Cloning and sequencing of the secY homolog from Coryneform bacteria.

A conserved domain of the secY genes from Bacillus subtilis, Mycoplasma capricolum and Escherichia coli was used to design degenerate oligodeoxyribonucleotides. These synthetic DNA sequences were used to screen a lambda library of Brevibacterium flavum MJ233. A 1.5-kb KpnI fragment of a recombinant lambda phage containing the secY homology from Br. flavum MJ233 was subsequently subcloned into plasmid pUC118. The complete nucleotide (nt) sequence of the cloned fragment indicated that the deduced gene product of the Br. flavum secY homolog is composed of 440 amino acids (aa) with a deduced M(r) of 47,871. Comparison of this aa sequence to the corresponding sequences from E. coli and B. subtilis revealed a high degree of conservation, and suggested that the Br. flavum secY homolog is a membrane protein containing ten transmembrane segments. In addition, we could identify, downstream from secY, a putative coding sequence of the enzyme adenylate kinase. This gene organization is identical to that observed in the B. subtilis genome.

Presence of mrr- and mcr-like restriction systems in coryneform bacteria.

Efficient transformation of Brevibacterium flavum MJ233C and Corynebacterium glutamicum ATCC 31831 (up to 5.0 x 10(7) transformants/microgram DNA) depends on the source of plasmid DNA. The transformation efficiencies of B. flavum MJ233C and C. glutamicum ATCC 31831 increased nearly 10(3)-fold when plasmid DNA was isolated from the recipient strain itself or from a damdcm Escherichia coli mutant, as compared with DNA passed through a modification-proficient E. coli strain. These results suggest the presence of a methyl-specific restriction system in certain strains of coryneform bacteria. In addition, electroporation conditions were optimized.

Detection of circulating tumor cells in patients with non-small cell lung cancer undergoing lobectomy by video-assisted thoracic surgery: a potential hazard for intraoperative hematogenous tumor cell dissemination.

OBJECTIVE: We prospectively tested whether circulating tumor cells can be found in the preoperative, intraoperative, and postoperative peripheral blood of patients with resectable non-small cell lung cancer who undergo video-assisted lobectomy. METHODS: We assayed for carcinoembryonic antigen messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction in the peripheral blood taken before, during, just after the completion of the lobectomy and then 2 to 3 weeks, and again 5 to 6 weeks, after the operation in 29 patients with pathologic stage I non-small cell lung cancer who underwent video-assisted lobectomy. We also analyzed the prognostic value of carcinoembryonic antigen mRNA expression pattern in an additional 57 patients with stage I non-small cell lung cancer, whose blood samples were previously assayed for carcinoembryonic antigen mRNA. RESULTS: Of the 29 patients, the preoperative blood samples from 18 patients were negative for carcinoembryonic antigen mRNA. Of these 18 patients, 16 (89%) had positive test results during operation, although the remaining 2 patients (11%) consistently showed negative test results. The occurrence of this change from negative to positive tests results for carcinoembryonic antigen mRNA during video-assisted lobectomy was significantly higher than in patients who underwent open lobectomy in a previous study (18 of 35 patients; 51%; P <.001). In the 57 patients with stage I cancer whose blood samples were previously assayed for carcinoembryonic antigen mRNA, patients with persistently positive test results for carcinoembryonic antigen mRNA before and during operation had a significantly shorter survival when compared with those patients whose test results were persistently positive. CONCLUSIONS: Video-assisted lobectomy, as compared with open lobectomy, for non-small cell lung cancer may increase the risk of seeding tumor cells into the circulation during operation.

Tumor neutrophil elastase is closely associated with the direct extension of non-small cell lung cancer into the aorta.

OBJECTIVE: Neutrophil elastase (NE) is the only neutral protease that is able to degrade insoluble elastin and other extracellular matrix constituents, and thus, may be involved in tumor invasion and metastasis. Using a highly specific and sensitive enzyme immunoassay (EIA), we recently demonstrated that immunoreactive (ir)-NE is produced by non-small cell lung cancer cell lines. We have measured the ir-NE concentration in non-small cell lung cancer tumor extracts and have evaluated its association with disease stage. METHODS: We measured the ir-NE concentration in 144 non-small cell lung cancer tumor extracts using EIA, which permits the rapid measurement of both the free and alpha1-protease inhibitor (alpha1-PI) complexed form of ir-NE. In 15 clinical T4 (cT4) patients, we also determined the concentration of free ir-NE in tumor extracts using a kit that detects only NE complexed with alpha1-PI and subtracting that value from the total NE concentration. RESULTS: ir-NE was detected in tumor extracts from 115 of 144 patients, ranging from 0.21 to 23.35 microg/100 mg protein. When the 144 specimens were grouped according to the clinical stage of disease, the ir-NE concentration (mean+/-SE) was significantly higher in those with cT4 disease (n=15; 7.90+/-1.88 microg/100 mg protein) than in those with cT1 (n=29; 1.27+/-0.27; p<0.001), cT2 (n=64; 1.18+/-0.17; p<0.001), or cT3 disease (n=26; 1.99+/-0.38; p<0.003). There was no significant association between the ir-NE concentration and cN-factor or any other clinical features. When the ir-NE concentration in the tumor extracts of the cT4 patients was compared with respect to the tumor invasion sites, the ir-NE level was significantly higher in those with surgical T4 (sT4) disease with aortic invasion (n=4; 17.4+/-3.10) than in those who were down-staged postoperatively (n=5; 4.9+/-1.33; p=0.005) or those with sT4 disease with involvement of other sites (n=6; 4.07+/-1.83; p=0.004). Similar results were observed for the free form of ir-NE. CONCLUSIONS: These data suggest that NE may be involved in tumor progression of non-small cell lung cancer. Since the aorta is one of the richest sources of polymeric and insoluble elastin, this enzyme may play an active role in the direct extension of the tumor into the aorta.

The sequence of vessel ligation affects tumor release into the circulation.

OBJECTIVE: Whether the sequence of pulmonary vein and artery ligation in pulmonary lobectomy for carcinoma affects intraoperative hematogenous cancer cell dissemination is not known. We examined whether vessel ligation sequence affects the presence of circulating cancer cells as reflected by carcinoembryonic antigen messenger ribonucleic acid. METHODS: We assayed for the transcripts of carcinoembryonic antigen messenger ribonucleic acid by reverse-transcriptase polymerase chain reaction in peripheral blood taken before, during, and after operation from 30 patients with non-small-cell lung cancer who underwent a curative lobectomy and from six patients with limited-stage small-cell lung cancer who were treated initially with chemotherapy followed by lobectomy. Each patient was randomly assigned before the operation to have either pulmonary vein ligation or pulmonary artery ligation first. Blood taken from 10 patients with interstitial pulmonary fibrosis who underwent an open lung biopsy and 41 healthy subjects served as a control. RESULTS: No control samples were positive for transcripts. Sixteen of the preoperative blood samples from the 30 patients with non-small-cell cancers were positive. Of these 16, eight samples remained positive even after lobectomy was performed; the remaining eight samples (four in each ligation group) became negative. Of the 14 initially negative samples (seven in each ligation group), nine samples became positive during the operation. Such conversion during the operation was more common with arterial ligation first (six patients, 85.7%) than with venous ligation first (three patients, 42.9%). Samples from all six patients with small-cell cancer were positive before the operation, and five of six samples remained positive after the operation. CONCLUSIONS: Many patients with non-small-cell lung cancer have systemic disease even when they were thought to have resectable tumors. Ligating the pulmonary vein before ligating the artery may lessen intraoperative hematogenous dissemination. Most small-cell lung cancers represent systemic disease even when considered resectable.

Detection of circulating tumor cells by reverse transcriptase-polymerase chain reaction in patients with resectable non-small-cell lung cancer.

OBJECTIVE: We tested whether circulating tumor cells can be detected in the peripheral blood of patients with resectable non-small-cell lung cancer (NSCLC) by reverse transcriptase-polymerase chain reaction (RT-PCR) of carcinoembryonic antigen (CEA) mRNA. METHODS: We assayed for CEA mRNA by RT-PCR in the peripheral blood, taken at the time of diagnosis before surgical intervention and again 2 to 3 weeks postoperatively, from 103 patients with NSCLC who underwent curative lobectomy. Blood samples taken from 15 patients with interstitial pulmonary fibrosis who underwent an open-lung biopsy and from 32 healthy subjects served as controls. RESULTS: No control samples were positive for CEA by RT-PCR. Sixty-two (60%) of the preoperative blood samples from the 103 patients with NSCLC were positive. Of these 62 samples, 27 (44%) remained positive even after surgical intervention, whereas the remaining 35 samples (56%) became negative. The incidence of positive CEA mRNA correlated highly with pathologic TNM stage of disease in both the preoperative and postoperative blood samples. CONCLUSIONS: Many patients with resectable NSCLC have detectable levels of circulating cells expressing carcinoembryonic antigen even after surgical intervention. Such patients may have a higher rate of relapse.

No-touch isolation technique reduces intraoperative shedding of tumor cells into the portal vein during resection of colorectal cancer.

BACKGROUND: The mutant-allele-specific amplification (MASA) method is capable of detecting 1 genetically altered tumor cell among thousands of normal cells. The MASA enabled us to detect occult tumor cells undetectable by histopathologic examination of lymph nodes and blood samples. METHODS: To investigate whether tumor manipulation during operation enhances cancer cell dissemination into the portal vein with use of MASA and to assess the effect of the no-touch isolation technique in the treatment of colorectal cancers, 27 colorectal cancers (17 were operated on conventionally and 10 were operated on according to the no-touch isolation technique) were screened for mutations in K-ras or p53. We next examined blood samples of the portal vein collected before, during, and after manipulation of tumors, using MASA to look for the specific mutation found in the primary tumors. RESULTS: Somatic mutations were identified in 18 of these primary tumors (11 were in the conventional resection technique group and 7 were in the no-touch isolation technique group). In 8 of 11 (73%) conventional resection technique cases, we identified the same genetic alteration of the primary tumor in the portal blood during operation, whereas only 1 patient (14%) in the no-touch isolation technique group had a positive result. CONCLUSIONS: The no-touch isolation technique may be useful to prevent cancer cells from being shed into the portal vein during surgical manipulation.

Cloning and characterization of mdc genes encoding malonate decarboxylase from Pseudomonas putida.

The DNA fragment encoding malonate decarboxylase, involved in malonate assimilation, was cloned from Pseudomonas putida. The 11-kb DNA fragment contained nine open reading frames, which were designated mdcABCDEGHLM in the given order. N-terminal protein sequencing established that the mdcA, mdcC, mdcD, mdcE and mdcH genes encoded subunits alpha, delta, beta, gamma and epsilon of the malonate decarboxylase, respectively. Malonate decarboxylase was functionally expressed in Escherichia coli from plasmid harboring the entire gene cluster or the mdc genes lacking the mdcL and mdcM genes. The mdcL and mdcM genes encode membrane proteins and disruption of the genes of P. putida by the insertion of a kanamycin resistance cassette reduced the malonate uptake activity of the organism. Thus, we conclude that MdcLM is a malonate transporter.

Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B. flavum and Escherichia coli.

A promoter probe shuttle vector suitable for the isolation of promoter elements from coryneform bacteria was constructed. This vector carried the neomycin phosphotransferase (NPTII) gene from transposon Tn5 as a reporter gene, and was capable of replication in both Escherichia coli and Brevibacterium flavum. The vector was used in the construction of a B. flavum library of 899 independently isolated promoter clones. Promoters with a wide range of activities in B. flavum, including some very strong promoter elements, were isolated. Comparative analysis suggests that significant differences between B. flavum and E. coli may exist in the determinants of promoter strength.

Regio- and diastereocontrol in carbonyl allylation by 1-halobut-2-enes with Tin(II) halides

Regio- and diastereoselective carbonyl allylations of 1-halobut-2-enes with tin(II) halides are described. Tin(II) bromide in a dichloromethane-water biphasic system is an effective reagent for unusual alpha-regioselective carbonyl allylation of 1-bromobut-2-ene to produce 1-substituted pent-3-en-1-ols. The addition of tetrabutylammonium bromide (TBABr) to the biphasic system produces 1-substituted 2-methylbut-3-en-1-ols via usual gamma-addition which is opposite to the alpha-addition without TBABr. The gamma-addition to aromatic aldehydes exhibits anti-diastereoselectivity, while that to aliphatic aldehydes is not diastereoselective. The allylation of benzaldehyde by 1-chlorobut-2-ene in 1,3-dimethylimidazolidin-2-one (DMI) does not occur with tin(II) chloride or bromide but does proceed with tin(II) iodide and exhibits gamma-syn selectivity which is unusual for a Barbier-type carbonyl allylation. In the carbonyl allylation by 1-chlorobut-2-ene with any tin(II) halide, the addition of tetrabutylammonium iodide (TBAI) accelerates the reaction and enhances gamma-syn selectivity. The use of tin(II) iodide and TBAI produces 2-methyl-1-phenylbut-3-en-1-ol with high yield and high syn-diastereoselectivity. The syn-diastereoselective carbonyl allylation of 1-chlorobut-2-ene using tin(II) iodide, a catalytic amount of TBAI, and NaI in DMI-H(2)O is applied to various aldehydes.

Genetic transformation system for a psychrotrophic deep-sea bacterium: isolation and characterization of a psychrotrophic plasmid.

A versatile system that permits genetic manipulation of a psychrotrophic deep-sea bacterium, Pseudoalteromonas sp. PS1M3, has been developed. A cryptic indigenous plasmid, pPS1M3, of 3.1 kb from the above strain was isolated and characterized. The nucleotide sequence analysis of plasmid pPS1M3 revealed the presence of one open reading frame, and its deduced amino acid sequence was identified as the essential protein for plasmid maintenance. Transformation with the pPS1M3 harboring antibiotic resistance genes by electroporation was fully successful using the pPS1M3-cured strain as a host. This plasmid was quite stable under nonselective culture conditions for about 100 generations at 4 degrees C. The copy number of this plasmid in the cell was about 5 copies per chromosome.

Cloning and sequence determination of the acetohydroxy acid synthase genes from Brevibacterium flavum MJ233 by using the polymerase chain reaction.

Taking advantage of highly conserved domains present in the three acetohydroxy acid synthase (AHAS) isozymes from E. coli K-12, we designed degenerate oligonucleotides corresponding to these regions. These synthetic DNA sequences were used as primers in polymerase chain reactions in order to amplify DNA sequences from Brevibacterium flavum MJ233 chromosomal DNA. The polymerase chain reaction product was used as a probe to recover genomic fragments from a lambda library of B. flavum MJ233. A 5.8-kb EcoRI fragment hybridizing to the probe was isolated and amplification of this fragment in a B. flavum strain resulted in increased AHAS-specific activity. Sequence analysis revealed two open reading frames (ilvL and ilvS) highly homologous at the amino acid level to the corresponding domains of the three AHAS isozymes of E. coli K-12. Moreover, disruption of the putative ilvL gene by a kanamycin resistance cassette resulted in L-isoleucine and L-valine auxotrophy. These observations demonstrate that the cloned fragment encodes the AHAS gene of B. flavum MJ233.

Identification and sequence determination of the acetohydroxy acid isomeroreductase gene from Brevibacterium flavum MJ233.

The enzyme acetohydroxy acid isomeroreductase (AHAIR) is the second enzyme in the parallel isoleucine-valine biosynthetic pathway. We previously reported the cloning and sequencing of the acetohydroxy acid synthase (AHAS) genes from Brevibacterium flavum MJ233. Analysis of the sequence downstream of the AHAS genes identified another open reading frame highly homologous at the amino acid level to the AHAIR gene from Escherichia coli (ilvC). We subcloned the B. flavum AHAIR gene on a 2.1 kb Bg/II-EcoRI fragment by complementation of an E. coli ilvC mutant. The nucleotide sequence of the B. flavum AHAIR gene consists of 338 codons (molecular weight of 36158). Comparison of the deduced protein sequence revealed a high degree of identity with the sequences of ilvC genes from other organisms. Disruption of the B. flavum ilvC gene by a kanamycin resistance cassette resulted in L-isoleucine and L-valine auxotrophy.

Analysis of the biotin biosynthesis pathway in coryneform bacteria: cloning and sequencing of the bioB gene from Brevibacterium flavum.

The biotin biosynthetic pathway of three coryneform bacteria, Brevibacterium flavum, Brevibacterium lactofermentum, and Corynebacterium glutamicum were analysed by cross-feeding experiments using several Escherichia coli biotin-requiring mutants. The three strains of coryneform bacteria tested were able to convert 7-keto-8-aminopelargonic acid to biotin, through a biotin synthetic pathway identical to that from E. coli. The biotin biosynthetic gene, bioB, of B. flavum was cloned by phenotypic complementation of E. coli bioB mutants. The bioB gene was located on a 1.7 kb HindIII-SacI DNA fragment. Nucleotide sequence analysis of this fragment revealed that the bioB gene of B. flavum consists of a 1005 bp open reading frame. Its deduced amino acid sequence is 35.7% and 31.5% identical to that of the E. coli and Bacillus sphaericus bioB gene products, respectively. B. flavum mutants obtained by in vivo disruption of the bioB gene lost their ability to grow on minimal medium containing dethiobiotin, indicating that the bioB gene product is necessary for the conversion of dethiobiotin to biotin.

Genomic organization of the biotin biosynthetic genes of coryneform bacteria: cloning and sequencing of the bioA-bioD genes from Brevibacterium flavum.

Three coryneform bacteria, Brevibacterium flavum, Brevibacterium lactofermentum and Corynebacterium glutamicum have been shown to be able to convert 7-keto-8-aminopelargonic acid to biotin through a biotin synthetic pathway identical to that from Escherichia coli (Hatakeyama et al., DNA Sequence, in press, 1993). We report in this paper the cloning and sequencing of the biotin biosynthetic genes encoding the 7,8-diaminopelargonic acid aminotransferase (bioA) and the dethiobiotin synthetase (bioD) of B. flavum MJ233, by complementation of E. coli bioA and bioD mutants. Both bioA and bioD genes from B. flavum were located on a 4.0-kb SalI DNA fragment. Nucleotide sequence analysis of this fragment revealed that these genes consist of a 1272 bp and a 675 bp open reading frame, respectively. The deduced amino acid sequence of the 7,8-diaminopelargonic acid aminotransferase (BioA) is 51.3% and 31.9% identical to that of the E. coli and Bacillus spaericus bioA gene products, respectively. The deduced amino acid sequence of the dethiobiotin synthetase (BioD) is 25.9% and 32.7% identical to that of the E. coli and B. sphaericus bioD gene products, respectively. In addition, the genomic organization of the bioA, bioB and bioD genes in B. flavum has been shown to be different from that in E. coli and B. sphaericus.

Analysis of histidine-dependent antitermination in Bacillus subtilis hut operon.

We have previously shown that a positive regulator, HutP, of Bacillus subtilis hut operon is a RNA binding protein. Here, we report precise binding site of HutP in cis-regulatory region on hut mRNA and the role of HutP in histidine-dependent antitermination of hut expression. Ethylnitrosourea modification interference assay showed that four binding sites of HutP were found in the cis-regulatory sequences and were located at the stem and the internal loop of an antiterminator structure. In vitro transcription assay indicated that HutP suppressed transcription termination in the presence of histidine. These results suggest that HutP function as an antiterminator in response to the presence of histidine.

Genetic analysis of an incomplete mutS gene from Pseudomonas putida.

We genetically characterized the Pseudomonas putida mutS gene and found that it encodes a smaller MutS protein than do the genes of other bacteria. This gene is able to function in the mutS mutants of Escherichia coli and Bacillus subtilis. A P. putida mutS mutant has a mutation frequency 1,000-fold greater than that of the wild-type strain.

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene in Escherichia coli.

The application of an inducible regulation system using the tryptophanase operon promoter (TPase promoter; Ptna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of Ptna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, L-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-Ptna TS was increased 500-fold compared to that of the cultured host strain. About 1 mol L-tryptophan/l reaction mixture was formed from indole and L-serine at 37 degrees C for 3.5 h.

Electrotransformation of intact cells of Brevibacterium flavum MJ-233.

Electroporation allowed transformation of intact cells of Brevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobase) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin in B. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was improved by the addition of 1.0 U/ml of penicillin G to the culture medium. The optimum yield of transformants per micrograms DNA was 5 x 10(4) when the cell suspension was pulsed at a cell density of 1 x 10(10)/ml and at a DNA amount of 1.0 micrograms.