have access to more abundant and diverse molecules, producing a highly competitive and selective environment. In the rhizosphere, as in other microbiomes, little is known about the genetic basis for individual species' behaviors within the community. In this study, we characterized competition between Pseudomonas koreensis and Flavobacterium johnsoniae, two common rhizosphere inhabitants. We identified a widespread gene cluster in several Pseudomonas spp. that is necessary for the production of a novel family of tetrahydropyridine alkaloids that are structural analogs of plant alkaloids. We expand the known repertoire of antibiotics produced by Pseudomonas in the rhizosphere and demonstrate the role of the metabolites in interactions with other rhizosphere bacteria. [ABSTRACT FROM AUTHOR]

The type IX secretion system (T9SS) is specific to the Bacteroidetes phylum. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilises the T9SS to transport many proteins—including its gingipain virulence factors—across the outer membrane and attach them to the cell surface. Additionally, the T9SS is also required for gliding motility in motile organisms, such as Flavobacterium johnsoniae. At least nineteen proteins have been identified as components of the T9SS, including the three transcription regulators, PorX, PorY and SigP. Although the components are known, the overall organisation and the molecular mechanism of how the T9SS operates is largely unknown. This review focusses on the recent advances made in the structure, function, and organisation of the T9SS machinery to provide further insight into this highly novel secretion system.

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Flavobacterium johnsoniae is a free-living member of the Bacteroidota phylum that is found in soil and water. It is frequently used as a model species for studying a type of gliding motility dependent on the type IX secretion system (T9SS). O -Glycosylation has been reported in several Bacteroidota species, and the O -glycosylation of S-layer proteins in Tannerella forsythia was shown to be important for certain virulence features. In this study, we characterized the O -glycoproteome of F. johnsoniae and identified 325 O -glycosylation sites within 226 glycoproteins. The structure of the major glycan was found to be a hexasaccharide with the sequence Hex-(Me-dHex)-Me-HexA-Pent-HexA-Me-HexNAcA. Bioinformatic localization of the glycoproteins predicted 68 inner membrane proteins, 60 periplasmic proteins, 26 outer membrane proteins, 57 lipoproteins, and 9 proteins secreted by the T9SS. The glycosylated sites were predominantly located in the periplasm, where they are postulated to be beneficial for protein folding/stability. Six proteins associated with gliding motility or the T9SS were demonstrated to be O -glycosylated. IMPORTANCE Flavobacterium johnsoniae is a Gram-negative bacterium that is found in soil and water. It is frequently used as a model species for studying gliding motility and the T9SS. In this study, we characterized the O -glycoproteome of F. johnsoniae and identified 325 O -glycosylation sites within 226 glycoproteins. The glycosylated domains were mainly localized to the periplasm. The function of O -glycosylation is likely related to protein folding and stability; therefore, the finding of the glycosylation sites has relevance for studies involving expression of the proteins. Six proteins associated with gliding motility or the T9SS were demonstrated to be O -glycosylated, which may impact the structure and function of these components., Competing Interests: The authors declare no conflict of interest.

Microbial decomposition of organic matter is an essential process in the global carbon cycle. The soil bacteria Pseudopedobacter saltans and Flavobacterium johnsoniae are both able to degrade complex organic molecules, but it is not fully known how their membrane structures are adapted to their environmental niche. The membrane lipids of these species were extracted and analyzed using high performance liquid chromatography-electrospray ionization/ion trap/mass spectrometry (HPLC-ESI/IT/MS) and high resolution accurate mass/mass spectrometry (HRAM/MS). Abundant unknown intact polar lipids (IPLs) from P. saltans were isolated and further characterized using amino acid analysis and two dimensional nuclear magnetic resonance (NMR) spectroscopy. Ornithine IPLs (OLs) with variable (hydroxy) fatty acid composition were observed in both bacterial species. Lysine-containing IPLs (LLs) were also detected in both species and were characterized here for the first time using HPLC-MS. Novel LLs containing hydroxy fatty acids and novel hydroxylysine lipids with variable (hydroxy) fatty acid composition were identified in P. saltans. The confirmation of OL and LL formation in F. johnsoniae and P. saltans and the presence of OlsF putative homologues in P. saltans suggest the OlsF gene coding protein is possibly involved in OL and LL biosynthesis in both species, however, potential pathways of OL and LL hydroxylation in P. saltans are still undetermined. Triplicate cultures of P. saltans were grown at three temperature/pH combinations: 30°C/pH 7, 15°C/pH 7 and 15°C/pH 9. The fractional abundance of total amino acid containing IPLs containing hydroxylated fatty acids was significantly higher at higher temperature, and the fractional abundance of lysine-containing IPLs was significantly higher at lower temperature and higher pH. These results suggest that these amino acid-containing IPLs, including the novel hydroxylysine lipids, could be involved in temperature and pH stress response

Glycoside hydrolase family (GH) 31 enzymes exhibit various substrate specificities, although the majority of members are α-glucosidases. Here, we constructed a heterologous expression system of a GH31 enzyme, Fjoh_4430, fromFlavobacterium johnsoniaeNBRC 14942, usingEscherichia coli, and characterized its enzymatic properties. The enzyme hydrolyzed dextran and pullulan to produce isomaltooligosaccharides and isopanose, respectively. When isomaltose was used as a substrate, the enzyme catalyzed disproportionation to form isomaltooligosaccharides. The enzyme also acted, albeit inefficiently, onp-nitrophenyl α-D-glucopyranoside, andp-nitrophenyl α-isomaltoside was the main product of the reaction. In contrast, Fjoh_4430 did not act on trehalose, kojibiose, nigerose, maltose, maltotriose, or soluble starch. The optimal pH and temperature were pH 6.0 and 60 °C, respectively. Our results indicate that Fjoh_4430 is a novel GH31 dextranase with high transglucosylation activity. TLC analysis of the reaction products generated by Fjoh_4430 enzyme from isomaltose. The left lane shows maltooligosaccharide markers. [ABSTRACT FROM PUBLISHER]

Bacteria of the family Flavobacteriaceae (flavobacteria) primarily comprise nonpathogenic bacteria that inhabit soil and water (both marine and freshwater). However, some bacterial species in the family, including Flavobacterium psychrophilum and Flavobacterium columnare , are known to be pathogenic to fish. Flavobacteria, including the abovementioned pathogenic bacteria, belong to the phylum Bacteroidota and possess two phylum-specific features, gliding motility and a protein secretion system, which are energized by a common motor complex. Herein, we focused on Flavobacterium collinsii (GiFuPREF103) isolated from a diseased fish ( Plecoglossus altivelis ). Genomic analysis of F. collinsii GiFuPREF103 revealed the presence of a type IX secretion system and additional genes associated with gliding motility and spreading. Using transposon mutagenesis, we isolated two mutants with altered colony morphology and colony spreading ability; these mutants had transposon insertions in pep25 and lbp26 . The glycosylation material profiles revealed that these mutants lacked the high-molecular-weight glycosylated materials present in the wild-type strain. In addition, the wild-type strains exhibited fast cell population movement at the edge of the spreading colony, whereas reduced cell population behavior was observed in the pep25- and lbp26 -mutant strains. In the aqueous environment, the surface layers of these mutant strains were more hydrophobic, and they formed biofilms with enhanced microcolony growth compared to those with the wild-type. In Flavobacterium johnsoniae , the Fjoh_0352 and Fjoh_0353 mutant strains were generated, which were based on the ortholog genes of pep25 and lbp26 . In these F. johnsoniae mutants, as in F. collinsii GiFuPREF103, colonies with diminished spreading capacity were formed. Furthermore, cell population migration was observed at the edge of the colony in wild-type F. johnsoniae , whereas individual cells, and not cell populations, migrated in these mutant strains. The findings of the present study indicate that pep25 and lbp26 contribute to the colony spreading of F. collinsii., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kondo, Ohara, Fujii, Nakai, Sato, Naito, Tsukuba, Kadowaki and Sato.)

A novel attenuated Flavobacterium johnsoniae M170 vaccine was developed from a pathogenic F. johnsoniae M168 isolate through a streptomycin-resistant strategy. The safety of the attenuated F. johnsoniae M170 strain was evaluated in grass carp ( Ctenopharyngodon idella ) through intraperitoneal (IP) injection and bath immersion. When grass carp (mean weight 10 g) were challenged by IP injection with 1 × 10 8 colony-forming units (CFU) per fish, or by immersion with 1 × 10 7 CFU/ml of the attenuated F. johnsoniae M170, no mortality, adverse behavior, or signs of disease were observed. However, when grass carp were challenged by IP injection of 1 × 10 7 CFU per fish, or by immersion with 1 × 10 7 CFU/ml of F. johnsoniae virulent strain M168, the mortality of challenged fish were 100% and 90%. Back-passage safety studies indicated that the M170 strain cannot revert back to a pathogenic state after 5 passages. The values of relative percent survival (RPS) in the IP injection group were 100% and 60% when challenged at 28 and 240 days post vaccination (dpv), while RPS in the bath immersion group were 73.1% and 34.8% when challenged at 28 and 240 dpv. ELISA (enzyme-linked immuno sorbent assay) results revealed that F. johnsoniae M170 could induce an antibody mediated immunity response. The above results suggest that the attenuated F. johnsoniae vaccine M170 could be used to protect grass carp from F. johnsoniae infections. Statement of relevance The live attenuated F. johnsoniae vaccine can offer excellent protection against infections by virulent F. johnsoniae in freshwater fish aquaculture. [ABSTRACT FROM AUTHOR]

Microbial decomposition of organic matter is an essential process in the global carbon cycle. The soil bacteria Pseudopedobacter saltans and Flavobacterium johnsoniae are both able to degrade complex organic molecules, but it is not fully known how their membrane structures are adapted to their environmental niche. The membrane lipids of these species were extracted and analyzed using high performance liquid chromatography-electrospray ionization/ion trap/mass spectrometry (HPLC-ESI/IT/MS) and high resolution accurate mass/mass spectrometry (HRAM/MS). Abundant unknown intact polar lipids (IPLs) from P. saltans were isolated and further characterized using amino acid analysis and two dimensional nuclear magnetic resonance (NMR) spectroscopy. Ornithine IPLs (OLs) with variable (hydroxy) fatty acid composition were observed in both bacterial species. Lysine-containing IPLs (LLs) were also detected in both species and were characterized here for the first time using HPLC-MS. Novel LLs containing hydroxy fatty acids and novel hydroxylysine lipids with variable (hydroxy) fatty acid composition were identified in P. saltans. The confirmation of OL and LL formation in F. johnsoniae and P. saltans and the presence of OlsF putative homologs in P. saltans suggest the OlsF gene coding protein is possibly involved in OL and LL biosynthesis in both species, however, potential pathways of OL and LL hydroxylation in P. saltans are still undetermined. Triplicate cultures of P. saltans were grown at three temperature/pH combinations: 30°C/pH 7, 15°C/pH 7, and 15°C/pH 9. The fractional abundance of total amino acid containing IPLs containing hydroxylated fatty acids was significantly higher at higher temperature, and the fractional abundance of lysine-containing IPLs was significantly higher at lower temperature and higher pH. These results suggest that these amino acid-containing IPLs, including the novel hydroxylysine lipids, could be involved in temperature and pH stress response of soil bacteria. [ABSTRACT FROM AUTHOR]

Aims: Previously, we selected a bacterial strain (GSE09) antagonistic to Phytophthora capsici on pepper, which produced a volatile compound (2,4-di- tert-butylphenol), inhibiting the pathogen. In this study, we identified strain GSE09 and characterized some of the biological traits of this strain in relation to its antagonistic properties against P. capsici. In addition, we examined bacterial colonization on the root surface or in rhizosphere soil and the effect of various concentrations of the volatile compound and strain GSE09 on pathogen development and radicle infection as well as radicle growth. Methods and Results: Strain GSE09 was identified as Flavobacterium johnsoniae, which forms biofilms and produces indolic compounds and biosurfactant but not hydrogen cyanide (HCN) with little or low levels of antifungal activity and swimming and swarming activities. Fl. johnsoniae GSE09 effectively colonized on pepper root, rhizosphere, and bulk (pot) soil, which reduced the pathogen colonization in the roots and disease severity in the plants. Various concentrations of 2,4-di- tert-butylphenol or strain GSE09 inhibited pathogen development (mycelial growth, sporulation, and zoospore germination) in I-plate (a plastic plate containing a center partition). In addition, germinated seeds treated with the compound (1-100 μg ml−1) or the strain (102-1010 cells ml−1) significantly reduced radicle infection by P. capsici without radicle growth inhibition. Conclusions: These results indicate that colonization of pepper root and rhizosphere by the Fl. johnsoniae strain GSE09, which can form biofilms and produce indolic compounds, biosurfactant, and 2,4-di -tert-butylphenol, might provide effective biocontrol activity against P. capsici. Significance and Impact of the Study: To our knowledge, this is the first study demonstrating that the Fl. johnsoniae strain GSE09, as a potential biocontrol agent, can effectively protect pepper plants against P. capsici infection by colonizing the roots. [ABSTRACT FROM AUTHOR]

Aims: To test whether the addition of Flavobacterium johnsoniae could increase the strength of saturated Ottawa 30 sand. Methods and Results: A box model was built that simulates groundwater-like flow through a main sand compartment. Strength tests were performed at seven locations and at two depths, 10·8 and 20·3 cm below the top of the tank, using a vane shear device before and after the addition of bacteria. After the addition of Fl. johnsoniae, sand samples were obtained from multiple sampling ports on the vertical sides of the box model. The presence of a bacterial biofilm was confirmed by staining these sand samples with SYTO-9 and Alexa Fluor 633 and viewing with a confocal microscope. The average shear strength increases after the addition of Fl. johnsoniae were 15·2–87·5%, depending on the experimental conditions. Conclusions: Flavobacterium johnsoniae caused a statistically significant increase in the strength of saturated Ottawa 30 sand. Significance and Impact of the Study: Biofilm-forming bacteria can increase the shear strength of saturated sand. The addition of biofilm-forming bacteria to a building site may be an alternate method to mitigate the effects of liquefaction. [ABSTRACT FROM AUTHOR]

Species-specific polymerase chain reaction (PCR) primers have been designed to identify the causative agent of columnaris disease, Flavobacterium columnare. The 16S rRNA gene sequences of F. columnare (eight sequences representing the different genotypes of the species) and related species (18 sequences) were aligned and compared to choose specific regions that are unique to F. columnare and do not have significant intraspecies variability. The species-specific regions in the 16S rRNA gene were used to design a pair of species-specific PCR primers, ColF and ColR. The PCR technique produced a specific amplicon of about 675 base pairs (bp) in 27 isolates of F. columnare and there was no amplification in the closely related species. The specificity of the amplified product was confirmed by digesting with HhaI. The PCR primers did not produce a 675bp product with F. columnare ATCC43622 strain. This ATCC43622 strain was characterized by biochemical and ribotyping methods and renamed Flavobacterium johnsoniae. The American Type Culture Collection has confirmed these findings and made the change. [Copyright &y& Elsevier]

Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes. We found that FjGH65A does not exhibit phosphorolytic activity, but it does hydrolyze kojibiose (α-1,2-glucobiose) and oligosaccharides containing a kojibiosyl moiety without requiring inorganic phosphate. In addition, stereochemical analysis demonstrated that FjGH65A catalyzes this hydrolytic reaction via an anomer-inverting mechanism. The three-dimensional structures of FjGH65A in native form and in complex with glucose were determined at resolutions of 1.54 and 1.40 Å resolutions, respectively. The overall structure of FjGH65A resembled those of other GH65 GPs, and the general acid catalyst Glu 472 was conserved. However, the amino acid sequence forming the phosphate-binding site typical of GH65 GPs was not conserved in FjGH65A. Moreover, FjGH65A had the general base catalyst Glu 616 instead, which is required to activate a nucleophilic water molecule. These results indicate that FjGH65A is an α-1,2-glucosidase and is the first bacterial GH found in the GH65 family., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacterial alternative complex III (ACIII) catalyzes menaquinol (MKH 2 ) oxidation, presumably fulfilling the role of cytochromes bc 1 in organisms that lack these enzymes. The molecular mechanism of ACIII is unknown and so far the complex has remained inaccessible for genetic modifications. The recently solved cryo-electron microscopy (cryo-EM) structures of ACIII from Flavobacterium johnsoniae, Rhodothermus marinus, and Roseiflexus castenholzii revealed no structural similarity to b 6 f in organisms that lack these enzymes. The molecular mechanism of ACIII is unknown and so far the complex has remained inaccessible for genetic modifications. The recently solved cryo-electron microscopy (cryo-EM) structures of ACIII from Flavobacterium johnsoniae, Rhodothermus marinus, and Roseiflexus castenholzii revealed no structural similarity to cytochrome bc 1 / b 6 f and there were variations in the heme-containing subunits ActA and ActE. These data implicated intriguing alternative electron transfer paths connecting ACIII with its redox partner, and left the contributions of ActE and the terminal domain of ActA to the catalytic mechanism unclear. Here, we report genetic deletion and complementation of F. johnsoniae actA and actE and the functional implications of such modifications. Deletion of actA led to the loss of activity of cytochrome aa 3 Energy conversion is a fundamental process of all organisms, realized by specialized protein complexes, one of which is alternative complex III (ACIII). ACIII is a functional analogue of well-known mitochondrial complex III, but operates according to a different, still unknown mechanism. To understand how ACIII interacts functionally with its protein partners, we developed a genetic system to mutate the Flavobacterium johnsoniae genes encoding ACIII subunits. Deletion and complementation of heme-containing subunits revealed that ACIII is the sole source of electrons for cytochrome actE did not impair the activity of cytochrome aa 3 , revealing that ActE is not required for electron transfer between ACIII and cytochrome aa 3 . Nevertheless, absence of ActE negatively impacted the cell growth rate, pointing toward another, yet unidentified, function of this subunit. Possible explanations for these observations, including a proposal of a split in electron paths at the ActA/ActE interface, are discussed. The described system for genetic manipulations in F. johnsoniae ACIII offers new tools for studying the molecular mechanism of operation of this enzyme. IMPORTANCE Energy conversion is a fundamental process of all organisms, realized by specialized protein complexes, one of which is alternative complex III (ACIII). ACIII is a functional analogue of well-known mitochondrial complex III, but operates according to a different, still unknown mechanism. To understand how ACIII interacts functionally with its protein partners, we developed a genetic system to mutate the Flavobacterium johnsoniae genes encoding ACIII subunits. Deletion and complementation of heme-containing subunits revealed that ACIII is the sole source of electrons for cytochrome aa 3 and that one of the redox-active subunits (ActE) is dispensable for electron transfer between these complexes. This study sheds light on the operation of the supercomplex of ACIII and cytochrome aa 3 and suggests a division in the electron path within ACIII. It also shows a way to manipulate protein expression levels for application in other members of the Bacteroidetes phylum.

The type IX secretion system (T9SS) is specific to the Bacteroidetes phylum. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilises the T9SS to transport many proteins—including its gingipain virulence factors—across the outer membrane and attach them to the cell surface. Additionally, the T9SS is also required for gliding motility in motile organisms, such as Flavobacterium johnsoniae. At least nineteen proteins have been identified as components of the T9SS, including the three transcription regulators, PorX, PorY and SigP. Although the components are known, the overall organisation and the molecular mechanism of how the T9SS operates is largely unknown. This review focusses on the recent advances made in the structure, function, and organisation of the T9SS machinery to provide further insight into this highly novel secretion system. [ABSTRACT FROM AUTHOR]

Flavobacterial infections are among the causes of fish losses in farms with the emergence of antibiotic-resistant isolates. Silver nanoparticles (AgNPs) are known for their potent antimicrobial activity against different types of bacteria. In this study, we evaluated the antibacterial properties of AgNPs (diameter: 23 nm) against Flavobacterium johnsoniae infection in common carp Cyprinus carpio. The assays included both in vitro and in vivo antibacterial tests in addition to evaluation of cell toxicity effects on the fish cell lines. The in vitro results revealed potent inhibitory effects of AgNPs on the growth of F. johnsoniae with a minimum inhibitory concentration of 34 µg ml-1. Fish cell (epithelioma papulosum cyprini and koi carp fin) viability was 95-100% after exposure to 500 ng ml-1 (and lower concentrations) of AgNPs. In the exposure experiment, mortality rates decreased from 45% in the infected non-treated group to 30 and 15% in the intraperitoneal injection and immersion-treated groups, respectively. Neither of the treated groups showed any clinical signs or histopathological lesions. The single-dose treatment with AgNPs during early infection with F. johnsoniae aided in minimizing fish losses.