Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC

Abstract

The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 degrees C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC.

Figure 1. Location of cloned DNA fragments and cat genes

Open arrows indicate ORFs and the directions of their transcripts. The probe synthesized by PCR using catB-N and catB-I primers was used for colony hybridization. The primers used for RT-PCR and primer extension analyses are indicated by horizontal arrows. The downward solid triangle indicates a transcriptional start site of cat gene cluster. A, ApaI; C, ClaI; E, EcoNI; N, NcoI; S, SacI.

Figure 2. Production of CatA, CatB and CatC from Rhodococcus sp. AN-22 on succinate medium

The bacterium was incubated at 30 °C with shaking. At appropriate intervals, cell growth (◆), CatA (▲), CatB (■) and CatC (●) were measured. Results are indicated as means±S.D. for three determinations.

Figure 3. Native PAGE (lanes 1) and SDS/PAGE (lanes 2) of CatB (A) and CatC (B) from Rhodococcus sp. AN-22

Lanes 1, native PAGE. The purified enzymes (5 μg each) were run on 7.5% (w/v) gels of pH 8.9 at 2 mA/tube for 2.5 h in a running buffer (pH 8.3) of Tris/glycine. Lanes 2, SDS/PAGE. The purified enzymes (4 μg each) denatured with SDS were run on 7.5% (w/v) gels containing 0.1% (w/v) SDS at 6 mA/tube for 3 h (A) or 2 h (B) in a running buffer (pH 7.2) of 0.1% (w/v) SDS/0.1 M sodium phosphate. Molecular-mass size markers used for SDS/PAGE were α-lactalbumin (14 kDa), trypsin inhibitor (20 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 and 45 kDa), BSA (67 and 66 kDa) and phosphorylase b (94 and 97 kDa). The gels used for the native PAGE and SDS/PAGE were stained with 0.25% (w/v) Coomassie Brilliant Blue R-250 in a solvent of ethanol/ethanoic (acetic) acid/water (9:2:9, v/v). The molecular masses of three markers were different in (A) and (B), because they were purchased from Pharmacia and Amersham Biosciences respectively. See the text for details.

Figure 4. Determination of molecular mass of CatB by gel filtration

The enzyme (24 units) and markers (2 mg of each) were placed on the top of a column (3.0 cm×60 cm) of Toyopearl HW-55S, and then eluted with buffer A containing 0.2 M NaCl. 1, Cytochrome c (12.5 kDa); 2, chymotrypsinogen A (25 kDa); 3, ovalbumin (45 kDa); 4, BSA (68 kDa); ●, CatB.

Figure 5. Primer extension analysis of cat gene transcripts

Total RNA prepared from aniline- and succinate-grown cells of Rhodococcus sp. AN-22 was used for the reverse-transcription reaction with the PE primer. This primer was also used for a dideoxy sequencing reaction using plasmid p42E6 as a template. The reverse-transcription products obtained (lanes 1 and 2) corresponded in size to the thymine residue (T) marked by an asterisk (*) in the coding strand.

Figure 6. Agarose gel electrophoresis of RT-PCR product

Total RNA prepared from succinate-grown cells of Rhodococcus sp. AN-22 was used for the synthesis of cDNA as a template. PCR was performed with the synthesized cDNA using OF and OR primers as amplification primers (lane 3). Control experiments were as follows: PCR was carried out using total RNA as a template without a reverse transcription step (lane 2) and using total DNA (lane 4) as a template. A 1-kb DNA ladder (New England Biolabs) was used as markers (lane 1). Sizes are given in kb.