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Two Gram-stain-negative, aerobic, rod-shaped, orange-coloured bacterial strains, designated strain C216 T and strain M2295, were isolated from mature mushroom compost from composting facilities in Victoria and South Australia, Australia, respectively. External structures such as flagella or pili were not observed on the cells under scanning electron microscopy. Optimal growth was found to occur at 45 °C, at pH 7.25 and in the absence of NaCl on Emerson's 350 YpSs medium. The genome sequence of strain C216 T was 3 342 126 bp long with a G+C content of 40.5 mol%. Functional analysis of the genome of strain C216 T revealed genes encoding chitinolytic and hemi-cellulolytic functions, with 166 predicted genes associated with carbohydrate metabolism (8.9% of the predicted genes). These functions are important for survival in the mushroom compost environment, which is rich in hemicelluloses. No antibiotic resistance genes were found in the genome sequence. The major fatty acids of strain C216 T were iso-C 15 : 0 (56.7%), iso-C 17 : 0 3-OH (15.6%), C 16 : 1  ω 7 c /iso-C 15 : 0 2-OH (7.3%) and iso-C 15 : 1 G (6.1%). The only respiratory quinone was MK-7. The major polar lipid of strain C216 T was phosphatidylethanolamine, but three unidentified phospholipids, four unidentified aminophospholipids/aminolipids and one unidentified glycolipid were also detected. Phylogenetic analysis based on proteins encoded by the core genome (bac120, 120 conserved bacterial genes) showed that strain C216 T forms a distinct lineage in the family Chitinophagaceae and that the closest identified relative is Niabella soli (69.69% ANI). These data demonstrate that strain C216 T represents a novel genus and novel species within the family Chitinophagaceae , for which we propose the name Mycovorax composti . The type strain is C216 T (=DSM 114558 T =LMG 32998 T ).

Contamination of aquifers by a combination of vanadate [V(V)] and nitrate (NO 3 - ) is widespread nowadays. Although bioremediation of V(V)- and nitrate-contaminated environments is possible, only a limited number of functional species have been identified to date. The present study demonstrates the effectiveness of V(V) reduction and denitrification by a denitrifying bacterium Acidovorax sp. strain BoFeN1. The V(V) removal efficiency was 76.5 ± 5.41 % during 120 h incubation, with complete removal of NO 3 - within 48 h. Inhibitor experiments confirmed the involvement of electron transport substances and denitrifying enzymes in the bioreduction of V(V) and NO 3 - . Cyt c and riboflavin were important for extracellular V(V) reduction, with quinone and EPS more significant for NO 3 - removal. Intracellular reductive compounds including glutathione and NADH directly reduce V(V) and NO 3 - . Reverse transcription quantitative PCR confirmed the important roles of nirK and napA genes in regulating V(V) reduction and denitrification. Bioaugmentation by strain BoFeN1 increased V(V) and NO 3 - removal efficiency by 55.3 % ± 2.78 % and 42.1 % ± 1.04 % for samples from a contaminated aquifer. This study proposes new microbial resources for the bioremediation of V(V) and NO 3 - contaminated aquifers, and contributes to our understanding of coupled vanadium, nitrogen, and carbon biogeochemical processes., 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 © 2024 Elsevier Ltd. All rights reserved.)

The main metabolic product of the pyridinecarboxamide insecticide flonicamid, N-(4-trifluoromethylnicotinyl)glycinamide (TFNG-AM), has been shown to have very high mobility in soil, leading to its accumulation in the environment. Catabolic pathways of flonicamid have been widely reported, but few studies have focused on the metabolism of TFNG-AM. Here, the rapid transformation of TFNG-AM and production of the corresponding acid product N-(4-trifluoromethylnicotinoyl) glycine (TFNG) by the plant growth-promoting bacterium Variovorax boronicumulans CGMCC 4969 were investigated. With TFNG-AM at an initial concentration of 0.86 mmol/L, 90.70 % was transformed by V. boronicumulans CGMCC 4969 resting cells within 20 d, with a degradation half-life of 4.82 d. A novel amidase that potentially mediated this transformation process, called AmiD, was identified by bioinformatic analyses. The gene encoding amiD was cloned and expressed recombinantly in Escherichia coli, and the enzyme AmiD was characterized. Key amino acid residue Val154, which is associated with the catalytic activity and substrate specificity of signature family amidases, was identified for the first time by homology modeling, structural alignment, and site-directed mutagenesis analyses. When compared to wild-type recombinant AmiD, the mutant AmiD V154G demonstrated a 3.08-fold increase in activity toward TFNG-AM. The activity of AmiD V154G was greatly increased toward aromatic L-phenylalanine amides, heterocyclic TFNG-AM and IAM, and aliphatic asparagine, whereas it was dramatically lowered toward benzamide, phenylacetamide, nicotinamide, acetamide, acrylamide, and hexanamid. Quantitative PCR analysis revealed that AmiD may be a substrate-inducible enzyme in V. boronicumulans CGMCC 4969. The mechanism of transcriptional regulation of AmiD by a member of the AraC family of regulators encoded upstream of the amiD gene was preliminarily investigated. This study deepens our understanding of the mechanisms of metabolism of toxic amides in the environment, providing new ideas for microbial bioremediation., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The cold-adapted bacterium Variovorax sp. PAMC28711 possesses two distinct glycoside hydrolase (GH) families of trehalase, GH15 and GH37. While numerous studies have explored bacterial trehalase, the presence of two different trehalase genes within a single strain has not been reported until now. Interestingly, despite both GH37 and GH15 trehalases serving the same purpose of degrading trehalose, but do not share the sequence similarity. The substrate specificity assay confirmed that Vtre37 and Vtre15 displayed hydrolytic activity on α, α-trehalose. The key catalytic sites were identified as D280 and E469 in Vtre37 and E389 and E554 in Vtre15 through site-directed mutation and confirmed these two enzymes belong to trehalase. In addition, Vtre37 exhibited a relatively high level of enzyme activity of 1306.33 (±53.091) μmolmg -1 , whereas Vtre15 showed enzyme activity of 408.39 (±12.503) μmolmg -1 . Moreover, Vtre37 performed admirably showing resistance to ethanol (10 %), with high stable at acidic pH range. Furthermore, both prediction and experimental results indicate that validoxylamine A showed a potent inhibitory activity against Vtre37 trehalase with a K i value of 16.85 nM. Therefore, we postulate that Vtre37 could be utilized as an ethanol enhancer and designed for screening inhibitors related to the trehalose degradation pathway. Additionally, we believe that characterizing these bacterial trehalase contributes to a better understanding of trehalose metabolism and its biological importance in bacteria., 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 © 2023. Published by Elsevier B.V.)

Pteris vittata (P. vittata), an arsenic (As) hyperaccumulator commonly used in the phytoremediation of As-contaminated soils, contains root-associated bacteria (RAB) including those that colonize the root rhizosphere and endosphere, which can adapt to As contamination and improve plant health. As(III)-oxidizing RAB can convert the more toxic arsenite (As(III)) to less toxic arsenate (As(V)) under As-rich conditions, which may promote plant survial. Previous studies have shown that microbial As(III) oxidation occurs in the rhizospheres and endospheres of P. vittata. However, knowledge of RAB of P. vittata responsible for As(III) oxidation remained limited. In this study, members of the Comamonadaceae family were identified as putative As(III) oxidizers, and the core microbiome associated with P. vittata roots using DNA-stable isotope probing (SIP), amplicon sequencing and metagenomic analysis. Metagenomic binning revealed that metagenome assembled genomes (MAGs) associated with Comamonadaceae contained several functional genes related to carbon fixation, arsenic resistance, plant growth promotion and bacterial colonization. As(III) oxidation and plant growth promotion may be key features of RAB in promoting P. vittata growth. These results extend the current knowledge of the diversity of As(III)-oxidizing RAB and provide new insights into improving the efficiency of arsenic phytoremediation., 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 © 2024 Elsevier Ltd. All rights reserved.)

A Gram-stain-negative, rod-shaped, non-motile, catalase-positive, denitrifying bacterium, designated strain Y-1 T , was isolated from an aeration tank of a sewage treatment plant in China and characterized using polyphasic taxonomic approaches. Strain Y-1 T could grow at 10-37 °C (optimum 25 °C), at pH 5.0-10.0 (optimum 7.0) and in the presence of 0-3.0% (w/v) NaCl (optimum 0.5%). The phylogenetic tree based on the 16S rRNA gene sequences revealed that strain Y-1 T was a member of genus Diaphorobacter, and showed the highest sequence similarities with Diaphorobacter oryzae RF3 T (97.50%), Diaphorobacter nitroreducens NA10B T (97.38%) and Diaphorobacter aerolatus 8604S-37 T (96.56%). In terms of carbon source utilization and enzyme activities, strain Y-1 T was significantly different from its similar strains. The major respiratory quinone was Q-8, and the main polar lipid was phosphatidylethanolamine. Comparative genomic analysis of strain Y-1 T and other Diaphorobacter species was conducted to explore the mechanisms underlying the differences among these strains. Strain Y-1 T encoded 3957 genes, consisting of 3813 protein-coding genes and 144 RNA coding genes, and encoded 652 enzymes with 31 unique enzymes compared with other related species. The DNA G + C content was 69.95 mol%. Strain Y-1 T exhibited 41.71% DNA-DNA relatedness and 95% ANIb with the most related type strains.On the basis of the evidence presented from polyphasic analysis, strain Y-1 T was suggested as a novel species within the genus Diaphorobacter, for which the name Diaphorobacter limosus sp. nov. is proposed, with the type strain Y-1 T (= KCTC 92852 T  = CCTCC AB 2023032 T )., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Acidovorax citrulli is the main pathogen causing bacterial fruit blotch, which seriously threatens the global watermelon industry. At present, rapid, sensitive, and low-cost detection methods are urgently needed. The established CRISPR/LbCas12a visual detection method can specifically detect A. citrulli and does not cross-react with other pathogenic bacteria such as Erwinia tracheiphila , Pseudomonas syringae , and Xanthomonas campestris . The sensitivity of this method for genomic DNA detection is as low as 0.7 copies/μL, which is higher than conventional PCR and real-time PCR. In addition, this method only takes 2.5 h from DNA extraction to quantitative detection and does not require complex operation and sample treatment. Additionally, the technique was applied to test real watermelon seed samples for A. citrulli , and the results were contrasted with those of real-time fluorescence quantitative PCR and conventional PCR. The high sensitivity and specificity have broad application prospects in the rapid detection of bacterial fruit blotch bacterial pathogens of watermelon.IMPORTANCEBacterial fruit blotch, Acidovorax citrulli , is an important seed-borne bacterial disease of watermelon, melon, and other cucurbits. The lack of rapid, sensitive, and reliable pathogen detection methods has hampered research on fruit spot disease prevention and control. Here, we demonstrate the CRISPR/Cas12a system to analyze aspects of the specificity and sensitivity of A. citrulli and to test actual watermelon seed samples. The results showed that the CRISPR/Cas12a-based free-amplification method for detecting bacterial fruit blotch pathogens of watermelons was specific for A. citrulli target genes and 100-fold more sensitive than conventional PCR with quantitative real-time PCR. This method provides a new technical tool for the detection of A. citrulli ., Competing Interests: The authors declare no conflict of interest.

In recent years Acidovorax avenae subsp. avenae was identified as a major cause of bacterial etiolation and decline (BED) in turfgrasses and has become a growing economical concern for the turfgrass industry. The symptoms of BED resemble those of "bakanae," or foolish seedling disease, of rice ( Oryzae sativa ), in which the gibberellins produced by the infecting fungus, Fusarium fujikuroi , contribute to the symptom development. Additionally, an operon coding for the enzymes necessary for bacterial gibberellin production was recently characterized in plant-pathogenic bacteria belonging to the γ-proteobacteria. We therefore investigated whether this gibberellin operon might be present in A. avenae subsp. avenae . A homolog of the operon has been identified in two turfgrass-infecting A. avenae subsp. avenae phylogenetic groups but not in closely related phylogenetic groups or strains infecting other plants. Moreover, even within these two phylogenetic groups, the operon presence is not uniform. For that reason, the functionality of the operon was examined in one strain of each turfgrass-infecting phylogenetic group ( A. avenae subsp. avenae strains KL3 and MD5). All nine operon genes were functionally characterized through heterologous expression in Escherichia coli and enzymatic activities were analyzed by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. All enzymes were functional in both investigated strains, thus demonstrating the ability of phytopathogenic β-proteobacteria to produce biologically active GA 4 . This additional gibberellin produced by A. avenae subsp. avenae could disrupt phytohormonal balance and be a leading factor contributing to the pathogenicity on turf grasses. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

In recent years, extremophilic microorganisms have been employed as producers of the microbial bioplastics polyhydroxyalkanoates (PHA), which are of great biotechnological value. Nevertheless, cold-loving or psychrophilic (cryophilic) bacteria have been neglected in this regard. Here, we present an investigation of the Arctic glacier-derived PHA producer Acidovorax sp. A1169. Biolog GEN III Microplates were used as a screening tool to identify the most suitable carbon substrate concerning PHA synthesis. The strain produced homopolymer poly(3-hydroxybutyrate) (PHB) most efficiently (2 g/L) at a temperature of 15 °C when supplied with fructose or mannitol as carbon sources with a substantial decrease of PHB biosynthesis at 17.5 °C. The PHB yield did not increase considerably or even decreased when carbon source concentration exceeded 10 g/L hinting that the strain is oligotrophic in nature. The strain was also capable of introducing 3-hydroxyvalerate (3HV) into the polymer structure, which is known to improve PHA thermoplastic properties. This is the first investigation providing insight into a PHA biosynthesis process by means of a true psychrophile, offering guidelines on polar-region bacteria cultivation, production of PHA and also on the methodology for genetic engineering of psychrophiles., (© 2023. The Author(s).)

Acidovorax citrulli is a seed-borne bacterial pathogen that causes bacterial fruit blotch in cucurbits and severely affects the production of cucumbers and watermelons globally. In this study, we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP) on the growth, metabolism, and virulence of A. citrulli . Bacterial population was not affected by DEHP exposure; moreover, significant changes were not observed in lipid peroxidation, membrane permeability, and nucleic acid leakage. However, palmitoleic acid content was increased in the cell membrane of DEHP-exposed A. citrulli . Further, DEHP exposure increased the activity of TCA cycle-related enzymes, including α-ketoglutarate dehydrogenase and succinyl-CoA synthetase, along with increase in the content of glutamate, succinate, fumarate, and malate in TCA cycle. Additionally, total 270 genes were differentially expressed by the treatment, of which 28 genes were upregulated and 242 genes, including those related to translation, flagellum-dependent cell motility, and flagellum assembly, were downregulated. Regarding virulence traits, swimming activity was decreased in DEHP-exposed A. citrulli ; however, biofilm formation was not affected in in vitro assay. Moreover, relative expression of pathogenicity genes, including hrpX and hrpG , were decreased in DEHP-exposed A. citrulli compared to that of unexposed A. citrulli . Therefore, these results suggest that DEHP accumulation in soil could potentially influence the metabolism and virulence traits of A. citrulli ., 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 Kim and Sang.)

A non-canonical biosynthetic pathway furnishing the first natural brexane-type bishomosesquiterpene (chlororaphen, C 17 H 28 ) was elucidated in the γ-proteobacterium Pseudomonas chlororaphis O6. A combination of genome mining, pathway cloning, in vitro enzyme assays, and NMR spectroscopy revealed a three-step pathway initiated by C10 methylation of farnesyl pyrophosphate (FPP, C 15 ) along with cyclization and ring contraction to furnish monocyclic γ-presodorifen pyrophosphate (γ-PSPP, C 16 ). Subsequent C-methylation of γ-PSPP by a second C-methyltransferase furnishes the monocyclic α-prechlororaphen pyrophosphate (α-PCPP, C 17 ), serving as the substrate for the terpene synthase. The same biosynthetic pathway was characterized in the β-proteobacterium Variovorax boronicumulans PHE5-4, demonstrating that non-canonical homosesquiterpene biosynthesis is more widespread in the bacterial domain than previously anticipated., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Bacteria that inhabit soils and the leaves of trees partially mitigate the release of the abundant volatile organic compound, isoprene (2-methyl-1,3-butadiene). While the initial steps of isoprene metabolism were identified in Rhodococcus sp. AD45 two decades ago, the isoprene metabolic pathway still remains largely undefined. Limited understanding of the functions of isoG, isoJ and aldH and uncertainty in the route of isoprene-derived carbon into central metabolism have hindered our understanding of isoprene metabolism. These previously uncharacterised iso genes are essential in Variovorax sp. WS11, determined by targeted mutagenesis. Using combined 'omics-based approaches, we propose the complete isoprene metabolic pathway. Isoprene is converted to propionyl-CoA, which is assimilated by the chromosomally encoded methylmalonyl-CoA pathway, requiring biotin and vitamin B12, with the plasmid-encoded methylcitrate pathway potentially providing robustness against limitations in these vitamins. Key components of this pathway were induced by both isoprene and its initial oxidation product, epoxyisoprene, the principal inducer of isoprene metabolism in both Variovorax sp. WS11 and Rhodococcus sp. AD45. Analysis of the genomes of distinct isoprene-degrading bacteria indicated that all of the genetic components of the methylcitrate and methylmalonyl-CoA pathways are not always present in isoprene degraders, although incorporation of isoprene-derived carbon via propionyl-CoA and acetyl-CoA is universally indicated., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Many genera closest to the family Comamonadaceae have not been classified into any family; moreover, some of them are not monophyletic groups beyond the genus level. To resolve the taxonomic uncertainty of the closest-to-Comamonadaceae (CTC) group, we performed 16S rRNA gene- and genome-based phylogenetic analyses combined with genome relatedness indices and phenotypic traits comparison. Phylogenies based on the 16S rRNA gene and genome sequences demonstrated that the CTC group formed a coherent and robust monophyletic lineage and was sister to the family Comamonadaceae, thereby proposing the CTC group as a novel family, Sphaerotilaceae fam. nov. The resolved genus- and species-level taxonomic relationships of this new family were then validated by the phylogenomic reconstruction and comparisons of genome relatedness indices including digital DNA-DNA hybridization and average nucleotide identity (ANI) as well as comprehensive phenotypic analysis for type strains. Finally, we reclassified all misidentified genera and species, resulting in 19 new combinations, and proposed Sphaerotilaceae-specific thresholds of ANI and average amino acid identity for genus delineation. Collectively, this study has established a sound taxonomic framework of the novel family Sphaerotilaceae and will help guide future taxonomic efforts and prevent the propagation of taxonomic errors., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Bacterial fruit blotch (BFB) is a disease of cucurbit plants caused by Acidovorax citrulli . Although A. citrulli has great destructive potential, the molecular mechanisms of pathogenicity of A. citrulli are not clear, particularly with regard to its type III secreted effectors. In this study, we characterized the type III secreted effector protein, AopV, from A. citrulli strain Aac5. We show that AopV significantly inhibits reactive oxygen species and the expression of PTI marker genes, and helps the growth of Pseudomonas syringae D36E in Nicotiana benthamiana . In addition, we found that the aromatic dehydratase ADT6 from watermelon was a target of AopV. AopV interacts with ADT6 in vivo and in vitro. Subcellular localization indicated ADT6 and AopV were co-located at the cell membrane. Together, our results reveal that AopV suppresses plant immunity and targets ADT6 in the cell membrane. These findings provide an new characterization of the molecular interaction of A. citrulli effector protein AopV with host cells.

Since the role of biobased plastics increases every year, the search for alternatives to petrol-based polymers is very important. Variovorax paradoxus TBEA6 is able to grow with 3,3'-thiodipropionic acid (TDP) as sole source for carbon and energy. TDP can be used as a precursor substrate for the synthesis of polythioesters (PTE). To increase the feasibility of PTE synthesis, a good understanding of the degradation pathway of TDP in V. paradoxus TBEA6 is essential. Therefore, two putative 3-hydroxyisobutyryl-CoA hydrolases (VPARA_03110 & VPARA_05510) and two putative 3-hydroxypropionate dehydrogenases (VPARA_41140 & VPARA_54550) were investigated in this study. The deletion mutant V. paradoxus ∆VPARA_05510 showed a TDP-negative phenotype during growth experiments. The ability to grow with TDP as sole carbon source was successfully restored by complementation. Supernatant analysis revealed that the deletion mutant did not metabolize TDP or 3MP anymore. A specific enzyme activity up to 0.032 U/mg for the purified 3-hydroxyisobutyryl-CoA hydrolase VPARA_05510 was determined. A shift in the proteins (VPARA_54550) melting temperature of 6 °C with 2000 µM 3HP in comparison to protein without ligand was observed during thermal shift assays with the putative 3-hydroxypropionate dehydrogenase., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Folic acid (FA) is a synthetic vitamin (B 9 ) and the oxidized form of a metabolic cofactor that is essential for life. Although the biosynthetic mechanisms of FA are established, its environmental degradation mechanism has not been fully elucidated. The present study aimed to identify bacteria in soil that degrade FA and the mechanisms involved., Results: We isolated the soil bacterium Variovorax sp. F1 from sampled weed rhizospheres in a grassland and investigated its FA degradation mechanism. Cultured Variovorax sp. F1 rapidly degraded FA to pteroic acid (PA), indicating that FA hydrolysis to PA and glutamate. We cloned the carboxypeptidase G (CPG) gene and found widely distributed paralogs within the Variovorax genus. Recombinant CPG preferred FA and deaminofolic acid as substrates, indicating its involvement in FA degradation by Variovorax. Prolonged culture of Variovorax sp. F1 resulted in decreased rates of deaminofolic acid (DFA) and deaminopteroic acid (DPA) accumulation. This indicated that the deamination reaction also comprised a route of FA degradation. We also identified an F1 gene that was orthologous to the pterin deaminase gene (Arad3529) of Agrobacterium radiobacter. The encoded protein deaminated FA and PA to DFA and DPA, which was consistent with the deamination activity of FA and PA in bacterial cell-free extracts., Conclusion: We discovered that the two enzymes required for FA degradation pathways in isolates of Variovorax sp. F1 comprise CPG and pterin deaminase, and that DFA and PA are intermediates in the generation of DPA., (© 2022. The Author(s).)

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is a devastating watermelon disease that severely impacts the global watermelon industry. Like other Gram-negative bacteria, the type three secretion system (T3SS) is the main pathogenicity factor of A. citrulli . The T3SS apparatus gene hrpE codes for the Hrp pilus and serves as a conduit to secret effector proteins into host cells. In this study, we found that the deletion of hrpE in A. citrulli results in the loss of pathogenicity on hosts and the hypersensitive response on non-hosts. In addition, the A. citrulli hrpE mutant showed a reduction in in vitro growth, in planta colonization, swimming and twitching motility, and displayed increases in biofilm formation ability compared to the wild type. However, when HrpE was transiently expressed in hosts, the defense responses, including reactive oxygen species bursts, callose deposition, and expression of defense-related genes, were activated. Thus, the A. Citrulli growth in HrpE-pretreated hosts was suppressed. These results indicated that HrpE is essential for A. citrulli virulence but can also be used by hosts to help resist A. citrulli . Our findings provide a better understanding of the T3SS pathogenesis in A. citrulli , thus providing a molecular basis for biopesticide development, and facilitating the effective control of BFB.

Rhodoferax sp. PAMC 29310 was isolated from a surface marine sediment of the East Siberian Sea, Arctic. Whole-genome sequencing of the strain Rhodoferax sp. PAMC 29310 was achieved using PacBio RS II and Illumina platform. The resulting complete genome comprised of 4,593,249 base pairs (G + C content of 58.0%) with a single chromosome, 4546 protein-coding genes, 57 tRNAs and 6 rRNA operons. A complete set of genes encoding the enzymes of glycolysis and citric acid cycle were identified. No genes encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and nitrogenase reductase (nif) were present indicating that strain PAMC 29310 is not capable of fixing of carbon and nitrogen. PAMC 29310 genome contains genes for dissimilatory and assimilatory nitrate reduction. Gene encoding choline dehydrogenase enzyme which functions at the first step in the synthesis of betaine, one of the most effective osmoprotectants, was detected. In particular, among the genomes of the genus Rhodoferax strains, gene encoding nitrite reductase (nirK), which reduces nitrite to nitric oxide and tetA gene encoding tetracycline resistance protein involved in the resistance to tetracycline were identified only in the genome of Rhodoferax sp. PAMC 29310. As the first genome from the strain which was isolated from marine sediment in the genus Rhodoferax, investigation of physiological characteristics based on the complete genome sequences will help understand the adaptation of Rhodoferax sp. PAMC 29310 in the marine sediment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Schistosomiasis is a neglected tropical parasitic and chronic disease affecting hundreds of millions of people. Adult schistosomes reside in the blood stream of the definitive mammalian host. These helminth parasites possess two epithelial surfaces, the tegument and the gastrodermis, both of which interact with the host during immune evasion and in nutrient uptake., Methods: Female ARC Swiss mice (4-6 weeks old) were infected percutaneously with Schistosoma japonicum cercariae freshly shed from Oncomelania hupensis quadrasi snails (Philippines strain). Fluorescent in situ hybridisation (FISH) was performed by using fresh adult S. japonicum perfused from those infected mice. Adult S. japonicum worms were processed to isolate the tegument from the carcass containing the gastrodermis; blood and bile were collected individually from infected and uninfected mice. Total DNA extracted from all those samples were used for microbiome profiling., Results: FISH and microbiome profiling showed the presence of bacterial populations on two epithelial surfaces of adult worms, suggesting they were distinct not only from the host blood but also from each other. Whereas microbial diversity was reduced overall in the parasite epithelial tissues when compared with that of host blood, specific bacterial taxa, including Anoxybacillus and Escherichia, were elevated on the tegument. Minimal differences were evident in the microbiome of host blood during an active infection, compared with that of control uninfected blood. However, sampling of bile from infected animals identified some differences compared with controls, including elevated levels of Limnohabitans, Clostridium and Curvibacter., Conclusions: Using FISH and microbial profiling, we were able to demonstrate, for the first time, that bacteria are presented on the epithelial surfaces of adult schistosomes. These schistosome surface-associated bacteria, which are distinct from the host blood microenvironment, should be considered as a new and important component of the host-schistosome interaction. The importance of individual bacterial species in relation to schistosome parasitism needs further elucidation., Competing Interests: The authors have declared that no competing interests exist.

Caldimonas manganoxidans is a Gram-negative, thermophilic, bioplastic-producing bacterium that is a promising strain to overcome the drawbacks of existing bioplastic manufacturing methods. However, genetic manipulation of this species has not previously been studied. Here, we developed an optimized electrotransformation protocol for C. manganoxidans by screening conditions, including the bacterial growth phase, electroporation buffer, pulse strength, and recovery time. The optimized transformation protocol obtained (3.1 ± 0.78) × 10 8 colony-forming units/μg DNA of plasmid pBBR1MCS-2. High transformation efficiency was observed when using plasmid DNA isolated from C. manganoxidans. The DNA methylases of Escherichia coli did not affect the transformation efficiency of C. manganoxidans. The electrotransformation technique proposed here will be beneficial for the genetic manipulation of thermophilic Caldimonas species., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Beta-proteobacteria belonging to the genus Acidovorax have been described from various environments. Many strains can interact with a range of hosts, including humans and plants, forming neutral, beneficial or detrimental associations. In the frame of this study, we investigated the genomic properties of 52 bacterial strains of the genus Acidovorax , isolated from healthy roots of Lotus japonicus, with the intent of identifying traits important for effective plant-growth promotion. Based on single-strain inoculation bioassays with L. japonicus , performed in a gnotobiotic system, we distinguished seven robust plant-growth promoting strains from strains with no significant effects on plant-growth. We showed that the genomes of the two groups differed prominently in protein families linked to sensing and transport of organic acids, production of phytohormones, as well as resistance and production of compounds with antimicrobial properties. In a second step, we compared the genomes of the tested isolates with those of plant pathogens and free-living strains of the genus Acidovorax sourced from public repositories. Our pan-genomics comparison revealed features correlated with commensal and pathogenic lifestyle. We showed that commensals and pathogens differ mostly in their ability to use plant-derived lipids and in the type of secretion-systems being present. Most free-living Acidovorax strains did not harbour any secretion-systems. Overall, our data indicate that Acidovorax strains undergo extensive adaptations to their particular lifestyle by horizontal uptake of novel genetic information and loss of unnecessary genes.

Bdellovibrio bacteriovorus, a Gram-negative predatory bacterium belonging to the Bdellovibrio and like organisms (BALOs), predates on Gram-negative bacteria. BALO strains differ in prey range but so far, the genetic basis of resistance against BALO predation is hardly understood. We developed a loss-of-function approach to screen for sensitive mutants in a library of strain M6, a predation-resistant strain of the plant pathogen Acidovorax citrulli. The screen is based on tracking the growth of a B. bacteriovorus strain expressing the fluorescent reporter Tdtomato in mutant pools to reveal predation-sensitive variants. Two independent loci were identified in mutant strains exhibiting significant levels of susceptibility to the predator. Genes in the two loci were analysed using both protein sequence homology and protein structure modeling. Both were secretion-related proteins and thus associated to the bacterial cell wall. Successful complementation of gspK, a gene encoding for a minor pseudopilin protein confirmed the involvement of the type II secretion system in A. citrulli M6 resistance. This proof of concept study shows that our approach can identify key elements of the BALO-prey interaction, and it validates the hypothesis that mutational changes in a single gene can drastically impact prey resistance to BALO predation., Competing Interests: Declaration of competing interest None of the authors has a conflict of interest., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Fe(II)-oxidizing microorganisms and Fe(III)-reducing microorganisms, which drive the biogeochemical Fe cycle on the Earth's surface, are phylogenetically and ecologically diverse. However, no single organism capable of aerobic Fe(II) oxidation and anaerobic Fe(III) reduction at circumneutral pH have been reported so far. Here, we report a novel neutrophilic Fe(II)-oxidizing Rhodoferax bacterium, strain MIZ03, isolated from an iron-rich wetland in Japan. Our cultivation experiments demonstrate that MIZ03 represents a much more versatile metabolism for energy acquisition than previously recognized in the genus Rhodoferax . MIZ03 can grow chemolithoautotrophically at circumneutral pH by oxidation of Fe(II), H 2 , or thiosulfate as the sole electron donor under (micro)aerobic conditions (i.e., using O 2 as the sole electron acceptor). In addition, it can reduce Fe(III) or nitrate under anaerobic conditions. Thus, this is the first report demonstrating the presence of a single bacterium capable of both Fe(II) oxidation and Fe(III) reduction at circumneutral pH. The observed physiology was consistent with its 4.9-Mbp complete genome encoding key genes for iron oxidation/reduction ( foxEY and mtrABC ), for nitrate reduction ( narGHI ), for thiosulfate oxidation ( soxABCDXYZ ), and for carbon fixation via the Calvin cycle. Our metagenomic survey suggests that there are more Rhodoferax members capable of Fe(II) oxidation and Fe(III) reduction. Such bifunctional Rhodoferax may have an ecological advantage in suboxic/anoxic environments at circumneutral pH by recycling of Fe as the electron donor and acceptor. IMPORTANCE The biogeochemical cycle of iron (Fe) via reactions of oxidation, reduction, precipitation, and dissolution is involved in the cycle of other ecologically relevant elements, such as C, N, P, S, As, Co, Ni, and Pb. The Fe cycle on the Earth's surface is driven by a variety of Fe(II)-oxidizing microorganisms and Fe(III)-reducing microorganisms. Here, we discovered a novel bacterium, Rhodoferax sp. strain MIZ03, capable of both Fe(II) oxidation and Fe(III) reduction at circumneutral pH, and we report its physiological characteristics and complete genome sequence. The unexpected capability of this bacterium provides novel insights into the Fe cycle in the environment. Moreover, this bacterium will help to better understand the molecular mechanisms of microbial Fe redox cycling as a model organism.

Diaphorobacter sp. strain JS3051 utilizes 2,3-dichloronitrobenzene (23DCNB), a toxic anthropogenic compound, as the sole carbon, nitrogen, and energy source for growth, but the metabolic pathway and its origins are unknown. Here, we establish that a gene cluster ( dcb ), encoding a Nag-like dioxygenase, is responsible for the initial oxidation of the 23DCNB molecule. The 2,3-dichloronitrobenzene dioxygenase system (DcbAaAbAcAd) catalyzes conversion of 23DCNB to 3,4-dichlorocatechol (34DCC). Site-directed mutagenesis studies indicated that residue 204 of DcbAc is crucial for the substrate specificity of 23DCNB dioxygenase. The presence of glutamic acid at position 204 of 23DCNB dioxygenase is unique among Nag-like dioxygenases. Genetic, biochemical, and structural evidence indicate that the 23DCNB dioxygenase is more closely related to 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42 than to the 34DCNB dioxygenase from Diaphorobacter sp. strain JS3050, which was isolated from the same site as strain JS3051. A gene cluster ( dcc ) encoding the enzymes for 34DCC catabolism, homologous to a clc operon in Pseudomonas knackmussii strain B13, is also on the chromosome at a distance of 2.5 Mb from the dcb genes. Heterologously expressed DccA catalyzed ring cleavage of 34DCC with high affinity and catalytic efficiency. This work not only establishes the molecular mechanism for 23DCNB mineralization, but also enhances the understanding of the recent evolution of the catabolic pathways for nitroarenes. IMPORTANCE Because anthropogenic nitroaromatic compounds have entered the biosphere relatively recently, exploration of the recently evolved catabolic pathways can provide clues for adaptive evolutionary mechanisms in bacteria. The concept that nitroarene dioxygenases shared a common ancestor with naphthalene dioxygenase is well established. But their phylogeny and how they evolved in response to novel nitroaromatic compounds are largely unknown. Elucidation of the molecular basis for 23DCNB degradation revealed that the catabolic pathways of two DCNB isomers in different isolates from the same site were derived from different recent origins. Integrating structural models of catalytic subunits and enzymatic activities data provided new insight about how recently modified enzymes were selected depending on the structure of new substrates. This study enhances understanding and prediction of adaptive evolution of catabolic pathways in bacteria in response to new chemicals.

Objectives: Catalytic promiscuity, or the ability to catalyze a secondary reaction, provides new opportunities for industrial biocatalysis by expanding the range of biocatalytic reactions. Some nitrilases converting nitriles to amides, referred to as the secondary activity, show great potential for amides production. And our goal was exploiting the amide-forming potential of nitrilases., Results: In this study, we characterized and altered the secondary activity of nitrilase from Acidovorax facilis 72 W (Nit72W) towards different substrates. We increased the secondary activity of Nit72W towards 2-cyanopyridine by 196-fold and created activity toward benzonitrile and p-nitrophenylacetonitrile by modifying the active pocket. Surprisingly, the best mutant, W188M, completely converted 250 mM 2-cyanopyridine to more than 98% 2-picolinamide in 12 h with a specific activity of 90 U/mg and showed potential for industrial applications., Conclusions: Nit72W was modified to increase its secondary activity for the amides production, especially 2-picolinamide.

The expression of non-coding RNAs (ncRNAs) has been observed in a variety of bacteria. However, the function of ncRNAs and their regulatory targets are largely unknown, and few ncRNAs are found to be associated with bacterial virulence. The bacterial brown stripe pathogen Acidovorax oryzae (Ao) RS-1 shows a high level of condition-dependent differential expression of ncRNA, which we identified in a genome wide screen. We experimentally validated 66 differentially expressed ncRNAs using an integrative analysis of conservative genome sequences and transcriptomic data during in vivo interaction of the bacterial pathogen with the rice plant. To test the relevance of the differentially expressed ncRNAs, we chose four with different positions within the genome, and with different secondary structures and promoter activities. The results show that the overexpression of the four ncRNAs caused a significant change in virulence-related phenotypes, resistance to various environmental stresses, expression of secretion systems and effector proteins, while changing the expression of ncRNA putative target genes. We conclude that these ncRNAs are examples for the inherent regulatory roles for many of the observed ncRNAs in response to changing conditions such as host interaction or environmental adaption., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

LuxR-type transcriptional regulators are essential for many physiological processes in bacteria, including pathogenesis. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes great losses in melon and watermelon worldwide. However, the LuxR-type transcriptional factors in A. citrulli have not been well studied, except for the previously reported LuxR-type regulatory protein, AcrR, involved in regulating virulence and motility. Here, we characterized a second LuxR-type regulator, AclR, in the group II strain Aac-5 of A. citrulli by mutagenesis, virulence and motility assays, and transcriptomic analysis. Deletion of aclR resulted in impaired twitching and swimming motility and flagellar formation and diminished virulence but increased biofilm formation. Transcriptomic analysis revealed that 1,379 genes were differentially expressed in the aclR mutant strain, including 29 genes involved in flagellar assembly and 3 involved in pili formation, suggesting a regulatory role for AclR in multiple important biological functions of A. citrulli . Together, our results not only indicate that AclR plays a global role in transcriptional regulation in A. citrulli influencing motility, biofilm formation, and virulence but also provide perspective regarding the regulatory network of biological functions in A. citrulli .[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer-Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Furthermore, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, that is, isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO-containing P. taiwanensis VLB120. Biofilms showed less favorable values for K S (9.3-fold higher) and k cat (4.8-fold lower) compared with corresponding K M and k cat values of isolated CHMO, but a favorable K I for cyclohexanone (5.3-fold higher). The unfavorable K S and k cat values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, to exploit the high potential of biofilms regarding process stability. Suspended cells showed only 1.8-fold higher K S , but 1.3- and 4.2-fold higher k cat and K I values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

3,3'-Thiodipropionic acid (TDP) is an antioxidant, which can be used as precursor carbon source to synthesize polythioesters. The bacterium Variovorax paradoxus TBEA6 strain can use TDP as a single source of carbon and energy. In the present study, experiments were carried out to identify proteins involved in the transport of TDP into the cells of strain TBEA6. Hence, eight putative tctC genes, which encode for the TctC proteins, were amplified from genomic DNA of TBEA6 strain using polymerase chain reaction and expressed in E. coli BL21 cells. Cells were grown in auto-induction medium, and protein purification was done using His Spin Trap affinity columns. Purity and molecular weight of each protein were confirmed by SDS-PAGE analysis. Protein-ligand interactions were monitored in thermoshift assays using the real-time PCR system. Two TctC proteins (locus tags VPARA-44430 and VPARA-01760) out of eight proteins showed a significant shift in their melting temperatures when they interact with the ligand (TDP or gluconate). The responsible genes were deleted in the genome of TBEA6 using suicide plasmid pJQ200mp18Tc, and single deletion mutants of the two candidate genes were subsequently generated. Finally, growth of the wild-type strain (TBEA6) and the two mutant strains (ΔVPARA-44430 and ΔVPARA-01760) were monitored and compared using TDP or gluconate as carbon sources. Wild type strains were successfully grown with TDP or gluconate. From the two mutant strains, one (ΔVPARA-44430) was unable to grow with TDP indicating that the tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.Key Points• Putative tctC genes from V. paradoxus TBEA6 were heterologously expressed in E. coli.• Protein-ligand interactions monitored in thermoshift assays using the real-time PCR.• tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.

Schlegelella thermodepolymerans is a moderately thermophilic bacterium capable of producing polyhydroxyalkanoates-biodegradable polymers representing an alternative to conventional plastics. Here, we present the first complete genome of the type strain S. thermodepolymerans DSM 15344 that was assembled by hybrid approach using both long (Oxford Nanopore) and short (Illumina) reads. The genome consists of a single 3,858,501-bp-long circular chromosome with GC content of 70.3%. Genome annotation identified 3,650 genes in total, whereas 3,598 open reading frames belonged to protein-coding genes. Functional annotation of the genome and division of genes into clusters of orthologous groups revealed a relatively high number of 1,013 genes with unknown function or unknown clusters of orthologous groups, which reflects the fact that only a little is known about thermophilic polyhydroxyalkanoates-producing bacteria on a genome level. On the other hand, 270 genes involved in energy conversion and production were detected. This group covers genes involved in catabolic processes, which suggests capability of S. thermodepolymerans DSM 15344 to utilize and biotechnologically convert various substrates such as lignocellulose-based saccharides, glycerol, or lipids. Based on the knowledge of its genome, it can be stated that S. thermodepolymerans DSM 15344 is a very interesting, metabolically versatile bacterium with great biotechnological potential., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

A non-motile, Gram-stain-negative, rod-shaped bacterium, designated strain S4 T , was obtained from soil sampled at Wonju, Gyeonggi-do, Republic of Korea. Cells were white-coloured, aerobic, grew optimally at 25-32 °C on R2A agar plate. A phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain S4 T formed a lineage within the family Comamonadaceae. The closest members were Caenimonas terrae SGM1-15 T (98.1% sequence similarity), Caenimonas koreensis EMB320 T (97.5%) and Ramlibacter solisilvae 5-10 T (97.8%). The sequence similarities of strain S4 T with other members of the family Comamonadaceae were ≤ 97.5%. The sole respiratory quinone was ubiquinone-8 (Q-8) and the principal polar lipid was phosphatidylethanolamine. The predominant cellular fatty acids were summed feature 3 (iso-C 15 :0  2-OH/C 16 :1 ω7c), C 16:0 and summed feature 8 (C 18:1 ω7c and/or C 18:1 ω6c). The DNA G + C content was 65.1 mol%. In addition, the average nucleotide identity (ANIu) and in silico DNA-DNA hybridization (dDDH) relatedness values between strain S4 T and Caenimonas koreensis were 77.6 and 21%, respectively. Based on genomic, chemotaxonomic, phenotypic, and phylogenetic analyses, strain S4 T represents a novel species in the genus Caenimonas, for which the name Caenimonas soli sp. nov. is proposed. The type strain is S4 T (= KCTC 72742 T  = NBRC 114610 T ).

Brown stripe, incited by Acidovorax oryzae is one of the most important and widespread diseases in rice (Oryza sativa) nurseries in Iran. Rice seedlings showing brown stripes were collected from suburb areas in southern Sari. Bacteria were isolated on plates of sorbitol neutral red agar. Species-specific PCR using trpB1/trpB2 and SEQID1/SEQID2 primer pairs resulted in amplification of the expected 478 bp and 514 bp long fragments, respectively. The 32 isolates subjected to REP-PCR analysis displayed 15 different banding profiles, with just one being shared by 10 isolates and 10 profiles were solitary and not shared by any isolates. Nonetheless, the isolates could not be different phenotypically. They appeared to be biochemically and nutritionally rather homogeneous while the genetic diversity as depicted in REP-PCR analysis was remarkable among the strains isolated from the closely located and even neighboring rice seedbeds. The implication of the findings in breeding programs is briefly discussed.

Microbial pathogens deliver effectors into plant cells to suppress plant immune responses and modulate host metabolism in order to support infection processes. We sought to determine if the Acidovorax avenae rice-virulent K1 strain can suppress pathogen-associated molecular pattern-triggered immunity (PTI) induced by flagellin isolated from the rice-avirulent N1141 strain. The flagellin-triggered PTI, including H 2 O 2 generation, callose deposition, and expression of several immune-related genes were strongly suppressed in K1 preinoculated cultured rice cells in a type III secretion system (T3SS)-dependent manner. By screening 4,562 transposon-tagged mutants based on their suppression ability, we found that 156 transposon-tagged K1 mutants lost the ability to suppress PTI induction. Mutant sequence analysis, comprehensive expression analysis using RNA sequencing, and the prediction of secretion through T3SS showed that a protein named A. avenae K1 suppression factor 1 (AKSF1) suppresses flagellin-triggered PTI in rice. Translocation of AKSF1 protein into rice cells is dependent on the T3SS during infection, an AKSF1 -disruption mutant lost the ability to suppress PTI responses, and expression of AKSF1 in the AKSF1 -disruption mutant complemented the suppression activity. When AKSF1 -disruption mutants were inoculated into the host rice plant, reduction of the disease symptoms and suppression of bacterial growth were observed. Taken together, our results demonstrate that AKSF1 is a novel effector that can suppress the PTI in a host rice plant.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.

Polaromonas vacuolata KCTC 22033 T is an obligate aerobic, Gram-negative, psychrophilic and rod-shaped bacterium isolated from beneath the sea ice off the coast of the Palmer Peninsula, Anvers Islands, Antarctica. P. vacuolata is the type species of Polaromonas genus and the first example of gas vacuolate Betaproteobacteria isolated from marine habitats. Here, we report a complete genome of P. vacuolata KCTC 22033 T , which consists of 3,837,686 bp (G + C content of 52.07%) with a single chromosome, 3461 protein-coding genes, 56 tRNAs and 6 rRNA operons. Genomic analysis revealed the presence of genes involved in bacterial adaptation under saline conditions, cold adaptation via the production of gas vesicles and cell adhesion proteins, and a photoheterotrophic lifestyle when challenged by starvation. Intriguingly, several of these genes were likely acquired from species outside the Polaromonas genus. The genomic information therefore describes the unique evolution and adaptation of P. vacuolata to its extraordinary habitat, i.e., beneath the Antarctic sea ice., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The chemical synthesis intermediate 3,4-dichloronitrobenzene (3,4-DCNB) is an environmental pollutant. Diaphorobacter sp. strain JS3050 utilizes 3,4-DCNB as a sole source of carbon, nitrogen and energy. However, the molecular determinants of its catabolism are poorly understood. Here, the complete genome of strain JS3050 was sequenced and key genes were expressed heterologously to establish the details of its degradation pathway. A chromosome-encoded three-component nitroarene dioxygenase (DcnAaAbAcAd) converted 3,4-DCNB stoichiometrically to 4,5-dichlorocatechol, which was transformed to 3,4-dichloromuconate by a plasmid-borne ring-cleavage chlorocatechol 1,2-dioxygenase (DcnC). On the chromosome, there are also genes encoding enzymes (DcnDEF) responsible for the subsequent transformation of 3,4-dichloromuconate to β-ketoadipic acid. The fact that the genes responsible for the catabolic pathway are separately located on plasmid and chromosome indicates that recent assembly and ongoing evolution of the genes encoding the pathway is likely. The regiospecificity of 4,5-dichlorocatechol formation from 3,4-DCNB by DcnAaAbAcAd represents a sophisticated evolution of the nitroarene dioxygenase that avoids misrouting of toxic intermediates. The findings enhance the understanding of microbial catabolic diversity during adaptive evolution in response to xenobiotics released into the environment., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Omega hydroxycarboxylic acids (ω-HAs) possess two functional groups, a hydroxyl group and a carboxyl group, and are essential precursors for the production of biodegradable polyester polymers. In this work, an Acidovorax mutant was investigated as a whole-cell biocatalyst for the conversion of cycloalkanes to their respective ω-hydroxycarboxylic acids. This Acidovorax sp. strain CHX100 originated from a wastewater treatment plant and uses cyclohexane as the sole source of carbon and energy with excellent growth rates (0.199 h -1 ). The metabolic efficiency of Acidovorax CHX100 is based on a highly efficient enzyme cascade used for the mineralization of cyclohexane. A deletion of 6-hydroxyhexanoate dehydrogenase in the native cycloalkane pathway resulted in the Acidovorax sp. strain CHX100 Δ6HX mutant, which accumulated short ω-hydroxycarboxylic acids (C5 to C10) from cycloalkanes. This mutant transformed cyclopentane and cyclohexane (5 mM) to 5-hydroxypentanoic acid and 6-hydroxyhexanoic acid, respectively, with a molar conversion above 98% in 6 h. An elementary environmental and economical assessment based on E-factor and biocatalyst yield suggests the use of inexpensive electron donor and carbon sources, with subsequent efforts to minimize waste generation. Such an early-stage analysis highlights the main bottlenecks that need to be solved in developing a sustainable bioprocess., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In a conserved culture of the purple sulfur bacterium Thiospirillum jenense DSM216 T , cells of this species were easily recognized by cell morphology, large-size spirilla and visible flagellar tuft. The Tsp. jenense genome is 3.22 Mb in size and has a GC content of 48.7 mol%. It was readily identified as a member of the Chromatiaceae by the complement of proteins in its genome. A whole genome comparison clearly placed Tsp. jenense near Thiorhodovibrio and Rhabdochromatium species and somewhat more distant from Thiohalocapsa and Halochromatium species. This relationship was also found with the sequences of the photosynthetic reaction center protein PufM. The genome sequence supported important properties of this bacterium: the presence of ribulose-bisphosphate carboxylase and enzymes of the Calvin cycle of autotrophic carbon dioxide fixation but the absence of carboxysomes, an incomplete tricarboxylic acid cycle and the lack of malate dehydrogenase, the presence of a sulfur oxidation pathway including adenylylsulfate reductase (aprAB) but absence of assimilatory sulfate reduction, the presence of hydrogenase (hoxHMFYUFE), nitrogenase and a photosynthetic gene cluster (pufBALMC). The FixNOP type of cytochrome oxidase was notably lacking, which may be the reason that renders the cells highly sensitive to oxygen. Two minor phototrophic contaminants were found using metagenomic binning: one was identified as a strain of Rhodopseudomonas palustris and the second one has an average nucleotide identity of 82% to the nearest neighbor Rhodoferax antarcticus. It should be considered as a new species of this genus and Rhodoferax jenense is proposed as the name.

Flagellins are the protein components of bacterial flagella and assemble in up to 20,000 copies to form extracellular flagellar filaments. An unusual family of flagellins was recently discovered that contains a unique metalloprotease domain within its surface-exposed hypervariable region. To date, these proteolytic flagellins (also termed flagellinolysins) have only been characterized in the Gram-positive organism Clostridium haemolyticum, where flagellinolysin was shown to be proteolytically active and capable of cleaving extracellular protein substrates. The biological function of flagellinolysin and its activity in other organisms, however, remain unclear. Here, using molecular biochemistry and proteomics, we have performed an initial characterization of a novel flagellinolysin identified from Hylemonella gracilis, a Gram-negative organism originally isolated from pond water. We demonstrate that H. gracilis flagellinolysin (HgrFlaMP) is an active calcium-dependent zinc metallopeptidase and characterize its cleavage specificity profile using both trypsin and GluC-derived peptide libraries and protein substrates. Based on high-throughput degradomic assays, HgrFlaMP cleaved 784 unique peptides and displayed a cleavage site specificity similar to flagellinolysin from C. haemolyticum. Additionally, by using a set of six protein substrates, we identified 206 protein-embedded cleavage sites, further refining the substrate preference of HgrFlaMP, which is dominated by large hydrophobic amino acids in P1', and small hydrophobic or medium-sized polar residues on the amino-terminal side of the scissile bond. Intriguingly, recombinant HgrFlaMP was also capable of cleaving full-length flagellins from another species, suggesting its potential involvement in interbacterial interactions. Our study reports the first experimentally characterized proteolytic flagellin in a Gram-negative organism, and provides new insights into flagellum-mediated enzymatic activity.

Plants grow within a complex web of species that interact with each other and with the plant 1-10 . These interactions are governed by a wide repertoire of chemical signals, and the resulting chemical landscape of the rhizosphere can strongly affect root health and development 7-9,11-18 . Here, to understand how interactions between microorganisms influence root growth in Arabidopsis, we established a model system for interactions between plants, microorganisms and the environment. We inoculated seedlings with a 185-member bacterial synthetic community, manipulated the abiotic environment and measured bacterial colonization of the plant. This enabled us to classify the synthetic community into four modules of co-occurring strains. We deconstructed the synthetic community on the basis of these modules, and identified interactions between microorganisms that determine root phenotype. These interactions primarily involve a single bacterial genus (Variovorax), which completely reverses the severe inhibition of root growth that is induced by a wide diversity of bacterial strains as well as by the entire 185-member community. We demonstrate that Variovorax manipulates plant hormone levels to balance the effects of our ecologically realistic synthetic root community on root growth. We identify an auxin-degradation operon that is conserved in all available genomes of Variovorax and is necessary and sufficient for the reversion of root growth inhibition. Therefore, metabolic signal interference shapes bacteria-plant communication networks and is essential for maintaining the stereotypic developmental programme of the root. Optimizing the feedbacks that shape chemical interaction networks in the rhizosphere provides a promising ecological strategy for developing more resilient and productive crops.

Dead fungal biomass is an abundant source of nutrition in both litter and soil of temperate forests largely decomposed by bacteria. Here, we have examined the utilization of dead fungal biomass by the five dominant bacteria isolated from the in situ decomposition of fungal mycelia using a multiOMIC approach. The genomes of the isolates encoded a broad suite of carbohydrate-active enzymes, peptidases and transporters. In the extracellular proteome, only Ewingella americana expressed chitinases while the two Pseudomonas isolates attacked chitin by lytic chitin monooxygenase, deacetylation and deamination. Variovorax sp. expressed enzymes acting on the side-chains of various glucans and the chitin backbone. Surprisingly, despite its genomic potential, Pedobacter sp. did not produce extracellular proteins to decompose fungal mycelia but presumably feeds on simple substrates. The ecological roles of the five individual strains exhibited complementary features for a fast and efficient decomposition of dead fungal biomass by the entire bacterial community., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli , seriously affects watermelon and other cucurbit crops, resulting in significant economic losses. However, the pathogenicity mechanism of A. citrulli is not well understood. Plant pathogenic bacteria often suppress the plant immune response by secreting effector proteins. Thus, identifying A. citrulli effector proteins and determining their functions may improve our understanding of the underlying pathogenetic mechanisms. In this study, a novel effector, AopN, which is localized on the cell membrane of Nicotiana benthamiana, was identified. The functional analysis revealed that AopN significantly inhibited the flg22-induced reactive oxygen species burst. AopN induced a programmed cell death (PCD) response. Unlike its homologous protein, the ability of AopN to induce PCD was dependent on two motifs of unknown functions (including DUP4129 and Cpta_toxin), but was not dependent on LXXLL domain. More importantly, the virulence of the aopN mutant of A. citrulli in N. benthamiana significantly decreased, indicating that it was a core effector. Further analysis revealed that AopN interacted with watermelon ClHIPP and ClLTP, which responds to A. citrulli strain Aac5 infection at the transcription level. Collectively, these findings indicate that AopN suppresses plant immunity and activates the effector-triggered immunity pathway.

Epothilones, as a new class of microtubule-stabilizing anticancer drugs, exhibit strong bioactivity against taxane-resistant cells and show clinical activity for the treatment of advanced breast cancer. Additionally, they also show great potential for a central nervous system injury and Alzheimer's disease. However, due to the long fermentation period of the original producer and challenges of genetic engineering of nonribosomal peptide/polyketide (NRP/PK) megasynthase genes, the application of epothilones is severely limited. Here, we addressed these problems by reassembling a novel 56-kb epothilone biosynthetic gene cluster, optimizing the promoter of each gene based on RNA-seq profiling, and completing precursor synthetic pathways in engineered Schlegella brevitalea . Furthermore, we debottlenecked the cell autolysis by optimizing culture conditions. Finally, the yield of epothilones in shake flasks was improved to 82 mg/L in six-day fermentation. Overall, we not only constructed epothilone overproducers for further drug development but also provided a rational strategy for high-level NRP/PK compound production.

Polyhydroxyalkanoates (PHAs), the intracellular polymers produced by various microorganisms as carbon and energy storage, are of great technological potential as biodegradable versions of common plastics. PHA-producing microbes are therefore in great demand and a plethora of different environments, especially extreme habitats, have been probed for the presence of PHA-accumulators. However, the polar region has been neglected in this regard, probably due to the low accessibility of the sampling material and unusual cultivation regime. Here, we present the results of a screening procedure involving 200 bacterial strains isolated from 25 habitats of both polar regions. Agar-based tests, microscopy, and genetic methods were conducted to elucidate the biodiversity and potential of polar-region PHA-accumulators. Microscopic observation of Nile Red stained cells proved to be the most reliable screening method as it allowed to confirm the characteristic bright orange glow of the Nile Red-PHA complex as well as the typical morphology of the PHA inclusions. Psychrophilic PHA-producers belonged mostly to the Comamonadaceae family (Betaproteobacteria) although actinobacterial PHA synthesizers of the families, Microbacteriaceae and Micrococcaceae also featured prominently. Glacial and postglacial habitats as well as developed polar region soils, were evaluated as promising for PHA-producer bioprospection. This study highlights the importance of psychrophiles as biodiverse and potent polyhydroxyalkanoate sources for scientific and application-aimed research.

Cytochrome P450 monooxygenases as powerful biocatalysts catalyze a wide range of chemical reactions to facilitate exogenous substances metabolism and biosynthesis of natural products. In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology, a new self-sufficient cytochrome P450 monooxygenase (P450(VpMO)), belongs to CYP116B class, was mined from Variovorax paradoxus S110 genome and expressed in Escherichia coli. Based on characterization of the enzymatic properties, it shows that the optimal pH and temperature for P450(VpMO) reaction activity are 8.0 and 45 °C, respectively. P450(VpMO) is relatively stable at temperatures below 35 °C. The Km and kcat of P450(VpMO) toward 4-Methoxyacetophenone are 0.458 mmol/L and 2.438 min⁻¹, respectively. Importantly, P450(VpMO) was able to catalyze the demethylation reaction for a range of substrates containing methoxy group. Its demethylation reactivity is reasonably better than other P450s belongs to CYP116B class, particularly, for 4-methoxyacetophenone with a great conversion efficiency at 91%, showing that P450(VpMO) could be used as a great biocatalyst candidate for further analysis.

A bacterium, designated HX2-24  T , was isolated from activated sludge treating pesticide-manufacturing wastewater. Colonies of the strain on nutrient agar were circular, transparent, and colorless. Strain HX2-24  T shared 98.1% 16S rRNA gene sequence similarity with Extensimonas vulgaris S4 T , and less than 97% similarities with other type strains. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain formed a clade with E. vulgaris S4 T . The major cellular fatty acids were C 16:0 , summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c) and C 17:0 cyclo, the major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), aminophospholipid (APL), glycophospholipid (GPL), and aminoglycolipid (AGL). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between HX2-24  T and E. vulgaris S4 T were 92% and 41%, respectively. The G + C content of strain HX2-24  T was 64.4 mol%. Thus, based on the phenotypic, chemotaxonomic, and genotypic characteristics, strain HX2-24  T represents a novel species in the genus Extensimonas, for which the name Extensimonas perlucida HX2-24  T sp. nov. is proposed. The type strain is HX2-24  T (= KCTC 72472  T  = CCTCC AB 2019178  T ).

Biodegradation of the phenylurea herbicide linuron appears a specialization within a specific clade of the Variovorax genus. The linuron catabolic ability is likely acquired by horizontal gene transfer but the mechanisms involved are not known. The full-genome sequences of six linuron-degrading Variovorax strains isolated from geographically distant locations were analyzed to acquire insight into the mechanisms of genetic adaptation toward linuron metabolism. Whole-genome sequence analysis confirmed the phylogenetic position of the linuron degraders in a separate clade within Variovorax and indicated that they unlikely originate from a common ancestral linuron degrader. The linuron degraders differentiated from Variovorax strains that do not degrade linuron by the presence of multiple plasmids of 20-839 kb, including plasmids of unknown plasmid groups. The linuron catabolic gene clusters showed 1) high conservation and synteny and 2) strain-dependent distribution among the different plasmids. Most of them were bordered by IS1071 elements forming composite transposon structures, often in a multimeric array configuration, appointing IS1071 as a key element in the recruitment of linuron catabolic genes in Variovorax. Most of the strains carried at least one (catabolic) broad host range plasmid that might have been a second instrument for catabolic gene acquisition. We conclude that clade 1 Variovorax strains, despite their different geographical origin, made use of a limited genetic repertoire regarding both catabolic functions and vehicles to acquire linuron biodegradation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Maltotetraohydrolase, widely used in food and medical fields, possesses the ability to hydrolyze starch to produce maltooligosaccharides with maltotetraose as the main product. It also has the potential usage in delaying bread aging., Results: Pseudomonas saccharophila maltotetraohydrolase was expressed in Bacillus subtilis WS11. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed obvious bands at 57 kDa (maltotetraohydrolase I) and 47 kDa (maltotetraohydrolase II). Both showed similar enzymatic properties, although the catalytic efficiency of maltotetraohydrolase I was 4.93 fold higher than that of maltotetraohydrolase II using soluble starch as substrate. In addition, the maltotetraohydrolase production was further scaled up in a 3-L fermentor, and the highest activity reached 1907 U mL -1 . Then, the recombinant maltotetraohydrolase was used to produce maltotetraose. The maltotetraose yields catalyzed by maltotetraohydrolase I and II reached 73.2% and 69.7%, respectively. Finally, when recombinant maltotetraohydrolase was used in bread-making, texture profile analysis of the bread indicated recombinant maltotetraohydrolase I exhibited a significant anti-aging effect., Conclusion: This is the first describing high-efficient expression of P. saccharophila maltotetraohydrolase in the food safety strain B. subtilis, and the yield represented the highest level ever reported. Excellent results were also obtained with respect to the preparation of maltotetraose and delaying bread aging using the recombinant maltotetraohydrolase. The present study will help lay the foundation for the industrial production and application of maltotetraohydrolase. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Hydrogenopahaga sp. strain UMI-18 is an alginolytic bacterium that can produce poly(3-hydroxybutylate) (PHB) using alginate as its sole carbon source. Genome analysis indicated that this strain harbors both PHB-synthesizing and alginate-assimilating gene clusters. In the present study, we cloned HyAly-I gene that encodes a PL-17 exolytic alginate lyase and investigated its enzymatic properties using recombinant HyAly-I (recHyAly-I) that was produced by Escherichia coli. The recHyAly-I preferably depolymerized poly(β-D-mannuronate) block of alginate in an exolytic manner at an optimal temperature and a pH at 40 °C and pH 6.0, respectively. It released 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from the non-reducing terminus of polymer and oligomer substrates. Interestingly, recHyAly-I was found to produce a novel unsaturated disaccharide, i.e., dimeric DEH (diDEH), along with monomeric DEH. Production of diDEH was prominent in the degradation of trisaccharides., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Most freshwater bacterial communities are characterized by a few dominant taxa that are often ubiquitous across freshwater biomes worldwide. Our understanding of the genomic diversity within these taxonomic groups is limited to a subset of taxa. Here, we investigated the genomic diversity that enables Limnohabitans , a freshwater genus key in funneling carbon from primary producers to higher trophic levels, to achieve abundance and ubiquity. We reconstructed eight putative Limnohabitans metagenome-assembled genomes (MAGs) from stations located along broad environmental gradients existing in Lake Michigan, part of Earth's largest surface freshwater system. De novo strain inference analysis resolved a total of 23 strains from these MAGs, which strongly partitioned into two habitat-specific clusters with cooccurring strains from different lineages. The largest number of strains belonged to the abundant LimB lineage, for which robust in situ strain delineation had not previously been achieved. Our data show that temperature and nutrient levels may be important environmental parameters associated with microdiversification within the Limnohabitans genus. In addition, strains predominant in low- and high-phosphorus conditions had larger genomic divergence than strains abundant under different temperatures. Comparative genomics and gene expression analysis yielded evidence for the ability of LimB populations to exhibit cellular motility and chemotaxis, a phenotype not yet associated with available Limnohabitans isolates. Our findings broaden historical marker gene-based surveys of Limnohabitans microdiversification and provide in situ evidence of genome diversity and its functional implications across freshwater gradients. IMPORTANCE Limnohabitans is an important bacterial taxonomic group for cycling carbon in freshwater ecosystems worldwide. Here, we examined the genomic diversity of different Limnohabitans lineages. We focused on the LimB lineage of this genus, which is globally distributed and often abundant, and its abundance has shown to be largely invariant to environmental change. Our data show that the LimB lineage is actually comprised of multiple cooccurring populations for which the composition and genomic characteristics are associated with variations in temperature and nutrient levels. The gene expression profiles of this lineage suggest the importance of chemotaxis and motility, traits that had not yet been associated with the Limnohabitans genus, in adapting to environmental conditions., (Copyright © 2020 American Society for Microbiology.)

LuxR-type regulators regulate many bacterial processes and play important roles in bacterial motility and virulence. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes great losses in melon and watermelon worldwide. We identified a LuxR-type, nonquorum sensing-related regulator, AcrR, in the group II strain Aac-5 of A. citrulli. We found that the acrR mutant lost twitching and swimming motilities, and flagellar formation. It also showed reduced virulence, but increased biofilm formation and growth ability. Transcriptomic analysis revealed that 394 genes were differentially expressed in the acrR mutant of A. citrulli, including 33 genes involved in flagellar assembly. Our results suggest that AcrR may act as a global regulator affecting multiple important biological functions of A. citrulli., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)