Complete genome sequence of Paraburkholderia terrae strain KU-46, a 2,4-dinitrophenol-degrading bacterium.

Paraburkholderia terrae strain KU-46 has been studied for its capability to degrade 2,4-dinitrophenol. Here, we present the complete 10,833,180bp genome of this microorganism, comprising five circular chromosomes housing 9,797 protein-coding sequences. The genes responsible for 2,4-dinitrophenol and 4-nitrophenol degradation are located on chromosome 2.

Precise microbiome engineering using natural and synthetic bacteriophages targeting an artificial bacterial consortium.

In natural microbiomes, microorganisms interact with each other and exhibit diverse functions. Microbiome engineering, which enables bacterial knockdown, is a promising method to elucidate the functions of targeted bacteria in microbiomes. However, few methods to selectively kill target microorganisms in the microbiome without affecting the growth of nontarget microorganisms are available. In this study, we focused on the host-specific lytic ability of virulent phages and validated their potency for precise microbiome engineering. In an artificial microbiome consisting of Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Lactiplantibacillus plantarum, the addition of bacteriophages infecting their respective host strains specifically reduced the number of these bacteria more than 102 orders. Remarkably, the reduction in target bacteria did not affect the growth of nontarget bacteria, indicating that bacteriophages were effective tools for precise microbiome engineering. Moreover, a virulent derivative of the λ phage was synthesized from prophage DNA in the genome of λ lysogen by in vivo DNA assembly and phage-rebooting techniques, and E. coli-targeted microbiome engineering was achieved. These results propose a novel approach for precise microbiome engineering using bacteriophages, in which virulent phages are synthesized from prophage DNA in lysogenic strains without isolating phages from environmental samples.

Identification and characterization of a pab gene cluster responsible for the 4-aminobenzoate degradation pathway, including its involvement in the formation of a γ-glutamylated intermediate in Paraburkholderia terrae strain KU-15.

Paraburkholderia terrae strain KU-15 grows on 2- and 4-nitrobenzoate and 2- and 4-aminobenzoate (ABA) as the sole nitrogen and carbon sources. The genes responsible for the potential degradation of 2- and 4-nitrobenzoate and 2-ABA have been predicted from its genome sequence. In this study, we identified the pab operon in P. terrae strain KU-15. This operon is responsible for the 4-ABA degradation pathway, which involves the formation of a γ-glutamylated intermediate. Reverse transcription-polymerase chain reaction revealed that the pab operon was induced by 4-ABA. Herein, studying the deletion of pabA and pabB1 in strain KU-15 and the examining of Escherichia coli expressing the pab operon revealed the involvement of the operon in 4-ABA degradation. The first step of the degradation pathway is the formation of a γ-glutamylated intermediate, whereby 4-ABA is converted to γ-glutamyl-4-carboxyanilide (γ-GCA). Subsequently, γ-GCA is oxidized to protocatechuate. Overexpression of various genes in E. coli and purification of recombinant proteins permitted the functional characterization of relevant pathway proteins: PabA is a γ-GCA synthetase, PabB1-B3 functions in a multicomponent dioxygenase system responsible for γ-GCA dioxygenation, and PabC is a γ-GCA hydrolase that reverses the formation of γ-GCA by PabA.

Cloning of two gene clusters involved in the catabolism of 2,4-dinitrophenol by Paraburkholderia sp. strain KU-46 and characterization of the initial DnpAB enzymes and a two-component monooxygenases DnpC1C2.

Little is currently known about the metabolism of the industrial pollutant 2,4-dinitrophenol (DNP), particularly among gram-negative bacteria. In this study, we identified two non-contiguous genetic loci spanning 22 kb of Paraburkholderia (formerly Burkholderia) sp. strain KU-46. Additionally, we characterized four key initial genes (dnpA, dnpB, and dnpC1C2) responsible for DNP degradation, providing molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription polymerase chain reaction (PCR) analysis indicated that dnpA, which encodes the initial hydride transferase, and dnpB which encodes a nitrite-eliminating enzyme, were induced by DNP and organized in an operon. Moreover, we purified DnpA and DnpB from recombinant Escherichia coli to demonstrate their effect on the transformation of DNP to NP through the formation of a hydride-Meisenheimer complex of DNP, designated as H--DNP. The function of DnpB appears new since all homologs of the DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designated dnpC1C2DXFER, and DnpC1 and DnpC2 were functionally characterized as the FAD reductase and oxygenase components of the two-component DNP monooxygenase, respectively. By elucidating the hqdA1A2BCD gene cluster, we are now able to delineate the final degradation pathway of hydroquinone to ß-ketoadipate before it enters the tricarboxylic acid cycle.

Subtractive modification of bacterial consortium using antisense peptide nucleic acids.

Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of the microbiome allows us to create an artificial microbiome without the microorganism of interest and to evaluate its functions and interactions with other constituent bacteria. However, few techniques that can specifically remove only a single species from a large number of microorganisms and can be applied universally to a variety of microorganisms have been developed. Antisense peptide nucleic acid (PNA) is a potent designable antimicrobial agent that can be delivered into microbial cells by conjugating with a cell-penetrating peptide (CPP). Here, we tested the efficacy of the conjugate of CPP and PNA (CPP-PNA) as microbiome modifiers. The addition of CPP-PNA specifically inhibited the growth of Escherichia coli and Pseudomonas putida in an artificial bacterial consortium comprising E. coli, P. putida, Pseudomonas fluorescens, and Lactiplantibacillus plantarum. Moreover, the growth inhibition of P. putida promoted the growth of P. fluorescens and inhibited the growth of L. plantarum. These results indicate that CPP-PNA can be used not only for precise microbiome engineering but also for analyzing the growth relationships among constituent microorganisms in the microbiome.

Complete Genome Sequence of Paraburkholderia terrae Strain KU-15, a 2-Nitrobenzoate-Degrading Bacterium.

Paraburkholderia terrae strain KU-15 has been investigated for its ability to degrade 2-nitrobenzoate. Here, we report the complete 10,422,345-bp genome of this microorganism, which consists of six circular replicons containing 9,483 protein-coding sequences. The genome carries genes that are potentially responsible for 2-nitrobenzoate and 4-nitirobenzoate degradation.

Bactericidal effect of nanostructures via lytic transglycosylases of Escherichia coli.

Nanostructures exhibit a bactericidal effect owing to physical interaction with the bacterial cell envelope. Here, we aimed to identify the mechanism underlying the bactericidal effect of nanostructures based on bacterial autolysis, in contrast to previous reports focusing on structural characteristics. The time profiles of active cell ratios of the Escherichia coli strains (WT, ΔmltA, ΔmltB, Δslt70), incubation time of the wild-type (WT) strains, and autolysis inhibition of WT strains were evaluated with respect to the bactericidal effect of the applied nanostructures. Addition of Mg2+, an autolysis inhibitor, was not found to cause significant cell damage. The incubation phase was significantly associated with envelope damage. The lytic transglycosylase-lacking strain of Slt70 (Δslt70) also showed only minimal envelope damage. Our results indicate that nanostructures may act by triggering bacterial autolysis.

Identification and characterization of a novel class of self-sufficient cytochrome P450 hydroxylase involved in cyclohexanecarboxylate degradation in Paraburkholderia terrae strain KU-64.

Cytochrome P450 monooxygenases play important roles in metabolism. Here, we report the identification and biochemical characterization of P450CHC, a novel self-sufficient cytochrome P450, from cyclohexanecarboxylate-degrading Paraburkholderia terrae KU-64. P450CHC was found to comprise a [2Fe-2S] ferredoxin domain, NAD(P)H-dependent FAD-containing reductase domain, FCD domain, and cytochrome P450 domain (in that order from the N terminus). Reverse transcription-polymerase chain reaction results indicated that the P450CHC-encoding chcA gene was inducible by cyclohexanecarboxylate. chcA overexpression in Escherichia coli and recombinant protein purification enabled functional characterization of P450CHC as a catalytically self-sufficient cytochrome P450 that hydroxylates cyclohexanecarboxylate. Kinetic analysis indicated that P450CHC largely preferred NADH (Km = 0.011 m m) over NADPH (Km = 0.21 m m). The Kd, Km, and kcat values for cyclohexanecarboxylate were 0.083 m m, 0.084 m m, and 15.9 s-1, respectively. The genetic and biochemical analyses indicated that the physiological role of P450CHC is initial hydroxylation in the cyclohexanecarboxylate degradation pathway.

Identification and characterization of a chc gene cluster responsible for the aromatization pathway of cyclohexanecarboxylate degradation in Sinomonas cyclohexanicum ATCC 51369.

Cyclohexanecarboxylate (CHCA) is formed by oxidative microbial degradation of n-alkylcycloparaffins and anaerobic degradation of benzoate, and also known to be a synthetic intermediate or the starter unit of biosynthesis of cellular constituents and secondary metabolites. Although two degradation pathways have been proposed, genetic information has been limited to the ß-oxidation-like pathway. In this study, we identified a gene cluster, designated chcC1XTC2B1B2RAaAbAc, that is responsible for the CHCA aromatization pathway in Sinomonas (formerly Corynebacterium) cyclohexanicum strain ATCC 51369. Reverse transcription-PCR analysis indicated that the chc gene cluster is inducible by CHCA and that it consists of two transcriptional units, chcC1XTC2B1B2R and chcAaAbAc. Overexpression of the various genes in Escherichia coli, and purification of the recombinant proteins led to the functional characterization of ChcAaAbAc as subunits of a cytochrome P450 system responsible for CHCA hydroxylation; ChcB1 and ChcB2 as trans-4-hydroxyCHCA and cis-4-hydroxyCHCA dehydrogenases, respectively; ChcC1 was identified as a 4-oxoCHCA desaturase containing a covalently bound FAD; and ChcC2 was identified as a 4-oxocyclohexenecarboxylate desaturase. The binding constant of ChcAa for CHCA was found to be 0.37 mM. Kinetic parameters established for ChcB1 indicated that it has a high catalytic efficiency towards 4-oxoCHCA compared to trans- or cis-4-hydroxyCHCA. The Km and Kcat values of ChcC1 for 4-oxoCHCA were 0.39 mM and 44 s-1, respectively. Taken together with previous work on the identification of a pobA gene encoding a 4-hydroxybenzoate hydroxylase, we have now localized the remaining set of genes for the final degradation of protocatechuate before entry into the tricarboxylic acid cycle.

Cloning, expression, and characterization of Baeyer-Villiger monooxygenases from eukaryotic Exophiala jeanselmei strain KUFI-6N.

The fungus Exophiala jeanselmei strain KUFI-6N produces a unique cycloalkanone monooxygenase (ExCAMO) that displays an uncommon substrate spectrum of Baeyer-Villiger oxidation of 4-10-membered ring ketones. In this study, we aimed to identify and sequence the gene encoding ExCAMO from KUFI-6N and overexpress the gene in Escherichia coli. We found that the primary structure of ExCAMO is most closely related to the cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011, with 54.2% amino acid identity. ExCAMO was functionally expressed in E. coli and its substrate spectrum and kinetic parameters were investigated. Substrate profiling indicated that ExCAMO is unusual among known Baeyer-Villiger monooxygenases owing to its ability to accept a variety of substrates, including C4-C12 membered ring ketones. ExCAMO has high affinity and catalytic efficiency toward cycloalkanones, the highest being toward cyclohexanone. Five other genes encoding Baeyer-Villiger monooxygenases were also cloned and expressed in E. coli.

A novel piperidine degradation mechanism in a newly isolated piperidine degrader Pseudomonas sp. strain KU43P.

The degradation pathways in microorganisms for piperidine, a secondary amine with various applications, are not yet fully understood, especially in non-Mycobacterium species. In this study, we have identified a piperidine-degrading isolate (KU43P) from a soil sample collected in a cultivation field in Osaka, Japan, and characterized its mechanisms of piperidine degradation, thereby furthering current understanding of the process. The genome of isolate KU43P consists of a 5,869,691-bp circular chromosome with 62.67% GC content and with 5,294 predicted protein-coding genes, 77 tRNA genes, and 22 rRNA genes. 16S rRNA gene sequence analysis and average nucleotide identity analysis suggest that the isolate is a novel species of the Pseudomonas putida group in the genus Pseudomonas. The genomic region encoding the piperidine degradation pathway, designated as the pip gene cluster, was identified using transposon mutagenesis and reverse transcription polymerase chain reaction. Deletion analyses of pipA, which encodes a glutamine synthetase (GS)-like protein, and pipBa, which encodes a cytochrome P450 monooxygenase, indicate that pipA and pipBa are involved in piperidine metabolism and suggest that pipA is involved in the first step of the piperidine metabolic pathway. Escherichia coli whole cells overexpressing PipA converted piperidine and glutamate to γ-glutamylpiperidide, and crude cell extract enzyme assays of PipA showed that this reaction requires ATP and Mg2+. These results clearly show that pipA encodes γ-glutamylpiperidide synthetase and that piperidine is first glutamylated and then hydroxylated in the piperidine degradation pathway of Pseudomonas sp. strain KU43P. This study has filled a void in the general knowledge of the microbial degradation of amine compounds.

Complete Genome Sequence of Mameliella alba Strain KU6B, a Cyclohexylamine-Utilizing Marine Bacterium.

Here, we report the complete genome sequence of Mameliella alba strain KU6B, a bacterium newly isolated from seawater of Boso Peninsula in Japan that is capable of utilizing cyclohexylamine. The complete genome contained a 5,386,988-bp circular chromosome and three circular plasmids of 256,516, 112,434, and 76,727 bp.

Complete Genome Sequence of Pseudomonas sp. Strain KUIN-1, a Model Strain for Studies on the Production of Cell-Free Ice Nucleation Proteins.

Pseudomonas sp. (formerly Pseudomonas fluorescens) strain KUIN-1 is an ice-nucleating bacterium that was isolated from the leaves of field beans (Phaseolus vulgaris L.). This microorganism can release cell-free ice nucleation proteins and shows cold shock-induced freezing tolerance. Here, we report the 6,028,589-bp complete genome sequence of Pseudomonas sp. KUIN-1.

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase. Corrigendum.

A statement is amended in the article by Isupov et al. [(2015). Acta Cryst. D71, 2344-2353].

Isolation of marine xylene-utilizing bacteria and characterization of Halioxenophilus aromaticivorans gen. nov., sp. nov. and its xylene degradation gene cluster.

Seven xylene-utilizing bacterial strains were isolated from seawater collected off the coast of Japan. Analysis of 16S rRNA gene sequences indicated that six isolates were most closely related to the marine bacterial genera Alteromonas, Marinobacter or Aestuariibacter. The sequence of the remaining strain, KU68FT, showed low similarity to the 16S rRNA gene sequences of known bacteria with validly published names, the most similar species being Maricurvus nonylphenolicus strain KU41ET (92.6% identity). On the basis of physiological, chemotaxonomic and phylogenetic data, strain KU68FT is suggested to represent a novel species of a new genus in the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria, for which the name Halioxenophilus aromaticivorans gen. nov., sp. nov. is proposed. The type strain of Halioxenophilus aromaticivorans is KU68FT (=JCM 19134T = KCTC 32387T). PCR and sequence analysis revealed that strain KU68FT possesses an entire set of genes encoding the enzymes for the upper xylene methyl-monooxygenase pathway, xylCMABN, resembling the gene set of the terrestrial Pseudomonas putida strain mt-2.

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase.

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a ß-bulge at the C-terminus of ß-strand 3, which is a feature observed in many proteins of this superfamily.

Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action.

Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for 'disarming' the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD(+)-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp(3)-to-sp(2) transition during enzyme-mediated oxidation.

Human α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD): a structural and mechanistic unveiling.

Human α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase determines the fate of tryptophan metabolites in the kynurenine pathway by controlling the quinolinate levels for de novo nicotinamide adenine dinucleotide biosynthesis. The unstable nature of its substrate has made gaining insight into its reaction mechanism difficult. Our electron paramagnetic resonance (EPR) spectroscopic study on the Cu-substituted human enzyme suggests that the native substrate does not directly ligate to the metal ion. Substrate binding did not result in a change of either the hyperfine structure or the super-hyperfine structure of the EPR spectrum. We also determined the crystal structure of the human enzyme in its native catalytically active state (at 1.99 Å resolution), a substrate analogue-bound form (2.50 Å resolution), and a selected active site mutant form with one of the putative substrate binding residues altered (2.32 Å resolution). These structures illustrate that each asymmetric unit contains three pairs of dimers. Consistent with the EPR findings, the ligand-bound complex structure shows that the substrate analogue does not directly coordinate to the metal ion but is bound to the active site by two arginine residues through noncovalent interactions.

Substrate profiling of cyclohexylamine oxidase and its mutants reveals new biocatalytic potential in deracemization of racemic amines.

A cyclohexylamine oxidase (CHAO) of bacterial origin was previously shown to be a potentially useful catalyst in the deracemization of racemic primary amines. To further explore the properties and application of this enzyme, five single-amino acid substitution mutants (L199A, M226A, Y321A, Y321F, and L353M) were created based on superimposition of the tertiary structure of CHAO and the monoamine oxidase (MAO) B homolog. The substrate specificity of the purified wild-type and five mutant enzymes were examined towards 38 structurally diverse amines. All the enzymes exhibited better activity for primary amines than secondary and tertiary amines and in general exhibited high stereoselectivity. Among the mutant enzymes, M226A displayed an enhanced activity (5-400%) towards most substrates, and L353M showed 7-445% higher activity towards primary aliphatic amines with cycloalkane or aromatic moieties. Kinetic parameters revealed that both Y321 mutants showed higher catalytic efficiency towards cyclooctanamine, whereas the wild-type CHAO (wt CHAO) was most efficient towards cyclohexylamine. The wt CHAO or variant L353M in combination with a borane-ammonia complex as reducing agent was applied to the deracemization of 1-aminotetraline to give the (R)-enantiomer, a precursor of an antidepressant drug Norsertraline, in good yield (73-76%), demonstrating their application potential in chiral amine synthesis.

Isolation and characterization of marine nonylphenol-degrading bacteria and description of Pseudomaricurvus alkylphenolicus gen. nov., sp. nov.

Two novel aerobic p-n-nonylphenol-degrading bacterial strains were isolated from seawater obtained from the coastal region of Ogasawara Islands, Japan. The 16S rRNA gene sequence analysis indicated that the strains are affiliated with the order Alteromonadales within the class Gammaproteobacteria. One isolate, strain KU41G2, is most closely related to Maricurvus nonylphenolicus (99.2 % similarity), and is tentatively identified as M. nonylphenolicus. The other isolate, strain KU41G(T), is also most closely related to M. nonylphenolicus; however, the 16S rRNA gene sequence similarity was only 94.7 %. Cells of strain KU41G(T) are Gram-negative rods with a single polar flagellum. The predominant respiratory lipoquinone was ubiquinone-8, and the major cellular fatty acids were C17:1 ω8c (24.2 %); C15:0 iso 2-OH; and/or C16:1 ω7c (16.3 %), C15:0 (10.3 %), C11:0 3-OH (9.5 %), C9:0 3-OH (6.7 %), C10:0 3-OH (6.4 %), and C18:1 ω7c (5.5 %). The DNA G+C content was 53.3 mol%. On the basis of physiological, chemotaxonomic, and phylogenetic data, strain KU41G(T) is suggested to represent a novel species of a new genus, for which we propose the name Pseudomaricurvus alkylphenolicus gen. nov., sp. nov. The type strain of P. alkylphenolicus is KU41G(T) (=JCM 19135(T) = KCTC 32386(T)).