Shinji Takenaka

Adaptation of Pseudomonas sp. Strain 7-6 to Quaternary Ammonium Compounds and Their Degradation via Dual Pathways

ABSTRACT Pseudomonas sp. strain 7-6, isolated from active sludge obtained from a wastewater facility, utilized a quaternary ammonium surfactant, n-dodecyltrimethylammonium chloride (DTAC), as its sole carbon, nitrogen, and energy source. When initially grown in the presence of 10 mM DTAC medium, the isolate was unable to degrade DTAC. The strain was cultivated in gradually increasing concentrations of the surfactant until continuous exposure led to high tolerance and biodegradation of the compound. Based on the identification of five metabolites by gas chromatography-mass spectrometry analysis, two possible pathways for DTAC metabolism were proposed. In pathway 1, DTAC is converted to lauric acid via n-dodecanal with the release of trimethylamine; in pathway 2, DTAC is converted to lauric acid via n-dodecyldimethylamine and then n-dodecanal with the release of dimethylamine. Among the identified metabolites, the strain precultivated on DTAC medium could utilize n-dodecanal and lauric acid as sole carbon sources and trimethylamine and dimethylamine as sole nitrogen sources, but it could not efficiently utilize n-dodecyldimethylamine. These results indicated pathway 1 is the main pathway for the degradation of DTAC.

Purification and Characterization of Two Novel Halotolerant Extracellular Proteases from Bacillus subtilis Strain FP-133

Bacillus subtilis strain FP-133, isolated from a fermented fish paste, synthesized two novel halotolerant extracellular proteases (expro-I and expro-II), showing activity and stability at concentrations of 0–20% (w/v) NaCl. Each protease was purified to homogeneity and characterized. The purified expro-I was a non-alkaline serine protease with an optimum pH of 7.5, although most serine proteases from Bacillus strains act at the alkaline side. The molecular mass of expro-I was 29 kDa. The purified expro-II was a metalloprotease with a molecular mass of 34 kDa. It was activated by Fe2+, which has never been reported as a bacterial protease activator. At a concentration of 7.5% (w/v) NaCl, both proteases preferred animal proteins to vegetable proteins as natural substrates. In addition, under saline conditions, expro-I and II showed high catalytic activity toward gelatin and casein respectively.

The Metabolic Pathway of 4-Aminophenol in Burkholderia sp. Strain AK-5 Differs from That of Aniline and Aniline with C-4 Substituents

ABSTRACT Burkholderia sp. strain AK-5 utilized 4-aminophenol as the sole carbon, nitrogen, and energy source. A pathway for the metabolism of 4-aminophenol in strain AK-5 was proposed based on the identification of three key metabolites by gas chromatography-mass spectrometry analysis. Strain AK-5 converted 4-aminophenol to 1,2,4-trihydroxybenzene via 1,4-benzenediol. 1,2,4-Trihydroxybenzene 1,2-dioxygenase cleaved the benzene ring of 1,2,4-trihydroxybenzene to form maleylacetic acid. The enzyme showed a high dioxygenase activity only for 1,2,4-trihydroxybenzene, with Km and Vmax values of 9.6 μM and 6.8 μmol min−1 mg of protein−1, respectively.

Cloning and sequence analysis of two catechol-degrading gene clusters from the aniline-assimilating bacterium Frateuria species ANA-18

The aniline-assimilating bacterium Frateuria species ANA-18 produced two catechol 1,2-dioxygenases, CD I and CD II, and two muconate cycloisomerases, MC I and MC II. The catA genes catA1 and catA2 encoding CD I and CD II, respectively, were cloned from a gene library of this bacterium. The catA1 gene was clustered with catB1 encoding MC I, catC1 encoding muconolactone isomerase (MI), catD encoding beta-ketoadipate enol-lactone hydrolase (ELH), and ORFR1 encoding a putative LysR-type regulator. The organization of these genes was ORFR1catB1C1D. The catA2 gene also constructed a gene cluster involving catB2 encoding MC II, catC2 encoding MI, and ORFR2 encoding a putative LysR-type regulator with the alignment of ORFR2catB2A2C2. The intergenic regions of ORFR1-catB1 and ORFR2-catB2 contained homologous sequences with the catR-catB intergenic region containing a repression binding site and activation binding site of CatR in Pseudomonas putida. These findings suggest that the two cat clusters were regulated independently in their expression. When a product of cloned catD was added to a reaction mixture containing beta-ketoadipate enol-lactone, beta-ketoadipate was produced. This observation showed that the cloned catD encoded ELH and was expressed in Escherichia coli. We found that Frateuria sp. ANA-18 had a large plasmid with a molecular size more than 100kb. Polymerase chain reaction amplifying partial catA genes and Southern hybridization analyses with probes containing catA genes were conducted, to examine the localization of the two catA genes. We concluded that the catA1 and catA2 genes were located on the chromosomal and large plasmid DNAs, respectively, in Frateuria sp. ANA-18.

Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22 : production, purification, characterization and gene analysis of CatA, CatB and CatC

The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 degrees C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC.

Complete nucleotide sequence and functional analysis of the genes for 2-aminophenol metabolism from Pseudomonas sp. AP-3

Abstract. A 13.9-kb region, which contained the 2-aminophenol 1,6-dioxygenase genes (amnBA) reported before, was cloned from the 2-aminophenol-assimilating bacterium Pseudomonas sp. AP-3. The complete nucleotide sequence of this region was determined and six genes were found downstream of amnBA. The eight genes together were designated amnBACFEDHG. Each gene was similar to the corresponding gene operating in the meta-cleavage pathway, except for amnB, amnA, and amnD. The four 2-aminophenol-metabolizing enzymes, 2-aminomuconic 6-semialdehyde dehydrogenase, 2-aminomuconate deaminase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase, were purified and characterized. NH2-terminal amino acid sequences of each purified enzyme agreed with those deduced from amnC, amnF, amnE, and amnD, respectively. These genes were therefore assigned as the genes encoding these respective proteins. The tight clustering of the amn genes, which were all transcribed in the same direction, raised the possibility that these genes formed a single operon. The organization of the amn genes was entirely different from that of the atd, dmp, and xyl genes reported in the meta-cleavage pathway, although these latter genes clustered similarly.

Purification and Characterization of Two Alkaline, Thermotolerant α-Amylases from Bacillus halodurans 38C-2-1 and Expression of the Cloned Gene in Escherichia coli

A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant α-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named α-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50–60 °C and pH 10–11, and 42 and 38% relative activities at 30 °C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding α-amylase I was cloned and named amyI. Production of AmyI with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl β-D(−)-thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.

Constitutive synthesis, purification, and characterization of catechol 1,2-dioxygenase from the aniline-assimilating bacterium Rhodococcus sp. AN-22.

A catechol 1,2-dioxygenase (CD) was found, which was synthesized constitutively in the aniline-assimilating bacterium Rhodococcus sp. AN-22 grown on a medium without aniline, as well as on aniline medium. The bacterium synthesized CD in its cells grown on all the 21 non-aromatic substrates examined, including four natural media such as meat and yeast extracts, one sugar, six organic acids, and 10 amino acids as carbon, energy, and nitrogen sources. When the bacterium was incubated on a medium with D-glucose, L-malate, isoleucine, leucine, etc., it synthesized more CD than that in cells grown on aniline. Two CDs, which were prepared from cells grown on aniline and L-malate, were purified separately to homogeneity and characterized. The two enzymes were apparently identical in molecular and catalytic properties including molecular mass, optimal pH, stability to heating, and substrate specificity for catechol analogues. However, they differed in the substrate specificity and resistance to sulfhydryl and chelating agents from the inducible CDs produced by other aniline-assimilating bacteria reported previously.

Metabolism of 2-aminophenol by Pseudomonas sp. AP-3: modified meta-cleavage pathway

Abstract A novel pathway for 2-aminophenol metabolism by Pseudomonas sp. AP-3 is proposed. The proposed pathway is similar to that known for meta-cleavage of catechol except that one of the hydroxyl groups on the metabolites is replaced by an amino group. During the degradation of 2-aminophenol, 2-amino-2,4-pentadienoic acid is the last metabolite containing an amino group. We, therefore, propose a modified meta-cleavage pathway for the 2-aminophenol metabolism.

A cell factory of Bacillus subtilis engineered for the simple bioconversion of myo-inositol to scyllo-inositol, a potential therapeutic agent for Alzheimer's disease

BackgroundA stereoisomer of inositol, scyllo-inositol, is known as a promising therapeutic agent for Alzheimers disease, since it prevents the accumulation of beta-amyloid deposits, a hallmark of the disease. However, this compound is relatively rare in nature, whereas another stereoisomer of inositol, myo-inositol, is abundantly available.ResultsBacillus subtilis possesses a unique inositol metabolism involving both stereoisomers. We manipulated the inositol metabolism in B. subtilis to permit the possible bioconversion from myo-inositol to scyllo-inositol. Within 48 h of cultivation, the engineered strain was able to convert almost half of 10 g/L myo-inositol to scyllo-inositol that accumulated in the culture medium.ConclusionsThe engineered B. subtilis serves as a prototype of cell factory enabling a novel and inexpensive supply of scyllo-inositol.