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Methanotrophic bacteria oxidize their growth substrate methane to methanol via the methane monooxygenase (MMO). Two types of MMO are known, the particulate MMO (pMMO) and the soluble MMO (sMMO) (3). The genes encoding the three subunits of the pMMO (pmoCAB) are found in multiple copies in methanotrophs (2, 9, 11). In Methylococcus capsulatus Bath, a type I -proteobacterial methanotroph (3), two complete copies of pmoCAB and a third copy of pmoC are present (11). Mutant analysis has shown that neither copy of pmoCAB is essential but that copy 2 is more important than copy 1 for growth and whole-cell methane oxidation (11). The role of the third copy of pmoC is unknown, but it may be essential (11).

In order to assess the relative expression of each set of pmo genes under different growth conditions, we have carried out a study of transcripts in pmoC1 and pmoC2 mutants and compared these results to expression from promoter-reporter gene fusions in strains containing wild-type pmoC1 and pmoC2.

Escherichia coli strains DH5, DH5 MCR (Bethesda Research Laboratories, Inc.), Inv and Top10 (Invitrogen), and S17-1 (10) were grown in Luria-Bertani medium in the presence of appropriate antibiotics as described previously (8). M. capsulatus Bath wild-type and mutant strains (MCK60 and MCK62) (11) were grown as described previously (11).

Transcript analysis. RNA blots were analyzed for insertion mutations of pmoC1 and pmoC2 with probes for pmoC, pmoA, and pmoB (Fig. 1). RNA was isolated from cells using the Perfect RNA total RNA isolation kit (Eppendorf-5 Prime, Inc., Boulder, Colo.). RNA blots were made, and hybridization was carried out at 55°C as described previously (8). The membranes were washed twice with 0.5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 55°C. Hybridization probes were generated from PCR products labeled with a random-primed labeling kit (Boehringer Mannheim, Indianapolis, Ind.). The PCR products were generated from the following primers: pmoC1 and pmoC2, css2F (5'-CCTGTGGGTGCGGTGGTAC-3') and css9R (5'-GCCTTCGTCCACGGCTTC-3'); pmoA1 and pmoA2, ass1F (5'-CTGGGACTTCTGGTCGGACTG-3') and mb661 (5'-CCGG[A,C]GCAACGTC[C,T]TTACC-3'); pmoB1 and pmoB2, bss1F (5'-CCGCCGTGGCAGCGACCGCC-3') and ESSR (5'-CCTTGAACGTCTAAATCCAGC-3'). These probes are specific to pmo genes in this strain (9, 11). A transcript pattern similar to that previously reported for the wild type was detected, including a full-length pmoCAB transcript and less-distinct smaller transcripts (Fig. 1). Since the ratios of the smaller mRNAs to the full-length pmoCAB transcript were roughly proportional, we focused on the pmoCAB transcript as an indicator of pMMO transcription. Figure 1 shows that each gene copy was transcriptionally active and that each was transcribed as a pmoCAB operon.

View larger version (56K): [in this window] [in a new window]   FIG. 1.   Northern blot analysis of pmo mRNAs from pmoC mutants. Probes are pmoA (A), pmoB (B), pmoC (C), and 16S rDNA(D). Total RNAs from MCK60 (pmoC1 mutant) (lanes 1 to 3) and MCK62 (pmoC2 mutant) (lanes 4 to 6) were grown on medium without copper added (lanes 1 and 4), with 5 µM copper (lanes 2 and 5), or with 50 µM copper (lanes 3 and 6). Each lane contains 10 µg of RNA. The arrowhead indicates the transcripts of pmoCAB.

Transcription start site mapping for pmoC1 and pmoC2. The locations of the transcription start sites of pmoC1 and pmoC2 were determined by primer extension experiments, using ThermoScript cDNA and 10 µg of total RNA. Primers were labeled with [-³²P]ATP (6,000 Ci/mmol) (NEN) using T4 polynucleotide kinase (Boehringer Mannheim). Radioactive sequencing reactions for primer extension analyses were carried out using the T7 Sequenase kit (Amersham Pharmacia Biotech, Piscataway, N.J.). Primers for the reverse transcription and for sequencing reactions were css21R (5'-CCTAAAGTGATGGTTGAC-3') for pmoC1 and css222R (5'-CCAACTGTTATATCGATGTG-3') for pmoC2. The mRNA start point of pmoC1 was detected 135 bp upstream of the translation start site, at a single A preceded by 10 (TAGACT) and 35 (TTGACA) boxes separated by 16 bp (Fig. 2). The mRNA start point of pmoC2 was detected 132 bp upstream of the translation start site, also at a single A preceded by 10 and 35 sequences identical to those for pmoC1 (Fig. 2). These sequences are located in both cases in a region of conserved sequences, flanked by sequences that are not conserved between the two copies. Both putative promoters demonstrate high similarity to the E. coli ⁷⁰ 10 and 35 promoter consensus (4). Immediately upstream of each promoter sequence is a highly conserved AT-rich region (CCTGCGTCAAAATCt/aCTCAg/tATTTTTC). This conserved sequence is a candidate for a regulatory sequence or an upstream promoter element (7). Transcription start sites were determined to be the same for both operons under conditions with and without copper added to the growth medium (data not shown). A promoter for one of the copies of pmoCAB of the type II, -proteobacterial methanotroph Methylocystis strain M has been mapped, and it also resembled an E. coli ⁷⁰ promoter (6). However, it was different from the sequences that we report here and did not contain the AT-rich region.

View larger version (35K): [in this window] [in a new window]   FIG. 2.   pmoC1 and pmoC2 transcription start sites. Primer extension analysis for pmoC1 (A) and pmoC2 (B) is shown. RNA was isolated from M. capsulatus cells grown for 2 h in batch culture without CuSO4 (fourth lanes) or with 20 µM CuSO4 added (fifth lanes).

Chromosomal reporter gene fusions for pmoC1 and pmoC2. Promoter-reporter (xylE) transcriptional fusions were generated in the chromosome for pmoC1 and pmoC2 using a new integrative vector, pMFX1. This vector contains the xylE gene from pHX200 (14) inserted into the KpnI sites of pAYC61 (1), with the Km^r gene from pUC4K replacing the Ap^r gene of pAYC61. This vector can be used to insert promoter-xylE transcriptional fusions into the chromosome at the site of the promoter fragment, as single-crossover insertions generating a fused gene followed by an intact gene with the native promoter. A 1,548-bp fragment containing the pmoC1 promoter region and a 443-bp fragment containing the pmoC2 promoter region were used to generate the chromosomal insertion strains, designated MCX13-2 and MCX215, respectively. Each construct contained a promoter fragment that had the same 3' end (25 bp of the 5' region of pmoC), and the remainder was upstream DNA. These constructions were transferred to M. capsulatus Bath by conjugation as described previously (12) and were selected on kanamycin (50 mg/liter). Diagnostic PCR of chromosomal DNA (11) confirmed the expected constructions. These mutants grew at the same rate as the wild type. XylE (catechol dioxygenase) activities (5) were determined in crude extracts of the mutants in 100 mM phosphate buffer, which were obtained by passing cells through a French pressure cell at 1.2 × 10⁸ Pa followed by centrifugation for 10 min at approximately 15,000 × g, or in whole cells permeabilized by treatment with 2% (vol/vol) toluene for 30 min (Table 1). The protein concentration was assessed spectrophotometrically (13).

Nucleotide sequence accession numbers. The GenBank accession number for the fragment upstream of pmoC1 is L40804, and those for the fragment upstream of pmoC2 are U94337 and AF273026.