Identification of indigo-related pigments produced by Escherichia coli containing a cloned Rhodococcus gene.

Pigments produced by Escherichia coli containing a cloned piece of DNA from Rhodococcus sp. ATCC 21145 were extracted in chloroform and separated into blue and pink components. Evidence from TLC, NMR spectroscopy, absorption spectrum analysis and solubility behaviour suggested that the blue pigment was indigo and the pink pigment was indirubin, a structural isomer of indigo. The proposed pathway for pigment production on LB agar involves the conversion of tryptophan to indole by tryptophanase of E. coli and the oxidation of indole to indigo by the product of the cloned Rhodococcus DNA insert.     

Construction of an insertional-inactivation cloning vector for Escherichia coli using a Rhodococcus gene for indigo production.

pSLH8, an insertional-inactivation cloning vector for Escherichia coli has been constructed by inserting a pigment gene (probably encoding an indole dioxygenase) from Rhodococcus sp. ATCC 21145 into pUC18. Wild-type E. coli colonies containing pSLH8 produce insoluble indigo and turn dark blue on unsupplemented LB agar. Insertion of DNA fragments into the unique BamHI, EcoRI, EcoRV, HindIII, PstI, SphI and SstI polylinker cloning sites disrupts the reading frame of the fully sequenced pigment gene and results in unpigmented colonies. pSLH8 may be an attractive alternative to pUC18 and similar plasmids because it does not require specifically mutated host strains or an expensive substrate for colour development.     

Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp.

A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1(pMVS301) and transformed into Rhodococcus sp. strain AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. Thiostrepton-resistant transformants were also ampicillin resistant and were shown to contain pMVS301, which was subsequently isolated and transformed back into E. coli. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. Optimization of the transformation procedure resulted in transformation frequencies in the range of 10(5) transformants per micrograms of pMVS301 DNA in Rhodococcus sp. strain H13-A and derivative strains. The plasmid host range extends to strains of Rhodococcus erythropolis, R. globulerus, and R. equi, whereas stable transformants were not obtained with R. rhodochrous or with several coryneform bacteria tested as recipients. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.     

Cloning and expression of the s-triazine hydrolase gene (trzA) from Rhodococcus corallinus and development of Rhodococcus recombinant strains capable of dealkylating and dechlorinating the herbicide atrazine.

We used degenerate oligodeoxyribonucleotides derived from the N-terminal sequence of the s-triazine hydrolase from Rhodococcus corallinus NRRL B-15444R in an amplification reaction to isolate a DNA segment containing a 57-bp fragment from the trzA gene. By using the nucleotide sequence of this fragment, a nondegenerate oligodeoxyribonucleotide was synthesized and used to screen a genomic library of R. corallinus DNA for fragments containing trzA. A 5.3-kb PstI fragment containing trzA was cloned, and the nucleotide sequence of a 2,450-bp region containing trzA was determined. No trzA expression was detected in Escherichia coli or several other gram-negative bacteria. The trzA gene was subcloned into a Rhodococcus-E. coli shuttle vector, pBS305, and transformed into several Rhodococcus strains. Expression of trzA was demonstrated in all Rhodococcus transformants. Rhodococcus sp. strain TE1, which possesses the catabolic gene (atrA) for the N-dealkylation of the herbicides atrazine and simazine, was able to dechlorinate the dealkylated metabolites of atrazine and simazine when carrying the trzA gene on a plasmid. A plasmid carrying both atrA and trzA was constructed and transformed into three atrA- and trzA-deficient Rhodococcus strains. Both genes were expressed in the transformants. The s-triazine hydrolase activity of the recombinant strains carrying the trzA plasmid were compared with that of the R. corallinus strain from which it was derived.     

Cloning of the genes for degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1.

The degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-1,3,5-triazine) is associated with an indigenous plasmid in Rhodococcus sp. strain TE1. Plasmid DNA libraries of Rhodococcus sp. strain TE1 were constructed in a Rhodococcus-Escherichia coli shuttle vector, pBS305, and transferred into Rhodococcus sp. strain TE3, a derivative of Rhodococcus sp. strain TE1 lacking herbicide degradation activity, to select transformants capable of growing on EPTC as the sole source of carbon (EPTC+). Analysis of plasmids from the EPTC+ transformants indicated that the eptA gene, which codes for the enzyme required for EPTC degradation, residues on a 6.2-kb KpnI fragment. The cloned fragment also harbored the gene required for atrazine N dealkylation (atrA). The plasmid carrying the cloned fragment could be electroporated into a number of other Rhodococcus strains in which both eptA and atrA were fully expressed. No expression of the cloned genes was evident in E. coli strains. Subcloning of the 6.2-kb fragment to distinguish between EPTC- and atrazine-degrading genes was not successful.     

Development of a host-vector system in a Rhodococcus strain and its use for expression of the cloned nitrile hydratase gene cluster.

Two different types of plasmid were isolated from strains of Rhodococcus rhodochrous. Two plasmids, of the same type but from different strains, were combined with Escherichia coli plasmids carrying antibiotic resistance markers to develop E. coli-Rhodococcus shuttle vectors. The ampicillin and kanamycin resistance markers served for selection in Rhodococcus. Electroporation was used to introduce recombinant plasmid DNA into R. rhodochrous ATCC 12674 at a frequency of 5 x 10(7) transformants per microgram DNA. With these host-vector and transformation systems, the nitrile hydratase and amidase genes of a Rhodococcus strain were introduced into the host strain and were efficiently expressed.     

Characterization of the yellow-pigment genes of Erwinia herbicola

A 6.7 kb DNA fragment containing the pigment genes of Erwinia herbicola Eho13 has been cloned into Escherichia coli. These genes were chromosomally encoded in E. herbicola. The entire DNA fragment could be divided into at least three regions. Deletions in Region I resulted in a non-pigmented phenotype, a deletion in Region II resulted in a pink/yellow phenotype, deletions in Region III resulted in either a pink or a non-pigmented phenotype. Tn1000 insertions in the same regions, however, gave different phenotypes. Insertions in Region II produced a pink phenotype. Insertions in Region III resulted in either a light-yellow or a non-pigmented phenotype. Minicell studies showed that the 6.7 kb DNA fragment encoded at least five proteins (50 kDa, 42 kDa, 36 kDa, 35 kDa and 34 kDa). A 2.7 kb HindIII deletion in Region I caused the disappearance of these proteins, suggesting that this 2.7 kb fragment may play a regulatory role in pigment synthesis. Our results also showed that a 4.1 kb EcoRV fragment consisted of Region I and a part of Region II complemented a pink/yellow clone of Eho10 (pHL545), suggesting that the pigments of Eho13 and Eho10 were probably similar or identical.     

Cloning, sequencing, and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain 141-8 in Escherichia coli.

The gene coding for N-acyl-D-mannosamine dehydrogenase (NAM-DH) from Flavobacterium sp. strain 141-8 was cloned and expressed under the control of a lac promoter in Escherichia coli JM109. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide composed of 272 amino acid residues (Mr, 27,473) was identified. The E. coli transformants which showed over 200-fold higher NAM-DH activity than did the Flavobacterium strain produced the enzyme as a protein fused with beta-galactosidase. Despite being a fusion, NAM-DH produced by E. coli transformants appeared unchanged in pH optimum, Km, and substrate specificity from Flavobacterium sp. strain 141-8. This newly recombinant enzyme may be applicable to the quantitative determination of sialic acid in serum.     

Cloning, sequencing, and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain 141-8 in Escherichia coli.

The gene coding for N-acyl-D-mannosamine dehydrogenase (NAM-DH) from Flavobacterium sp. strain 141-8 was cloned and expressed under the control of a lac promoter in Escherichia coli JM109. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide composed of 272 amino acid residues (Mr, 27,473) was identified. The E. coli transformants which showed over 200-fold higher NAM-DH activity than did the Flavobacterium strain produced the enzyme as a protein fused with beta-galactosidase. Despite being a fusion, NAM-DH produced by E. coli transformants appeared unchanged in pH optimum, Km, and substrate specificity from Flavobacterium sp. strain 141-8. This newly recombinant enzyme may be applicable to the quantitative determination of sialic acid in serum.     

Characterization of production of cholesterol oxidases in three Rhodococcus strains

The production of cholesterol oxidase in two strains of Rhodococcus equi No. 23 from butter, and Rhodococcus sp. No. 33 from bacon, which had properties on biochemical and physiological tests almost similar to the strains of R. equi , was compared with that of the type strain (ATCC 6939) of R. equi. The intensity of cholesterol oxidase activity, both extracellular and membrane-bound, from the three strains was in the order No. 23, ATCC 6939 and No. 33. More extracellular enzyme was produced by strain No. 23 than by the other two strains. Halo formation on the agar medium containing cholesterol depended on the conversion of cholesterol to 4-cholesten-3-one by the extracellular cholesterol oxidase.     

Efficient electrotransformation of Enterococcus hirae with a new Enterococcus-Escherichia coli shuttle vector

In the present study, an Enterococcus-Escherichia coli shuttle vector, pC3, was constructed that allows efficient transformation by electroporation of Enterococcus hirae ATCC9790. 5 x 10(6) transformants per microgram of plasmid DNA were obtained, using a commercial capacitor discharge device with an improved circuitry and a home-made electrode assembly, delivering pulses of 24 kV/cm across the cell suspension. The transformants were stable without selective pressure and plasmid DNA reisolated from transformed cells displayed no alterations in restriction enzyme analysis. Chromosomal DNA from E coli or E hirae, carried by pC3, was stably maintained in E hirae, making cloning and genetic manipulation in this organism feasible.     

Construction and characterization of a phage-plasmid hybrid (phagemid), pCAK1, containing the replicative form of viruslike particle CAK1 isolated from Clostridium acetobutylicum NCIB 6444.

A bacteriophage-plasmid hybrid (phagemid) designated pCAK1 was constructed by ligating 5-kbp Escherichia coli plasmid pAK102 (AprEmr) and the 6.6-kbp HaeIII-linearized replicative form of the CAK1 viruslike particle from Clostridium acetobutylicum NCIB 6444. Phagemid pCAK1 (11.6 kbp) replicated via the ColE1 replication origin derived from pAK102 in E. coli. Single-stranded DNA (ssDNA) molecules complexed with protein in a manner which protected ssDNA from nucleases were recovered from the supernatant of E. coli DH11S transformants containing pCAK1 in the absence of cell lysis. This suggests that the viral-strand DNA synthesis replication origin of CAK1 and associated gene expression are functional in E. coli DH11S. The single-stranded form of pCAK1 isolated from E. coli supernatant was transformed into E. coli DH5 alpha' or DH11S by electroporation. Isolation of ampicillin-resistant E. coli transformants following transformation suggests that the complementary-strand DNA synthesis replication origin of CAK1 is also functional in E. coli. The coat proteins associated with ssDNA of pCAK1 demonstrated sensitivity to proteinase K and various solvents (i.e., phenol and chloroform), similar to the results obtained previously with CAK1. Following phagemid construction in E. coli, pCAK1 was transformed into C. acetobutylicum ATCC 824 and C. perfringens 13 by intact cell electroporation. Restriction enzyme analysis of pCAK1 isolated from erythromycin-resistant transformants of both C. acetobutylicum and C. perfringens suggested that it was identical to that present in E. coli transformants.     

Characterization of production of cholesterol oxidases in three Rhodococcus strains

The production of cholesterol oxidase in two strains of Rhodococcus equi No. 23 from butter, and Rhodococcus sp. No. 33 from bacon, which had properties on biochemical and physiological tests almost similar to the strains of R. equi, was compared with that of the type strain (ATCC 6939) of R. equi. The intensity of cholesterol oxidase activity, both extracellular and membrane-bound, from the three strains was in the order No. 23, ATCC 6939 and No. 33. More extracellular enzyme was produced by strain No. 23 than by the other two strains. Halo formation on the agar medium containing cholesterol depended on the conversion of cholesterol to 4-cholesten-3-one by the extracellular cholesterol oxidase.     

Cloning and characterization of a Streptomyces single module type non-ribosomal peptide synthetase catalyzing a blue pigment synthesis

In the present study, we cloned a gene, designated bpsA, which encodes a single module type non-ribosomal peptide synthetase (NRPS) from a D-cycloserine (DCS)-producing Streptomyces lavendulae ATCC11924. A putative oxidation domain is significantly integrated into the adenylation domain of the NRPS, and the condensation domain is absent from the module. When S. lividans was transformed with a plasmid carrying bpsA, the transformed cells produced a blue pigment, suggesting that bpsA is responsible for the blue pigment synthesis. However, to produce the blue pigment in Escherichia coli, the existence of the 4'-phosphopantetheinyl transferase (PPTase) gene from Streptomyces was necessary, in addition to bpsA. The chemical structure of the pigment was determined as 5,5'-diamino-4,4'-dihydroxy-3,3'-diazadiphenoquinone-(2,2'), called indigoidine. The bpsA gene product, designated BPSA, was overproduced in an E. coli host-vector system and purified to homogeneity, demonstrating that the recombinant enzyme prefers L-Gln as a substrate. The in vitro experiment using L-Gln also showed that the blue pigment was formed by the purified BPSA only when the enzyme was phosphopantetheinylated by adding a Streptomyces PPTase purified from E. coli cells. Each site-directed mutagenesis experiment of Lys(598), Tyr(601), Ser(603), and Tyr(608), which are seen in the oxidation domain of BPSA, suggests that these residues are essential for the binding of FMN to the protein and the synthesis of the blue pigment.     

Molecular cloning and expression of a xylanase gene of alkalophilic Aeromonas sp. no. 212 in Escherichia coli

A gene coding for a xylanase activity of alkalophilic Aeromonas sp. no. 212 (ATCC 31085) was cloned in Escherichia coli HB101 with pBR322. Plasmid pAX1 was isolated from transformants producing xylanase, and the xylanase gene was located in a 6.0 kb Hind III fragment. The pAX1-encoded xylanase activity in E. coli HB101 was about 80 times higher than that of xylanase L in alkalophilic Aeromonas sp. no. 212. About 40% of the enzyme activity was observed in the periplasmic space of E. coli HB101. The pAX1-encoded xylanase had the same enzymic properties as those of xylanase L produced by alkalophilic Aeromonas sp. no. 212, but its molecular weight was lower (135 000 vs 145 000, as estimated by SDS polyacrylamide gel electrophoresis).     

嗜碱细菌NTT36嗜碱基因的亚克隆及其在大肠杆菌和农杆菌中的表达

将大肠杆菌ＨＢ１０１嗜碱转化子中质粒ｐＧＣＡ所携带的嗜碱基因亚克隆至双元载体ｐＢＩ１２１质粒中,构建了植物表达载体ｐＬＧＣ重组质粒。用其转化大肠杆菌ＨＢ１０１获得了能在碱性和卡那霉素抗性平板上生长的转化子,再通过三亲交配法将亚克隆质粒ｐＬＧＣ转化进农杆菌ＬＢＡ４４０４,又获得能在碱性平板和卡那霉素及利福平双抗平板上生长的转化子,Ｓｏｕｔｈｅｒｎ杂交结果表明ＨＢ１０１转化子亚克隆质粒ｐＬＧＣ是由来自于嗜碱芽孢杆菌ＮＴＴ３６染色体ＤＮＡ和双元载体ｐＢＩ１２１组成,且农杆菌ＬＢＡ４４０４转化子含有来自大肠杆菌亚克隆转化子的ｐＬＧＣ质粒。     

Carotenoid pigments and the enhanced resistance of Pseudomonas methanolica to the action of ultraviolet radiation

The correlation was studied between the elevated resistance of Pseudomonas methanolica, a facultative methylotroph, to the bactericidal action of UV (254 nm) and the content of a pink pigment in this organism. Absorption spectra of cell extracts containing the pigment were recorded. The ethanol and acetone extracts had two absorption maxima at 420-430 and 500-510 nm, respectively, which was typical of carotenoids. The culture with the pigment and its variant without the pigment had nearly identical survival rates. Therefore, the elevated resistance of P. methanolica cells seems to stem from the activity of DNA reparation cellular systems rather than from the presence of carotenoid pigments in the cells.     

Cloning and expression in Escherichia coli of a recA-like gene from the acidophilic autotroph Thiobacillus ferrooxidans

A recombinant plasmid, pRSR100, containing the functional analogue of the Escherichia coli recA gene was isolated from a genomic library of Thiobacillus ferrooxidans ATCC 33020. The plasmid complemented defects in DNA repair and homologous recombination in E. coli recA mutant strains. Antiserum raised against E. coli RecA protein reacted with the native but defective E. coli HB101 RecA protein; it did not react with protein extracts from the recA deletion mutant E. coli JK696, but it reacted with two protein bands in extracts of E. coli JK696(pRSR100). A single band with an apparent Mr equal to the higher-Mr band in E. coli JK696(pRSR100) was detected in T. ferrooxidans cell extracts with the E. coli RecA antiserum.     

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.