The genes for benzene catabolism in Pseudomonas putida ML2 are flanked by two copies of the insertion element IS1489, forming a class-I-type catabolic transposon, Tn5542.

Two directly repeated sequences of the IS elements IS1489v1 and IS1489v2 flank the benzene dioxygenase (bedC1C2BA) and the cis-benzene dihydrodiol dehydrogenase (bedD) genes on the catabolic plasmid pHMT112 in Pseudomonas putida ML2, forming a Class-I-type composite transposon, Tn5542. Both IS1489v1 and IS1489v2 contain an identical 1371-bp open reading frame, tnpA, that is preceded by a possible ribosome binding site. The tnpA gene of IS1489v1 is bound by a pair of 40-bp imperfect inverted repeats while that of IS1489v2 is flanked only by the left inverted repeat. The tnpA gene codes for a putative 53-kDa polypeptide of 456 amino acids bearing similarity to transposases encoded on IS elements of P. alcaligenes, P. aeruginosa, P. stutzeri, and Serratia marcescens. The basic nature of the putative TnpA protein with a deduced pI of 8.93 is typical of IS-encoded transposases. Similar to other IS elements, an outward facing promoter was detected at the right end of IS1489v1. Experiments involving the suicide vector, pKNG101, failed to show transposition of Tn5542.

Concurrent chemoradiotherapy in patients with locally advanced nasopharyngeal carcinoma of the undifferentiated type.

The result with radiotherapy alone in patients with locally advanced nasopharyngeal carcinoma (NPC) was disappointing. Encouraging results have been reported with the use of concurrent chemoradiotherapy in patients with locally advanced squamous cell carcinoma of the head and neck. Hence, we decided to explore the use of this treatment schedule in patients who presented with locally advanced disease (UICC/AJCC classification system). Between July 1995 and March 1996, 14 patients with locally advanced NPC were treated with the following schedule: radiation therapy was given conventionally to a total of 66 to 70 Gy to both the nasopharynx and neck with or without parapharyngeal/intracavitary boost; chemotherapy consisted of intravenous cisplatin at 20 mg/m2/day and intravenous 5-flurouracil 1000 mg/m2/day, infused over 8 hours on days 1 to 4 during the first and fifth week of radiation therapy. Depending on the patient's tolerability and clinical assessment of toxicity, a third cycle of chemotherapy was planned 4 to 5 weeks after the second cycle, upon the completion of the radiotherapy. Twelve patients completed all intended treatment. Two patients failed to do so due to treatment-related mortality. The median follow-up duration was 30 months. Limiting toxicities were myelosuppression and oropharyngeal mucositis. The overall response rate was a 100% at both the primary and nodal sites of disease. The median disease-free survival was 21 months. Forty per cent of the patients were alive at 3 years. This treatment schedule was associated with an unacceptable treatment-related death rate. As a result, this protocol was terminated.

Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD+-dependent cis-benzene dihydrodiol dehydrogenase.

The catabolic plasmid pHMT112 in Pseudomonas putida ML2 contains the bed gene cluster encoding benzene dioxygenase (bedC1C2BA) and a NAD+-dependent dehydrogenase (bedD) required to convert benzene into catechol. Analysis of the nucleotide sequence upstream of the benzene dioxygenase gene cluster (bedC1C2BA) revealed a 1,098-bp open reading frame (bedD) flanked by two 42-bp direct repeats, each containing a 14-bp sequence identical to the inverted repeat of IS26. In vitro translation analysis showed bedD to code for a polypeptide of ca. 39 kDa. Both the nucleotide and the deduced amino acid sequences show significant identity to sequences of glycerol dehydrogenases from Escherichia coli, Citrobacter freundii, and Bacillus stearothermophilus. A bedD mutant of P. putida ML2 in which the gene was disrupted by a kanamycin resistance cassette was unable to utilize benzene for growth. The bedD gene product was found to complement the todD mutation in P. putida 39/D, the latter defective in the analogous cis-toluene dihydrodiol dehydrogenase. The dehydrogenase encoded by bedD) was overexpressed in Escherichia coli and purified. It was found to utilize NAD+ as an electron acceptor and exhibited higher substrate specificity for cis-benzene dihydrodiol and 1,2-propanediol compared with glycerol. Such a medium-chain dehydrogenase is the first to be reported for a Pseudomonas species, and its association with an aromatic ring-hydroxylating dioxygenase is unique among bacterial species capable of metabolizing aromatic hydrocarbons.