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Thermus spp. are gram-negative thermophilic microorganisms that grow at temperatures between 50 and 82°C (1, 11). Plasmid vectors that have been used in Thermus spp. have been constructed that encode tryptophan, leucine, or pyrimidine synthesis genes to complement auxotrophic and deleted hosts, providing for positive selection of transformants (4, 12). The host-vector system described here is an improvement on previously reported plasmid vectors for Thermus spp. because not only is the Escherichia coli-Thermus shuttle vector easily selected and maintained in Thermus spp., but malate dehydrogenase activity, encoded by the mdh gene present in this vector can also be readily and accurately quantified (2).

Construction of Thermus thermophilus HB27 and Thermus flavus hosts with deletions of the mdh gene. To create the mdh strain of T. thermophilus HB27, we constructed an integration vector designated pUC-S KmA. The construction is detailed in Fig. 1. A 2.3-kb PCR fragment containing a region spanning three separate genes, succinate coenzyme A ligase (scsA), malate dehydrogenase (mdh), and purine phosphoribosyltransferase (gpt), was amplified from the T. flavus chromosome and cloned into pUC18. A 10-ul PCR amplification reaction was conducted in an Idaho Technologies Rapid Air Thermo-Cycler in the presence of 8% glycerol and 1% dimethyl sulfoxide. The PCR amplification cycle was run for 40 cycles at 94°C, 55°C, and 1 min of holding at 72°C. The 984-bp mdh gene is located near the center of this 2.3-kb fragment, so that the chromosomal regions that flank mdh are 780 bp (5') and 560 bp (3'). The entire coding sequence of the mdh gene was removed by restriction enzymes and replaced with a thermotolerant kanamycin resistance cassette (Km^r) (7). This vector, designated pUC-SKmA, can replicate in E. coli but not in Thermus. However, homologous recombination between the scsA and gpt gene sequences allows pUC-SKmA to integrate into the chromosome when it was used to transform T. thermophilus HB27 (5). Transformants were screened at 55°C on TT rich medium (6) supplemented with kanamycin (40 µg/ml). Approximately 10⁴ kanamycin-resistant transformants per µg of DNA were observed. Two distinct colony types arose after 5 days of incubation. The majority of the colonies were very small (0.1 to 0.5 mm) even after 5 days. A few colonies (60 to 100 CFU) were much larger (1.5 to 2.8 mm), the same size as wild-type T. thermophilus HB27 colonies. The two colony types were subcultured, and total DNA (plasmid and chromosomal) was dot blotted onto a nylon membrane.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Construction of T. thermophilus mdh Kmr MM8-5. (A) The region surrounding the mdh gene from T. flavus was amplified from chromosomal DNA using PCR primers forward. (5'-ACAACAAAGCTTCGGGCAAAGGGGGAACGGAGGTCCT-3') and reverse (5'-ACAACAAAGCTTGAGCCTTTTGACCTCGTCCTGGGG-3'). These primers were designed to amplify the sequence of the malate dehydrogenase gene and the regions immediately flanking the malate dehydrogenase gene from T. flavus according to published DNA sequences (8-10). Restriction sites were added to the PCR primers to give 5' and 3' HindIII sites. The 2.3-kb PCR product containing the mdh gene and flanking regions was cloned into the HindIII site of pUC18. The resulting plasmid is 4,982 bp and was designated pUCS-M-A. (B) Plasmid pUCS-M-A was digested with BsiWI and NgoMI, removing the all but the first 20 bp of the mdh gene sequence. A kanamycin resistance cassette was amplified using plasmid pTEXJ17 as the template and using PCR primers forward (5'-ACAACACGTACGGATTACGCCAAGCTTCATGGCCTAA-3') and reverse (5'-ACAACAGCCGGCTCGTTCAAAATGGTATGCGTTTTG-3'). Restriction sites were added to the PCR primers to give a 5' BsiWI and 3' NgoMI site. The cassette contains a strong constitutive Thermus promoter (J17) upstream of the themostable kanamycin nucleotidyltransferase cassette (Kmr). To prevent transcription readthrough from the native mdh promoter, a transcription termination sequence was cloned upstream of the J17 promoter. The kanamycin resistance cassette was digested with BsiWI and NgoMI and ligated into BsiWI-NgoMI-digested pUCS-M-A. The mdh gene was replaced with a kanamycin resistance cassette, and the resulting plasmid was designated pUC-SKmA. (C) After transformation of plasmid pUC-SKmA into T. thermophilus HB27, a double-crossover homologous recombination event replaces mdh with the Kmr determinant. The mdh Kmr T. thermophilus HB27 strain subsequently isolated was designated MM8-5. RBS, ribosome-binding site; PRM, primer.

T. thermophilus mdh

Construction of Thermus vectors containing mdh as a reporter gene. The malate dehydrogenase (mdh) gene from T. flavus was amplified by PCR and cloned into Thermus-E. coli expression vector pTEXI. The expression vector pTEXI is capable of replication in both Thermus spp. and E. coli, and the promoter (J17) employed in this expression vector functions in both bacterial hosts. J17 is a constitutive promoter isolated in our lab from T. thermophilus chromosomal DNA. The expression vector containing the mdh gene, designated pTEXI-mdh, is diagramed in Fig. 2A.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   (A) Expression vector pTEX1-mdh contains the T. flavus malate dehydrogenase gene downstream of the strong constitutive Thermus promoter J17. The mdh gene from T. flavus was amplified from chromosomal DNA using PCR primers forward mdh (5'-ACACAGAATTCGCATGCTCAAGAAGGCCCTGGGCTAA-3') and reverse mdh (5'-ACACACGGATCCTGCGCCAGCATGGGGTGGTATAAA-3'). Restriction sites were added to the PCR primers to give 5' EcoRI and 3' BamHI sites. Plasmid pTEX1 was digested with EcoRI and BamHI, and an EcoRI-BamHI-digested mdh PCR product was ligated into the vector to create pTEX1-mdh. (B) Integrative vector pSJ17mdhA has the J17 promoter-mdh gene cassette flanked by the scsA and gpt chromosomal regions.

Malate dehydrogenase activity of Thermus constructs. The levels of malate dehydrogenase (2) being produced by plasmid and integrative expression vectors were evaluated in both T. thermophilus HB27 and MM8-5. Crude lysates prepared from each culture were assayed for enzyme activity at two temperatures (25 and 50°C), and the results are shown in Table 1. T. thermophilus MM8-5 had slight to no malate dehydrogenase activity, confirming complete deletion of the mdh gene from the chromosome of this strain. The activity observed in assays performed at 50°C reflect a slight amount of background due to the conversion of NADH to NAD by unidentified components of cell lysates rather than the malate dehydrogenase-dependent conversion of oxaloacetate and NADH to malate and NAD.

mdh

mdh