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Inborn errors of tyrosine metabolism result in patient's inability to degrade tyrosine. Current treatment consists of a phenylalanine and tyrosine restricted diet and nitisinone, causing a block in the tyrosine degradation pathway. However, tyrosine levels will increase, leading to acquired hypertyrosinemia, implying the need for an add-on treatment. Tyrosine ammonia lyases (TAL) can provide such an add-on treatment as they catalyze the deamination of tyrosine into p-coumaric acid and ammonia. In this study, we developed a robust high-throughput screening (HTS) assay to assess the capacity of bacterial TAL enzymes to decrease excessive tyrosine. The assay is based on the spectrophotometric quantification of p-coumaric acid after conversion of tyrosine by bacterial TAL. As a benchmark, TAL from Flavobacterium johnsoniae (FjTAL) was used to optimize the assay. Optimal growth conditions for high-level protein expression were determined by incubating transformed Escherichia coli BL21 (DE3) cells at different temperatures during various incubation times. Subsequently, assay temperature and pH were optimized followed by testing different ratios of tyrosine assay mixes to bacterial lysate. Finally, assay robustness and functionality were evaluated. Optimal FjTAL expression was obtained after incubation for 24 h at 22 °C. Ideal assay conditions consist of a 80/20 ratio of 1 mM tyrosine assay mix to FjTAL lysate performed at pH 9.2 and 37 °C. The robustness test showed Z' values > 0.4 and signal window values > 2 without edge or drift effects. As proof-of-principle, we successfully determined the catalytic activity of two other bacterial TAL enzymes RsTAL (5.718.10 -3  ± 0.21.10 -3 ) and SeSAM8 (4.658.10 -3  ± 0.37.10 -3 ). A robust, simple and reliable HTS assay was thus developed to evaluate the tyrosine degradation capacity of bacterial TAL enzymes., (© 2024. The Author(s).)

The α-1,3-glucanase Agl-EK14 from Flavobacterium sp. EK-14 comprises a signal peptide (SP), a catalytic domain (CAT), a first immunoglobulin-like domain (Ig1), a second immunoglobulin-like domain (Ig2), a ricin B-like lectin domain (RicinB), and a carboxy-terminal domain (CTD). SP and CTD are predicted to be involved in extracellular secretion, while the roles of Ig1, Ig2, and RicinB are unclear. To clarify their roles, domain deletion enzymes Agl-EK14ΔRicinB, Agl-EK14ΔIg2RicinB, and Agl-EK14ΔIg1Ig2RicinB were constructed. The insoluble α-1,3-glucan hydrolytic, α-1,3-glucan binding, and fungal cell wall hydrolytic activities of the deletion enzymes were almost the same and lower than those of Agl-EK14. Kinetic analysis revealed that the K m values of the deletion enzymes were similar and uniformly higher than those of Agl-EK14. These results suggest that the deletion of RicinB causes a decline in binding and hydrolytic activity and increases the K m value. To confirm the role of RicinB, Ig1, Ig2, and RicinB were fused with green fluorescent protein (GFP). As a result, RicinB-fused GFP (GFP-RicinB) showed binding to insoluble α-1,3-glucan and Aspergillus oryzae cell walls, whereas Ig1- and Ig2-fused GFP did not. These results indicated that RicinB is involved in α-1,3-glucan binding. The fusion protein GFP-Ig1Ig2RicinB was also constructed and GFP-Ig1Ig2RicinB showed strong binding to the cell wall of A. oryzae compared to GFP-RicinB. Gel filtration column chromatography suggested that the strong binding was due to GFP-Ig1Ig2RicinB loosely associated with itself., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Monolignols and their derivatives exhibit various pharmaceutical and physiological characteristics, such as antioxidant and anti-inflammatory properties. However, they remain difficult to synthesize. In this study, we engineered several whole-cell bioconversion systems with carboxylate reductase (CAR)-mediated pathways for efficient synthesis of p -coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine in Escherichia coli BL21 (DE3). By overexpressing the l-tyrosine ammonia lyase from Flavobacterium johnsoniae (FjTAL), carboxylate reductase from Segniliparus rugosus (SruCAR), alcohol dehydrogenase YqhD and hydroxylase HpaBC from E. coli , and caffeate 3-O-methyltransferase (COMT) from Arabidopsis thaliana , three enzyme cascades FjTAL-SruCAR-YqhD, FjTAL-SruCAR-YqhD-HpaBC, and FjTAL-SruCAR-YqhD-HpaBC-COMT were constructed to produce 1028.5 mg/L p -coumaryl alcohol, 1015.3 mg/L caffeyl alcohol, and 411.4 mg/L coniferyl alcohol from 1500, 1500, and 1000 mg/L l-tyrosine, with productivities of 257.1, 203.1, and 82.3 mg/L/h, respectively. This work provides an efficient strategy for the biosynthesis of p -coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine.

Plant root-associated environments such as the rhizosphere, rhizoplane, and endosphere, are notably different from non-root-associated soil environments. However, the microbial dynamics in these spatially divided compartments remain unexplored. In this study, we propose a combinational analysis of single-cell genomics with 16S rRNA gene sequencing. This method enabled us to understand the entire soil microbiome and individual root-associated microorganisms. We applied this method to soybean microbiomes and revealed that their composition was different between the rhizoplane and rhizosphere in the early growth stages, but became more similar as growth progressed. In addition, a total of 610 medium- to high-quality single-amplified genomes (SAGs) were acquired, including plant growth-promoting rhizobacteria (PGPR) candidates while genomes with high GC content tended to be missed by SAGs. The whole-genome analyses of the SAGs suggested that rhizoplane-enriched Flavobacterium solubilizes organophosphate actively and Bacillus colonizes roots more efficiently. Single-cell genomics, together with 16S rRNA gene sequencing, enabled us to connect microbial taxonomy and function, and assess microorganisms at a strain resolution even in the complex soil microbiome., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Environmental pollution stands as one of the significant global challenges we face today. Polycyclic aromatic hydrocarbons (PAHs), a class of stubborn organic pollutants, have long been a focal point of bioremediation research. This study aims to explore the impact and mechanisms of graphene oxide (GO) on the phytoremediation effectiveness of PAHs. The results underscore the significant efficacy of GO in accelerating the degradation of PAHs. Additionally, the introduction of GO altered the diversity and community structure of endophytic bacteria within the roots, particularly those genera with potential for PAH degradation. Through LEfSe analysis and correlation studies, we identified specific symbiotic bacteria, such as Mycobacterium , Microbacterium , Flavobacterium , Sphingomonas , Devosia , Bacillus , and Streptomyces , which coexist and interact under the influence of GO, synergistically degrading PAHs. These bacteria may serve as key biological markers in the PAH degradation process. These findings provide new theoretical and practical foundations for the application of nanomaterials in plant-based remediation of polluted soils and showcase the immense potential of plant-microbe interactions in environmental restoration.

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 PorX Fj , the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorX Fj 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, PorX Fj crystallized as a dimer identical to active PorX. The CheY-like domain of PorX Fj is in an active-like conformation, and PorX Fj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX., (© 2024. The Author(s).)

The glycoside hydrolase (GH) 87 α-1,3-glucanase (Agl-EK14) gene was cloned from the genomic DNA of the gram-negative bacterium Flavobacterium sp. EK14. The gene consisted of 2940 nucleotides and encoded 980 amino acid residues. The deduced amino acid sequence of Agl-EK14 included a signal peptide, a catalytic domain, a first immunoglobulin-like domain, a second immunoglobulin-like domain, a ricin B-like lectin domain, and a carboxyl-terminal domain (CTD) involved in extracellular secretion. Phylogenetic analysis of the catalytic domain of GH87 enzymes suggested that Agl-EK14 is distinct from known clusters, such as clusters composed of α-1,3-glucanases from bacilli and mycodextranases from actinomycetes. Agl-EK14 without the signal peptide and CTD hydrolyzed α-1,3-glucan, and the reaction residues from 1 and 2% substrates were almost negligible after 1440 min reaction. Agl-EK14 hydrolyzed the cell wall preparation of Aspergillus oryzae and released glucose, nigerose, and nigero-triose from the cell wall preparation. After treatment of A. oryzae live mycelia with Agl-EK14 (at least 0.5 nmol/ml), mycelia were no longer stained by red fluorescent protein-fused α-1,3-glucan binding domains of α-1,3-glucanase Agl-KA from Bacillus circulans KA-304. Results suggested that Agl-EK14 can be applied to a fungal cell wall lytic enzyme., (© 2023. The Author(s).)

Dextran is an α-(1→6)-glucan that is synthesized by some lactic acid bacteria, and branched dextran with α-(1→2)-, α-(1→3)-, and α-(1→4)-linkages are often produced. Although many dextranases are known to act on the α-(1→6)-linkage of dextran, few studies have functionally analyzed the proteins involved in degrading branched dextran. The mechanism by which bacteria utilize branched dextran is unknown. Earlier, we identified dextranase (FjDex31A) and kojibiose hydrolase (FjGH65A) in the dextran utilization locus (FjDexUL) of a soil Bacteroidota Flavobacterium johnsoniae and hypothesized that FjDexUL is involved in the degradation of α-(1→2)-branched dextran. In this study, we demonstrate that FjDexUL proteins recognize and degrade α-(1→2)- and α-(1→3)-branched dextrans produced by Leuconostoc citreum S-32 (S-32 α-glucan). The FjDexUL genes were significantly upregulated when S-32 α-glucan was the carbon source compared with α-glucooligosaccharides and α-glucans, such as linear dextran and branched α-glucan from L. citreum S-64. FjDexUL glycoside hydrolases synergistically degraded S-32 α-glucan. The crystal structure of FjGH66 shows that some sugar-binding subsites can accommodate α-(1→2)- and α-(1→3)-branches. The structure of FjGH65A in complex with isomaltose supports that FjGH65A acts on α-(1→2)-glucosyl isomaltooligosaccharides. Furthermore, two cell surface sugar-binding proteins (FjDusD and FjDusE) were characterized, and FjDusD showed an affinity for isomaltooligosaccharides and FjDusE for dextran, including linear and branched dextrans. Collectively, FjDexUL proteins are suggested to be involved in the degradation of α-(1→2)- and α-(1→3)-branched dextrans. Our results will be helpful in understanding the bacterial nutrient requirements and symbiotic relationships between bacteria at the molecular level., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Ribosomes of Bacteroidia (formerly Bacteroidetes) fail to recognize Shine-Dalgarno (SD) sequences even though they harbor the anti-SD (ASD) of 16S rRNA. Inhibition of SD-ASD pairing is due to sequestration of the 3' tail of 16S rRNA in a pocket formed by bS21, bS18, and bS6 on the 30S platform. Interestingly, in many Flavobacteriales, the gene encoding bS21, rpsU, contains an extended SD sequence. In this work, we present genetic and biochemical evidence that bS21 synthesis in Flavobacterium johnsoniae is autoregulated via a subpopulation of ribosomes that specifically lack bS21. Mutation or depletion of bS21 in the cell increases translation of reporters with strong SD sequences, such as rpsU'-gfp, but has no effect on other reporters. Purified ribosomes lacking bS21 (or its C-terminal region) exhibit higher rates of initiation on rpsU mRNA and lower rates of initiation on other (SD-less) mRNAs than control ribosomes. The mechanism of autoregulation depends on extensive pairing between mRNA and 16S rRNA, and exceptionally strong SD sequences, with predicted pairing free energies of < -13 kcal/mol, are characteristic of rpsU across the Bacteroidota. This work uncovers a clear example of specialized ribosomes in bacteria., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Flavobacterium johnsoniae cells move rapidly over solid surfaces by gliding motility. The collective migration of F. johnsoniae on the surfaces results in the formation of spreading colonies. Colony spreading is influenced by adhesin components on the cell surface and the concentrations of agar and glucose. For example, on nutrient-poor agar media, film-like, round spreading colonies are formed. F. johnsoniae displays at least two types of migration: small cell cluster movements leading to concentric colonies and individual cell movements leading to dendritic colonies. The methods for observing colony morphology are described in this chapter., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Many phylum Bacteroidetes bacteria are motile without either flagella or pili. These cells move on surfaces such as glass or agar, and a motor generates a propulsion force for the cells via a proton motive force across the cytoplasmic membrane. The gliding motility depends on the helical track of cell adhesin along the longer axis of the cell body. Here, we describe live-cell imaging of gliding motility under optical microscopy, as well as an immunofluorescent labeling method for visualizing helical trajectories., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Haloacid dehalogenases are potentially involved in bioremediation of contaminated environments and few have been biochemically characterized from marine organisms. The l-2-haloacid dehalogenase (l-2-HAD) from the marine Bacteroidetes Zobellia galactanivorans Dsij T (ZgHAD) has been shown to catalyze the dehalogenation of C2 and C3 short-chain l-2-haloalkanoic acids. To better understand its catalytic properties, its enzymatic stability, active site, and 3D structure were analyzed. ZgHAD demonstrates high stability to solvents and a conserved catalytic activity when heated up to 60°C, its melting temperature being at 65°C. The X-ray structure of the recombinant enzyme was solved by molecular replacement. The enzyme folds as a homodimer and its active site is very similar to DehRhb, the other known l-2-HAD from a marine Rhodobacteraceae. Marked differences are present in the putative substrate entrance sites of the two enzymes. The H179 amino acid potentially involved in the activation of a catalytic water molecule was confirmed as catalytic amino acid through the production of two inactive site-directed mutants. The crystal packing of 13 dimers in the asymmetric unit of an active-site mutant, ZgHAD-H179N, reveals domain movements of the monomeric subunits relative to each other. The involvement of a catalytic His/Glu dyad and substrate binding amino acids was further confirmed by computational docking. All together our results give new insights into the catalytic mechanism of the group of marine l-2-HAD., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

In the CAZy database, the α-amylase family GH13 has already been divided into 45 subfamilies, with additional subfamilies still emerging. The presented in silico study was undertaken in an effort to propose a novel GH13 subfamily represented by the experimentally characterized cyclomaltodxtrinase from Flavobacterium sp. No. 92. Although most cyclomaltodextrinases have been classified in the subfamily GH13&#95;20. This one has not been assigned any GH13 subfamily as yet. It possesses a non-specified immunoglobulin-like domain at its N-terminus mimicking a starch-binding domain (SBD) and the segment MPDLN in its fifth conserved sequence region (CSR) typical, however, for the subfamily GH13&#95;36. The searches through sequence databases resulted in collecting a group of 108 homologs forming a convincing cluster in the evolutionary tree, well separated from all remaining GH13 subfamilies. The members of the newly proposed subfamily share a few exclusive sequence features, such as the "aromatic" end of the CSR-II consisting of two well-conserved tyrosines with either glycine, serine, or proline in the middle or a glutamic acid succeeding the catalytic proton donor in the CSR-III. Concerning the domain N of the representative cyclomaltodextrinase, docking trials with α-, β- and γ-cyclodextrins have indicated it may represent a new type of SBD. This new GH13 subfamily has been assigned the number GH13&#95;46.

Latest solutions in biotechnologies and biosensing targeted cold-active extremozymes. Analysis of acetaldehyde as a relevant quality indicator of wine is one example of application that could benefit from using low-temperatures operating catalysts. In search of novel aldehyde dehydrogenases (ALDH) with high stability and activity at low temperatures, the recombinant S2-ALDH from the Antarctic Flavobacterium PL002 was obtained by cloning and expression in Escherichia coli BL21(DE3). Structural and phylogenetic analyses revealed strong protein similarities (95%) with psychrophilic homologs, conserved active residues and structural elements conferring enzyme flexibility. Arrhenius plot revealed a conformational shift at 30 °C, favoring catalysis (low activation energy) at lower temperatures. In addition to a broad substrate specificity with preference for acetaldehyde (Km = 1.88 mM), this enzyme showed a high tolerance for ethanol (15%) and several salts and chelators (an advantage for wine analysis), while being sensitive to mercury (I 50  = 1.21 µM). The neutral optimal pH (7.5) and the stability up to 40 °C and after lyophilization represent major assets for developing S2-ALDH-based sensors. An enzymatic electrochemical assay was developed for acetaldehyde detection in wines with proven accuracy in comparison with the reference spectrophotometric method, thus evidencing the potential of S2-ALDH as effective biocatalyst for industry and biosensing., (© 2022. The Author(s).)

Carbohydrate-active enzymes are important components of the polysaccharide metabolism system in marine bacteria. Carrageenase is indispensable for forming carrageenan catalytic pathways. Here, two GH16&#95;13 carrageenases showed likely hydrolysis activities toward different types of carrageenans (e.g., κ-, hybrid β/κ, hybrid α/ι, and hybrid λ), which indicates that a novel pathway is present in the marine bacterium Flavobacterium algicola to use κ-carrageenan (KC), ι-carrageenan (IC), and λ-carrageenan (LC). A comparative study described the different features with another reported pathway based on the specific carrageenans (κ, ι, and λ) and expanded the carrageenan metabolic versatility in F. algicola . A further comparative genomic analysis of carrageenan-degrading bacteria indicated different distributions of carrageenan metabolism-related genes in marine bacteria. The crucial core genes encoding the GH127 α-3,6-anhydro-d-galactosidase (ADAG) and 3,6-anhydro-d-galactose (d-AHG)-utilized cluster have been conserved during evolution. This analysis further revealed the horizontal gene transfer (HGT) phenomenon of the carrageenan polysaccharide utilization loci (CarPUL) from Bacteroidetes to other bacterial phyla, as well as the versatility of carrageenan catalytic activities in marine bacteria through different metabolic pathways. IMPORTANCE Based on the premise that the specific carrageenan-based pathway involved in carrageenan use by Flavobacterium algicola has been identified, another pathway was further analyzed, and it involved two GH16&#95;13 carrageenases. Among all the characterized carrageenases, the members of GH16&#95;13 accounted for only a small portion. Here, the functional analysis of two GH16&#95;13 carrageenases suggested their hydrolysis effects on different types of carrageenans (e.g., κ, hybrid β/κ, hybrid α/ι-, and hybrid λ-), which led to the identification of another pathway. Further exploration enabled us to elucidate the novel pathway that metabolizes KC and IC in F. algicola successfully. The coexistence of these two pathways may provide improved survivability by F. algicola in the marine environment.

Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish. F. columnare virulence mechanisms are not well understood, and current methods to control columnaris disease are inadequate. Iron acquisition from the host is important for the pathogenicity and virulence of many bacterial pathogens. F. columnare iron acquisition has not been studied in detail. We identified genes predicted to function in siderophore production for ferric iron uptake. Genes predicted to encode the proteins needed for siderophore synthesis, export, uptake, and regulation were deleted from F. columnare strain MS-FC-4. The mutants were examined for defects in siderophore production, for growth defects in iron-limited conditions, and for virulence against zebrafish and rainbow trout. Mutants lacking all siderophore activity were obtained. These mutants displayed growth defects when cultured under iron-limited conditions, but they retained virulence against zebrafish and rainbow trout similar to that exhibited by the wild type, indicating that the F. columnare MS-FC-4 siderophores are not required for virulence under the conditions tested. IMPORTANCE Columnaris disease, which is caused by Flavobacterium columnare, is a major problem for freshwater aquaculture. Little is known regarding F. columnare virulence factors, and control measures are limited. Iron acquisition mechanisms such as siderophores are important for virulence of other pathogens. We identified F. columnare siderophore biosynthesis, export, and uptake genes. Deletion of these genes eliminated siderophore production and resulted in growth defects under iron-limited conditions but did not alter virulence in rainbow trout or zebrafish. The results indicate that the F. columnare strain MS-FC-4 siderophores are not critical virulence factors under the conditions tested but may be important for survival under iron-limited conditions in natural aquatic environments or aquaculture systems.

Fucoidans are hetero-sulfated polysaccharides that are widely distributed in brown algae and have been extensively studied for their various biological activities. The structure-function relationship of fucoidans remains unclear but can be studied using fucoidan-degrading enzymes (fucoidanases). Here, we isolated and identified Flavobacterium sp. SW as a microbial strain that can grow on fucoidan from Cladosiphon okamuranus as the sole carbon source. Genomic analysis of this strain revealed the presence of two genes, swfct and swfcn2, that are homologous to fct114 from Luteolibacter algae H18 and fcnA from Psychromonas sp. SW5A, respectively. The gene products were produced in Escherichia coli and showed significantly different specificities for fucoidan. Swfct catalyzed the degradation of deacetylated fucoidan from C. okamuranus, and Swfcn2 degraded fucoidans from Saccharina sculpera and Macrocystis pyrifera. The general properties of Swfct were examined by measuring the amounts of reducing ends produced by the enzymatic reaction, and the enzyme properties of Swfcn2 were evaluated by carbohydrate-polyacrylamide gel electrophoresis. Our findings indicate that one microbial strain can harbor genes encoding two different types of fucoidanases., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Fucoidans represent a type of polyanionic fucose-containing sulfated polysaccharides (FCSPs) that are cleaved by fucoidan-degrading enzymes, producing low-molecular-weight fucoidans with multiple biological activities suitable for pharmacological use. Most of the reported fucoidan-degrading enzymes are glycoside hydrolases, which have been well studied for their structures and catalytic mechanisms. Little is known, however, about the rarer fucoidan lyases, primarily due to the lack of structural information. FdlA from Flavobacterium sp. SA-0082 is an endo-type fucoidan-degrading enzyme that cleaves the sulfated fuco-glucuronomannan (SFGM) through a lytic mechanism. Here, we report nine crystal structures of the catalytic N-terminal domain of FdlA (FdlA-NTD), in both its wild type (WT) and mutant forms, at resolutions ranging from 1.30 to 2.25 Å. We show that the FdlA-NTD adopts a right-handed parallel β-helix fold, and possesses a substrate binding site composed of a long groove and a unique alkaline pocket. Our structural, biochemical, and enzymological analyses strongly suggest that FdlA-NTD utilizes catalytic residues different from other β-helix polysaccharide lyases, potentially representing a novel polysaccharide lyase family.

Background: Melanins are a heterologous group of biopolymeric pigments synthesized by diverse prokaryotes and eukaryotes and are widely utilized as bioactive materials and functional polymers in the biotechnology industry. Here, we report the high-level melanin production using a new melanogenic Flavobacterium kingsejongi strain and a recombinant Escherichia coli overexpressing F. kingsejongi 4-hydroxyphenylpyruvate dioxygenase (HPPD)., Results: Melanin synthesis of F. kingsejongi strain was confirmed via melanin synthesis inhibition test, melanin solubility test, genome analysis, and structural analysis of purified melanin from both wild-type F. kingsejongi and recombinant E. coli expressing F. kingsejongi HPPD. The activity of F. kingsejongi HPPD was demonstrated via in vitro assays with 6 × His-tagged and native forms of HPPD. The specific activity of F. kingsejongi HPPD was 1.2 ± 0.03 μmol homogentisate/min/mg-protein. Bioreactor fermentation of F. kingsejongi produced a large amount of melanin with a titer of 6.07 ± 0.32 g/L, a conversion yield of 60% (0.6 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.03 g/L·h, indicating its potential for industrial melanin production. Additionally, bioreactor fermentation of recombinant E. coli expressing F. kingsejongi HPPD produced melanin at a titer of 3.76 ± 0.30 g/L, a conversion yield of 38% (0.38 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.04 g/L·h., Conclusions: Both strains showed sufficiently high fermentation capability to indicate their potential as platform strains for large-scale bacterial melanin production. Furthermore, F. kingsejongi strain could serve as a model to elucidate the regulation of melanin biosynthesis pathway and its networks with other cellular pathways, and to understand the cellular responses of melanin-producing bacteria to environmental changes, including nutrient starvation and other stresses., (© 2022. The Author(s).)

Marine bacteria usually contain polysaccharide utilization loci (PUL) for metabolizing red algae polysaccharides. They are of great significance in the carbon cycle of the marine ecosystem, as well as in supporting marine heterotrophic bacterial growth. Here, we described the whole κ-carrageenan (KC), ι-carrageenan (IC), and partial λ-carrageenan (LC) catabolic pathways in a marine Gram-negative bacterium, Flavobacterium algicola, which is involved carrageenan polysaccharide hydrolases, oligosaccharide sulfatases, oligosaccharide glycosidases, and the 3,6-anhydro-d-galactose (d-AHG) utilization-related enzymes harbored in the carrageenan-specific PUL. In the pathways, the KC and IC were hydrolyzed into 4-sugar-unit oligomers by specific glycoside hydrolases. Then, the multifunctional G4S sulfatases would remove their nonreducing ends' G4S sulfate groups, while the ι-neocarratetrose (Nι4) product would further lose the nonreducing end of its DA2S group. Furthermore, the neocarrageenan oligosaccharides (NCOSs) with no G4S and DA2S groups in their nonreducing ends would completely be decomposed into d-Gal and d-AHG. Finally, the released d-AHG would enter the cytoplasmic four-step enzymatic process, and an l-rhamnose-H + transporter (RhaT) was preliminarily verified for the function for transportation of d-AHG. Moreover, comparative analysis with the reported carrageenan metabolism pathways further implied the diversity of microbial systems for utilizing the red algae carrageenan. IMPORTANCE Carrageenan is the main polysaccharide of red macroalgae and is composed of d-AHG and d-Gal. The carrageenan PUL (CarPUL)-encoded enzymes exist in many marine bacteria for decomposing carrageenan to provide self-growth. Here, the related enzymes in Flavobacterium algicola for metabolizing carrageenan were characterized for describing the catabolic pathways, notably, although the specific polysaccharide hydrolases existed that were like previous studies. A multifunctional G4S sulfatase also existed, which was devoted to the removal of G4S or G2S sulfate groups from three kinds of NCOSs. Additionally, the transformation of three types of carrageenans into two monomers, d-Gal and d-AHG, occurred outside the cell with no periplasmic reactions that existed in previously reported pathways. These results help to clarify the diversity of marine bacteria using macroalgae polysaccharides.

Motile bacteria usually rely on external apparatus like flagella for swimming or pili for twitching. By contrast, gliding bacteria do not rely on obvious surface appendages to move on solid surfaces. Flavobacterium johnsoniae and other bacteria in the Bacteroidetes phylum use adhesins whose movement on the cell surface supports motility. In F. johnsoniae, secretion and helicoidal motion of the main adhesin SprB are intimately linked and depend on the type IX secretion system (T9SS). Both processes necessitate the proton motive force (PMF), which is thought to fuel a molecular motor that comprises the GldL and GldM cytoplasmic membrane proteins. Here, we show that F. johnsoniae gliding motility is powered by the pH gradient component of the PMF. We further delineate the interaction network between the GldLM transmembrane helices (TMHs) and show that conserved glutamate residues in GldL TMH2 are essential for gliding motility, although having distinct roles in SprB secretion and motion. We then demonstrate that the PMF and GldL trigger conformational changes in the GldM periplasmic domain. We finally show that multiple GldLM complexes are distributed in the membrane, suggesting that a network of motors may be present to move SprB along a helical path on the cell surface. Altogether, our results provide evidence that GldL and GldM assemble dynamic membrane channels that use the proton gradient to power both T9SS-dependent secretion of SprB and its motion at the cell surface., Competing Interests: The authors have declared that no competing interests exist.

The regeneration of bioavailable phosphate from immobilized organophosphorus represents a key process in the global phosphorus cycle and is facilitated by enzymes known as phosphatases. Most bacteria possess at least one of three phosphatases with broad substrate specificity, known as PhoA, PhoX, and PhoD, whose activity is optimal under alkaline conditions. The production and activity of these phosphatases is repressed by phosphate availability. Therefore, they are only fully functional when bacteria experience phosphorus-limiting growth conditions. Here, we reveal a previously overlooked phosphate-insensitive phosphatase, PafA, prevalent in Bacteroidetes , which is highly abundant in nature and represents a major route for the regeneration of environmental phosphate. Using the enzyme from Flavobacterium johnsoniae , we show that PafA is highly active toward phosphomonoesters, is fully functional in the presence of excess phosphate, and is essential for growth on phosphorylated carbohydrates as a sole carbon source. These distinct properties of PafA may expand the metabolic niche of Bacteroidetes by enabling the utilization of abundant organophosphorus substrates as C and P sources, providing a competitive advantage when inhabiting zones of high microbial activity and nutrient demand. PafA, which is constitutively synthesized by soil and marine flavobacteria, rapidly remineralizes phosphomonoesters releasing bioavailable phosphate that can be acquired by neighboring cells. The pafA gene is highly diverse in plant rhizospheres and is abundant in the global ocean, where it is expressed independently of phosphate availability. PafA therefore represents an important enzyme in the context of global biogeochemical cycling and has potential applications in sustainable agriculture., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Alginate-assimilating bacteria degrade alginate into an unsaturated monosaccharide, which is converted into 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEHU). DEHU is reduced to 2-keto-3-deoxy-D-gluconate by a DEHU-specific reductase using NAD(P)H. This is followed by pyruvate production via the Entner-Doudoroff pathway. Previously, we identified FlRed as a DEHU reductase in an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. Here, we showed that FlRed can also catalyze the oxidation of DEHU with NAD + , producing 2-keto-3-deoxy-D-glucarate (KDGR). FlRed showed a predilection for NADH and NAD + over NADPH and NADP + , respectively, and the K m value for NADH was approximately 2.6-fold less than that for NAD + . Furthermore, we identified two key enzymes, FlDet and FlDeg, for KDGR catabolism. FlDet was identified as an enzyme of the ribonuclease activity regulator A family, which converts KDGR to α-ketoglutaric semialdehyde (α-KGSA). FlDeg, a type II α-KGSA dehydrogenase, generated α-ketoglutaric acid by oxidizing the aldehyde group of α-KGSA using NAD(P) + . Consequently, unlike the conventional DEHU reduction pathway, DEHU can be directly converted to α-ketoglutaric acid without consuming NAD(P)H. Alginate upregulated the expression of not only FlRed and two enzymes of the DEHU-reduction pathway, but also FlDet and FlDeg. These results revealed dual pathways of DEHU metabolism involving reduction or oxidation by FlRed., (© 2021. The Author(s).)

Bacterial alternative complex III (ACIII) catalyzes menaquinol (MKH 2 ) oxidation, presumably fulfilling the role of cytochromes bc 1 / 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 (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 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.

Group II C-type lectin domain (CTLD) containing receptors belong to a large family of pattern recognition receptors which mainly act on the innate immunity. They are structurally related and consist of a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and a single extracellular CTLD. Although they have been described in teleost fish, their involvement in immune responses is not well understood. In this study, four immune-related lectin-like receptors (termed CiILLR1 and CiILLR5-7), belonging to the group II CTLD receptors, were identified in grass carp (Ctenopharyngodon idella). They contain a short cytoplasmic tail and a single CTLD in the extracellular region. The CiILLR1 has a WxHxxxxxY motif similar to the WxHxxxxY motif which is required for the recognition of β-glucans by some of the group II CTLD containing lectins in mammals. Further, a modified QPD motif (EPD) known to be involved in binding to carbohydrate ligands is present in the CiILLR1, 5 and 6. However, CiILLR7 lacks these motifs. Expression analysis revealed that they were constitutively expressed in the head kidney and spleen. Moreover, CiILLR1, 5 and 6 could be up-regulated in the head kidney and spleen of fish after infection with Flavobacterium columnare and in the primary head kidney leukocytes by LPS and PHA. Expression of CiILLR1, CiILLR5 and CiILLR6 were mainly detected in the enriched lymphocytes whilst CiILLR7 was expressed in the enriched monocytes/macrophages. The results expand existing knowledge on the immune responses of the C-type lectin receptors in teleost fish., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Iron is essential for growth and virulence in most pathogenic bacterial strains. In some cases, the hosts for these pathogenic bacteria develop specialized strategies to sequester iron and limit infectivity. This in turn may result in the invading pathogens utilizing high-affinity iron transport mechanisms, such as the use of iron-chelating siderophores, to extend beyond the host defences. Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD) in salmonids, relies on iron metabolism for infectivity, and the genome of the model CSF-259-93 strain has recently been made available. Further, this strain serves as a parent strain for a live-attenuated vaccine strain, B.17, which has been shown to provide rainbow trout with protection against BCWD. To elucidate specific gene expression responses to iron metabolism and compare strain differences, both F. psychrophilum strains were grown under iron-limiting conditions and 26 genes related to iron metabolism were mapped for 96 hr in culture via qPCR analyses. Results indicate increased production of the ferrous iron transport protein B (FITB; p =.008), and ferric receptor CfrA (FR 1; p =.012) in the wild-type CSF-259-93 strain at 72 hr and 96 hr post-exposure to iron-limiting media. In the B.17 vaccine strain, siderophore synthase (SS) expression was found to be downregulated at 72 hr, in comparison with 0h (p =.018). When strains were compared, FITB (p =.021), FR1 (p =.009) and SS (p =.016) were also elevated in B.17 at 0 hr and TonB outer protein membrane receptor 1 (TBomr1; p =.005) had a lower expression at 96 hr. Overall, this study demonstrated strain-related gene expression changes in only a fraction of the iron metabolism genes tested; however, results provide insight on potential virulence mechanisms and clarification on iron-related gene expression for F. psychrophilum., (© 2021 John Wiley & Sons Ltd.)

Modern aquaculture systems are designed for intensive rearing of fish or other species. Both land-based and offshore systems typically contain high loads of biomass and the water quality in these systems is of paramount importance for fish health and production. Microorganisms play a crucial role in removal of organic matter and nitrogen-recycling, production of toxic hydrogen sulfide (H 2 S), and can affect fish health directly if pathogenic for fish or exerting probiotic properties. Methods currently used in aquaculture for monitoring certain bacteria species numbers still have typically low precision, specificity, sensitivity and are time-consuming. Here, we demonstrate the use of Digital PCR as a powerful tool for absolute quantification of sulfate-reducing bacteria (SRB) and major pathogens in salmon aquaculture, Moritella viscosa, Yersinia ruckeri and Flavobacterium psychrophilum. In addition, an assay for quantification of Listeria monocytogenes, which is a human pathogen bacterium and relevant target associated with salmonid cultivation in recirculating systems and salmon processing, has been assessed. Sudden mass mortality incidents caused by H 2 S produced by SRB have become of major concern in closed aquaculture systems. An ultra-sensitive assay for quantification of SRB has been established using Desulfovibrio desulfuricans as reference strain. The use of TaqMan® probe technology allowed for the development of multi-plex assays capable of simultaneous quantification of these aquaculture priority bacteria. In single-plex assays, limit of detection was found to be at around 20 fg DNA for M. viscosa, Y. ruckeri and F. psychrophilum, and as low as 2 fg DNA for L. monocytogenes and D. desulfuricans., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

In the past, the potato plant microbiota and rhizosphere have been studied in detail to improve plant growth and fitness. However, less is known about the postharvest potato tuber microbiome and its role in storage stability. The storage stability of potatoes depends on genotype and storage conditions, but the soil in which tubers were grown could also play a role. To understand the ecology and functional role of the postharvest potato microbiota, we planted four potato varieties in five soil types and monitored them until the tubers started sprouting. During storage, the bacterial community of tubers was analysed by next-generation sequencing of the 16S rRNA gene amplicons. The potato tubers exhibited soil-dependent differences in sprouting behaviour. The statistical analysis revealed a strong shift of the tuber-associated bacterial community from harvest to dormancy break. By combining indicator species analysis and a correlation matrix, we predicted associations between members of the bacterial community and tuber sprouting behaviour. Based on this, we identified Flavobacterium sp. isolates, which were able to influence sprouting behaviour by inhibiting potato bud outgrowth.

Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor and a proton-driven motor that powers gliding motility and the type 9 protein secretion system in Bacteroidetes bacteria. Here, we present cryo-electron microscopy structures of the gliding motility/type 9 protein secretion system motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identify protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane.

The plant microbiota play a key role in plant productivity, nutrient uptake, resistance to stress and flowering. The flowering of moso bamboo has been a focus of study. The mechanism of flowering is related to nutrient uptake, temperature, hormone balance and regulation of key genes. However, the connection between microbiota of moso bamboo and its flowering is unknown. In this study, samples of rhizosphere soil, rhizomes, roots and leaves of flowering and nonflowering plants were collected, and 16S rRNA amplicon Illumina sequencing was utilized to separate the bacterial communities associated with different flowering stages of moso bamboo. We identified 5442 OTUs, and the number of rhizosphere soil OTUs was much higher than those of other samples. Principal component analysis (PCA) and hierarchical clustering (Bray Curtis dis) analysis revealed that the bacterial microorganisms related to rhizosphere soil and endophytic tissues of moso bamboo differed significantly from those in bulk soil and rhizobacterial and endosphere microbiomes. In addition, the PCA analyses of root and rhizosphere soil revealed different structures of microbial communities between bamboo that is flowering and not flowering. Through the analysis of core microorganisms, it was found that Flavobacterium, Bacillus and Stenotrophomonas played an important role in the absorption of N elements, which may affect the flowering time of moso bamboo. Our results delineate the complex host-microbe interactions of this plant. We also discuss the potential influence of bacterial microbiome in flowering, which can provide a basis for the development and utilization of moso bamboo.

Colony spreading of Flavobacterium johnsoniae is shown to include gliding motility using the cell surface adhesin SprB, and is drastically affected by agar and glucose concentrations. Wild-type (WT) and ΔsprB mutant cells formed nonspreading colonies on soft agar, but spreading dendritic colonies on soft agar containing glucose. In the presence of glucose, an initial cell growth-dependent phase was followed by a secondary SprB-independent, gliding motility-dependent phase. The branching pattern of a ΔsprB colony was less complex than the pattern formed by the WT. Mesoscopic and microstructural information was obtained by atmospheric scanning electron microscopy (ASEM) and transmission EM, respectively. In the growth-dependent phase of WT colonies, dendritic tips spread rapidly by the movement of individual cells. In the following SprB-independent phase, leading tips were extended outwards by the movement of dynamic windmill-like rolling centers, and the lipoproteins were expressed more abundantly. Dark spots in WT cells during the growth-dependent spreading phase were not observed in the SprB-independent phase. Various mutations showed that the lipoproteins and the motility machinery were necessary for SprB-independent spreading. Overall, SprB-independent colony spreading is influenced by the lipoproteins, some of which are involved in the gliding machinery, and medium conditions, which together determine the nutrient-seeking behavior.

Chitin is one of the most abundant renewable organic materials found on earth. The chitin utilization locus in Flavobacterium johnsoniae, which encodes necessary proteins for complete enzymatic depolymerization of crystalline chitin, has recently been characterized but no detailed structural information on the enzymes was provided. Here we present protein structures of the F. johnsoniae chitobiase (FjGH20) and chitinase B (FjChiB). FjGH20 is a multi-domain enzyme with a helical domain not before observed in other chitobiases and a domain organization reminiscent of GH84 (β-N-acetylglucosaminidase) family members. The structure of FjChiB reveals that the protein lacks loops and regions associated with exo-acting activity in other chitinases and instead has a more solvent accessible substrate binding cleft, which is consistent with its endo-chitinase activity. Additionally, small angle X-ray scattering data were collected for the internal 70 kDa region that connects the N- and C-terminal chitinase domains of the unique 158 kDa multi-domain chitinase A (FjChiA). The resulting model of the molecular envelope supports bioinformatic predictions of the region comprising six domains, each with similarities to either Fn3-like or Ig-like domains. Taken together, the results provide insights into chitin utilization by F. johnsoniae and reveal structural diversity in bacterial chitin metabolism.

Statistical experimental designs were used to formulate a culture medium for zeaxanthin production by an Antarctic Flavobacterium sp. P8 strain. Eleven nutritional factors were assayed in shaken flasks. The effect of temperature on zeaxanthin and carotenoid production was also studied. Peptone, yeast extract, and sodium chloride were the nutrients that caused the principal impact on the biomass growth. These components were further studied to enhance zeaxanthin and total carotenoid concentrations. Although a high production rate of zeaxanthin and carotenoids was achieved, the aerobic characteristics of the bacterial strain and the oxygen requirements for zeaxanthin biosynthesis incorporate a factor that requires additional consideration. Scaling up the process to a 5 L-bioreactor that increased dissolved oxygen availability resulted in a 4.5-fold increase in the total carotenoid content and an almost 9-fold increase in zeaxanthin, which represented 98% of the total carotenoids produced. The results reveal that Flavobacterium sp. P8 is a promising strain for zeaxanthin production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Phthalic acid esters (PAEs), especially dibutyl phthalate (DBP) pollution in the environment, have attracted worldwide attention. Four Phragmites australis-based, mesocosm-scale vertical flow constructed wetlands (VFCWs) with different hydraulic loading rates (HLRs) were operated for one year to study the removal efficiency and mechanisms of DBP in the reclaimed water. The average removal efficiencies for DBP were 93.77 ± 3.27%, 94.9 ± 2.60% and 97.0 ± 3.00% in the VFCWs under HLRs of 0.33, 0.22 and 0.11 m/d, respectively. DBP can be accumulated and degraded by wetland plants and its concentration in the roots (0.256-8.45 mg/kg) were higher than in the leaves (0.243-0.482 mg/kg). The concentrations of primary and secondary metabolites mono-n-butyl phthalate (MBP) and phthalic acid (PA) were 0.142-2.35 mg/kg and 0.113-0.545 mg/kg respectively in the plant tissues. The concentrations of DBP were 38.2-271 μg/kg in the substrates. Mass balance for DBP indicates that the estimated plant uptake and substrate adsorption of total DBP is negligible. This suggests that biodegradation and other process are the primary pathways for DBP removal in VFCWs. The results of 16S rDNA and ITS rDNA high-throughput sequencing indicated that both bacterial and fungal community diversity decreased with the exposure of DBP. Janthinobacterium, Flavobacterium and Curvularia genera may be the main participants in the biodegradation of DBP in the CWs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A Gram-negative, non-motile, rod-shaped bacterial strain, designated GS13 T , was isolated from sediments in a branch of the Nackdong River in Sangju, Korea. Optimal growth occurred at pH 7.0, 20 °C, and 0% NaCl. Phylogenetic analyses using 16S rRNA showed that strain GS13 T is a member of the genus Flavobacterium, with highest similarity to Flavobacterium soyangense IMCC26223 T (97.0%). The DNA G+C content of strain GS13 T was 36.2 mol%. The dominant fatty acids were summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c) and iso-C 15:0 . The major polar lipids were phosphatidylethanolamine, three unidentified aminolipids, three unidentified lipids, and one unidentified aminophospholipid. The predominant respiratory quinone was MK-6. Our data demonstrate that strain GS13 T can be distinguished from closely-related Flavobacterium species. Thus, strain GS13 T is a novel Flavobacterium species, and we propose the name Flavobacterium nackdongense sp. nov. The type strain is GS13 T (=KCTC 62569 T  = JCM 32765 T ).

Heterotrophic bacteria associated with microphytoplankton, particularly those colonizing the phycosphere, are major players in the remineralization of algal-derived carbon. Ocean warming might impact dissolved organic carbon (DOC) uptake by microphytoplankton-associated bacteria with unknown biogeochemical implications. Here, by incubating natural seawater samples at three different temperatures, we analysed the effect of experimental warming on the abundance and C and N uptake activity of Rhodobacteraceae and Flavobacteria, two bacterial groups typically associated with microphytoplankton. Using a nano-scale secondary ion mass spectrometry (nanoSIMS) single-cell analysis, we quantified the temperature sensitivity of these two taxonomic groups to the uptake of algal-derived DOC in the microphytoplankton associated fraction with 13 C-bicarbonate and 15 N-leucine as tracers. We found that cell-specific 13 C uptake was similar for both groups (~0.42 fg C h -1 μm -3 ), but Rhodobacteraceae were more active in 15 N-leucine uptake. Due to the higher abundance of Flavobacteria associated with microphytoplankton, this group incorporated fourfold more carbon than Rhodobacteraceae. Cell-specific 13 C uptake was influenced by temperature, but no significant differences were found for 15 N-leucine uptake. Our results show that the contribution of Flavobacteria and Rhodobacteraceae to C assimilation increased up to sixfold and twofold, respectively, with an increase of 3°C above ambient temperature, suggesting that warming may differently affect the contribution of distinct copiotrophic bacterial taxa to carbon cycling., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The gliding bacterium Flavobacterium johnsoniae is known to have an adhesin, SprB, that moves along the cell surface on a spiral track. Following viscous shear, cells can be tethered by the addition of an anti-SprB antibody, causing spinning at 3 Hz. Labeling the type 9 secretion system (T9SS) with a YFP fusion of GldL showed a yellow fluorescent spot near the rotation axis, indicating that the motor driving the motion is associated with the T9SS. The distance between the rotation axis and the track (90 nm) was determined after adding a Cy3 label for SprB. A rotary motor spinning a pinion of radius 90 nm at 3 Hz would cause a spot on its periphery to move at 1.5 μm/s, the gliding speed. We suggest the pinion drives a flexible tread that carries SprB along a track fixed to the cell surface. Cells glide when this adhesin adheres to the solid substratum., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Methomyl is a broad-spectrum oxime carbamate commonly used to control arthropods, nematodes, flies, and crop pests. However, extensive use of this pesticide in agricultural practices has led to environmental toxicity and human health issues. Oxidation, incineration, adsorption, and microbial degradation methods have been developed to remove insecticidal residues from soil/water environments. Compared with physicochemical methods, biodegradation is considered to be a cost-effective and ecofriendly approach to the removal of pesticide residues. Therefore, micro-organisms have become a key component of the degradation and detoxification of methomyl through catabolic pathways and genetic determinants. Several species of methomyl-degrading bacteria have been isolated and characterized, including Paracoccus , Pseudomonas , Aminobacter , Flavobacterium , Alcaligenes , Bacillus , Serratia , Novosphingobium , and Trametes . The degradation pathways of methomyl and the fate of several metabolites have been investigated. Further in-depth studies based on molecular biology and genetics are needed to elaborate their role in the evolution of novel catabolic pathways and the microbial degradation of methomyl. In this review, we highlight the mechanism of microbial degradation of methomyl along with metabolic pathways and genes/enzymes of different genera.

Increasing evidence shows that static magnetic fields (SMFs) can affect microbial growth metabolism, but the specific mechanism is still unclear. In this study, we have investigated the effect of moderate-strength SMFs on growth and vitamin K 2 biosynthesis of Flavobacterium sp. m1-14. First, we designed a series of different moderate-strength magnetic field intensities (0, 50, 100, 150, 190 mT) and exposure times (0, 24, 48, 72, 120 h). With the optimization of static magnetic field intensity and exposure time, biomass and vitamin K 2 production significantly increased compared to control. The maximum vitamin K 2 concentration and biomass were achieved when exposed to 100 mT SMF for 48 h; compared with the control group, they increased by 71.3% and 86.8%, respectively. Interestingly, it was found that both the cell viability and morphology changed significantly after SMF treatment. Second, the adenosine triphosphate (ATP) and glucose-6-phosphate dehydrogenase (G6PDH) metabolism is more vigorous after exposed to 100 mT SMF. This change affects the cell energy metabolism and fermentation behavior, and may partially explain the changes in bacterial biomass and vitamin K 2 production. The results show that moderate-strength SMFs may be a promising method to promote bacterial growth and secondary metabolite synthesis.

A yellow pigmented, Gram-stain-negative, strictly aerobic, rod-shaped, motile by means of gliding, catalase and oxidase positive bacterium, designated strain DS2-A T , was isolated from soil. Growth was observed at 4-32°C (optimum, 28°C), pH 6-9 (optimum, 7.0), and with 0-0.25% (w/v) NaCl (optimum, 0%). Phylogenetic analysis of 16S rRNA gene sequence revealed that strain DS2-A T belonged to the genus Flavobacterium and was most closely related to Flavobacterium aquatile LMG 4008 T (96.4%), Flavobacterium terrae DSM 18829 T (95.6%), Flavobacterium vireti THG-SM1 T (95.5%), Flavobacterium inkyongense IMCC27201 T (95.4%), Flavobacterium brevivitae TTM-43 T (95.2%), and Flavobacterium cucumis DSM 18830 T (95.2%). Strain DS2-A T produces flexirubin-type pigments. The major fatty acids were iso-C 15:0 , iso-C 17:0 3-OH, and iso-C 15:0 3-OH. The major respiratory quinone was identified as menaquinone-6. The major polar lipid was found to be phosphatidylethanolamine. The average nucleotide identity values between strain DS2-A T and selected taxa, F. aquatile LMG 4008 T , F. terrae DSM 18829 T , and F. cucumis DSM 18830 T , were 72, 72.7, and 71.6%, respectively. The draft genome of strain DS2-A T has a number of 14 contigs, scaffold N50 of 476,310 bp and a total size of 3,563,867 bp. Additionally, strain DS2-A T contains 3,127 of gene, 41 of tRNA, 6 of rRNA, and 3 of ncRNA. The DNA G + C content of stain DS2-A T was 40.7 mol%. Based on phylogenetic and phenotypic analyses, strain DS2-A T is considered as a novel species of the genus Flavobacterium, for which the name Flavobacterium humi sp. nov., (type strain DS2-A T = KACC 19715 T = JCM 32786 T ) has been proposed.

In all cells, initiation of translation is tuned by intrinsic features of the mRNA. Here, we analyze translation in Flavobacterium johnsoniae, a representative of the Bacteroidetes. Members of this phylum naturally lack Shine-Dalgarno (SD) sequences in their mRNA, and yet their ribosomes retain the conserved anti-SD sequence. Translation initiation is tuned by mRNA secondary structure and by the identities of several key nucleotides upstream of the start codon. Positive determinants include adenine at position -3, reminiscent of the Kozak sequence of Eukarya. Comparative analysis of Escherichia coli reveals use of the same Kozak-like sequence to enhance initiation, suggesting an ancient and widespread mechanism. Elimination of contacts between A-3 and the conserved β-hairpin of ribosomal protein uS7 fails to diminish the contribution of A-3 to initiation, suggesting an indirect mode of recognition. Also, we find that, in the Bacteroidetes, the trinucleotide AUG is underrepresented in the vicinity of the start codon, which presumably helps compensate for the absence of SD sequences in these organisms., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Discarded Polyethylene (PE) mulches have posed a persistent pollution in the agricultural field. Although PE degraded by microbes has been reported, the effect on in situ soil environment is not completely understood. To determine the biodegradability and ecological impacts of PE-based mulches, we tested the biodegradation related properties of screened strains and analyzed change in soil quality and microbial community after covering with different mulches. Strain Bacillus aryabhattai 5-3 showed 3.85 ± 0.50% gravimetric weight loss after a 30 days incubation with PE-based mulch as the sole carbon source; Considerable pits and vast cavities were found on the surface using AFM and SEM. The hydrophobicity of mulch sheet was decreased to 68.9 ± 0.8°, FTIR identified that new oxidized groups were formed on the surfaces of incubated mulch. Additionally, the fluctuations of microbial communities indicate that synthetic mulch could replace the current commercial product to minimise agricultural pollution., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Flavobacterium sp. AUG42 is a cellulase-producing bacterium isolated from the Antarctic oligochaete Grania sp. (Annelida). In this work, we report that AUG42 produces a glycoside hydrolase cocktail with CMCase, PASCase and cellobiase activities (optimum pHs and temperatures ranging from 5.5 to 6.5 and 40 to 50 °C, respectively). The time-course analyses of the bacterial growth and cellulase production showed that the cocktail has maximal activity at the stationary phase when growing at 16 °C with filter paper as a cellulosic carbon source, among the tested substrates. The analyses of the CAZome and the identification of secreted proteins by shotgun Mass Spectrometry analysis showed that five glycoside hydrolyses are present in the bacterial secretome, which probably cooperate in the degradation of the cellulosic substrates. Two of these glycoside hydrolyses may harbor putative carbohydrate binding modules, both with a cleft-like active site. The cellulolytic cocktail was assayed in saccharification experiments using carboxymethylcellulose as a substrate and results showed the release of glucose (a fermentable sugar) and other reducing-sugars, after 24 h incubation. The ecological relevance of producing cellulases in the Antarctic environment, as well as their potential use in the bio-refinery industry, are discussed., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

This study presents taxonomic description of two novel diesel-degrading, psychrophilic strains: Kopri-42 T and Kopri-43, isolated during screening of oil-degrading psychrotrophs from oil-contaminated Arctic soil. A preliminary 16S rRNA gene sequence and phylogenetic tree analysis indicated that these Arctic strains belonged to the genus Flavobacterium, with the nearest relative being Flavobacterium psychrolimnae LMG 22018 T (98.9% sequence similarity). The pairwise 16S rRNA gene sequence identity between strains Kopri-42 T and Kopri-43 was 99.7%. The DNA-DNA hybridization value between strain Kopri-42 T and Kopri-43 was 88.6 ± 2.1% indicating that Kopri-42 T and Kopri-43 represents two strains of the same genomospecies. The average nucleotide identity and in silico DNA-DNA hybridization values between strain Kopri-42 T and nearest relative F. psychrolimnae LMG 22018 T were 92.4% and 47.9%, respectively. These values support the authenticity of the novel species and confirmed the strain Kopri-42 T belonged to the genus Flavobacterium as a new member. The morphological, physiological, biochemical and chemotaxonomic data also distinguished strain Kopri-42 T from its closest phylogenetic neighbors. Based on the polyphasic data, strains Kopri-42 T and Kopri-43 represents a single novel species of the genus Flavobacterium, for which the name Flavobacterium petrolei sp. nov. is proposed. The type strain is Kopri-42 T (=KEMB 9005-710 T  = KACC 19625 T  = NBRC 113374 T ).

The rare ginsenoside Rg3 is attracting more attention because of its good physiological activity and urgent need. There are many pathways to obtain ginsenoside Rg3, including chemical and biological methods. Among these, the conversion of the protopanaxadiol-type ginsenosides by microbial hydrolysis is a trend due to its high efficiency and mild conditions. For effectively extracting from the other panaxadiol saponins, the conversion process for ginsenoside Rg3 was investigated using β-glycosidase-producing endophytic fungus in Panax ginseng in this study. The metabolic pathways are as follows: ginsenoside Rb1 → Gyp-XVII and ginsenoside Rb1 → ginsenoside Rd → ginsenoside Rg3. Phylogenetic analysis of 16S rDNA gene sequence, showed that GE 32 strain belonged to Flavobacterium species. These results suggest that the process of rare ginsenoside Rg3 production by endophytic bacteria GE 32 is efficient for the industrial production and application. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on cultivable β-glycosidase-producing endophytic bacteria from Panax ginseng. Flavobacterium sp. GE32 could convert major ginsenoside Rb1 into Gyp-XVII and minor ginsenoside Rg3. Strain GE 32 has potential to be applied on the preparation for minor ginsenoside Rg3 in pharmaceutical industry., (© 2018 The Society for Applied Microbiology.)

The type 9 secretion system (T9SS) is the protein export pathway of bacteria of the Gram-negative Fibrobacteres-Chlorobi-Bacteroidetes superphylum and is an essential determinant of pathogenicity in severe periodontal disease. The central element of the T9SS is a so-far uncharacterized protein-conducting translocon located in the bacterial outer membrane. Here, using cryo-electron microscopy, we provide structural evidence that the translocon is the T9SS protein SprA. SprA forms an extremely large (36-strand) single polypeptide transmembrane β-barrel. The barrel pore is capped on the extracellular end, but has a lateral opening to the external membrane surface. Structures of SprA bound to different components of the T9SS show that partner proteins control access to the lateral opening and to the periplasmic end of the pore. Our results identify a protein transporter with a distinctive architecture that uses an alternating access mechanism in which the two ends of the protein-conducting channel are open at different times.

A novel bacterium designated S-42 T was isolated from stream bank soil. Cells were found to be aerobic, Gram staining-negative, oxidase-positive, catalase-negative, non-motile, non-spore-forming, rod-shaped, and yellow-pigmented. The strain can grow at 15-35 °C, pH 6.0-10.0, and at 0.5% (w/v) NaCl concentration. Urea was hydrolysed. Flexirubin-type pigments were absent. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain S-42 T formed a lineage within the family Flavobacteriaceae of the phylum Bacteroidetes that is distinct from various species of the genus Flavobacterium, including Flavobacterium maotaiense T9 T (97.6% sequence similarity), Flavobacterium hibernum ATCC 51468 T (97.4%), and Flavobacterium granuli Kw05 T (97.1%). The 16S rRNA gene sequences identity between strain S-42 T and other members of the genus Flavobacterium were < 97.0%. Strain S-42 T contains MK-6 as sole respiratory quinone. The major polar lipids were identified as phosphatidylethanolamine and an unidentified aminolipid. The major cellular fatty acids were identified as iso-C 15:0 , summed feature 3 (C 16:1 ω7c and/or C 16: 1 ω6c), C 16:0 , anteiso-C 15:0 , iso-C 17:0 3-OH, iso-C 15:0 3-OH, and iso-C 15:1 G. The DNA G + C content of the strain was 35.8 mol%. The polyphasic characterization indicated that strain S-42 T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium ureilyticum sp. nov. is proposed. The type strain is S-42 T (= KEMB 9005-537 T  = KACC 19115 T  = NBRC 112683 T ).

Strain ZZ-8 T , a Gram-negative, aerobic, non-spore-forming, non-motile, yellow-pigmented, rod-shaped bacterium, was isolated from metolachlor-contaminated soil in China. The taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain ZZ-8 T is a member of the genus Flavobacterium and shows high sequence similarity to Flavobacterium humicola UCM-46 T (97.2%) and Flavobacterium pedocola UCM-R36 T (97.1%), and lower (< 97%) sequence similarity to other known Flavobacterium species. Chemotaxonomic analysis revealed that strain ZZ-8 T possessed MK-6 as the major respiratory quinone; and iso-C 15:0 (28.5%), summed feature 9 (iso-C 17:1 w9c/C 16:0 10-methyl, 22.9%), iso-C 17:0 3-OH (17.0%), iso-C 15:0 3-OH (8.9%), iso-C 15:1 G (8.6%) and summed feature 3 (C 16:1 w7c/C 16:1 w6c, 5.7%) as the predominant fatty acids. The polar lipids of strain ZZ-8 T were determined to be lipids, a glycolipid, aminolipids and phosphatidylethanolamine. Strain ZZ-8 T showed low DNA-DNA relatedness with F. pedocola UCM-R36 T (43.23 ± 4.1%) and F. humicola UCM-46 T (29.17 ± 3.8%). The DNA G+C content was 43.3 mol%. Based on the phylogenetic and phenotypic characteristics, chemotaxonomic data and DNA-DNA hybridization, strain ZZ-8 T is considered a novel species of the genus Flavobacterium, for which the name Flavobacterium zaozhuangense sp. nov. (type strain ZZ-8 T  = KCTC 62315 T  = CCTCC AB 2017243 T ) is proposed.

In this study, a Flavobacterium sp. is isolated from natural spring, and identified using molecular techniques. Extracellular and intracellular secondary metabolites are identified using solid phase microextraction gas chromatography-mass spectrometry and ultra performance liquid chromatography. Cytotoxic activity of the extracellular compounds produced by the Flavobacterium sp. and quercetin as the standard are measured using ECV304 human endothelial cells in vitro. Our results show that Flavobacterim sp. isolate has the highest percentage of similarity with Flavobacterium cheonhonense strain ARSA-15 (99%). Quercetin is detected as the major extracellular compound produced by the Flavobacterium sp. Methanol extract of Flavobacterium sp. resulted in a higher cell viability results when compared to DMSO extracts. Computational chemistry approach was used and it has been found that polar solvent (methanol) contributed to higher antioxidant activity. In conclusion, Flavobacterium sp. can be used to produce quercetin for industrial purposes., Competing Interests: The authors declare patent application (Turk Patent and Trademark Office, Ref. no. PT2017-02287 [Biological Quercetin Production]). There are no further patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.