Expression of etbA1 and ebdA1 in RHA1.
To discriminate between the etbA1
transcripts, RT-PCR analysis was performed with RNAs from RHA1 cells grown on biphenyl, ethylbenzene, or LB, using the sequences upstream from etbA1
as one of the primers, since the sequences upstream from these two genes are different. The etbA1
forward primers were designed using the sequences downstream from the transcriptional start site of each gene, as determined by Takeda et al. (18
). The primer pair for etbA1
did not produce any PCR product with ebdA1
plasmid DNA as the template, and that for ebdA1
produced no PCR product with etbA1
plasmid DNA as the template (data not shown). When total RNAs of the cells grown on biphenyl or ethylbenzene were used as templates, PCR products with the expected sizes for etbA1
were obtained. No PCR product was obtained from the cells grown in LB medium (data not shown). Takeda et al. indicated the induction of bphA1
gene transcription in the presence of biphenyl or ethylbenzene by RT-PCR (19
) and suggested that transcription from five adjacent promoters upstream from the degradation gene clusters containing etbA1
, and bphA1
is coordinately controlled by the BphST system (18
). The BphST system induced transcription beginning with each of the five promoters in response to ethylbenzene and other aromatic compounds as well as biphenyl (19
). Our results indicated that etbA1
as well as bphA1
are inducibly transcribed in the presence of biphenyl or ethylbenzene.
To verify the protein products of etbA1 and ebdA1 in RHA1 induced during incubation with biphenyl, we performed 2-D PAGE (Fig. ). The results for protein samples from the cells grown on biphenyl revealed the identity of a protein spot whose molecular mass and isoelectric point (pI) were 51 kDa and 5.5, respectively (Fig. ). These values were very close to those of 51.7 kDa and 5.2, respectively, which were estimated from the amino acid sequence of EtbA1/EbdA1. The N-terminal amino acid sequence of the protein from this spot was determined, and the obtained sequence, MLXSEXFSPG (X is an unknown), was correspondent with the sequence of EtbA1 and EbdA1 (MLRSERFSPG). These results indicated that either etbA1, ebdA1, or both are inducibly expressed as a protein product in RHA1 grown on biphenyl. In addition to EtbA1/EbdA1, 15 protein spots were unique to the cells grown on biphenyl in comparison to those grown in LB medium. One spot with a molecular mass of 34 kDa and a pI of 4.8 was assigned as the EtbC dioxygenase protein based on its deduced molecular mass, pI, and N-terminal amino acid sequence, AKVTELGYL. Another spot with a molecular mass of 45 kDa and a pI of 5.1 was estimated to be EtbA4, whose deduced molecular mass and pI are 44 kDa and 4.9, respectively (Fig. ).
FIG. 2. Two-dimensional polyacrylamide gel electrophoresis analysis of soluble proteins from RHA1 cells before (A) and after (B) incubation in the presence of biphenyl for 16 h. Proteins were separated in an isoelectric point (pI) gradient from 4 to 7 and then (more ...) Disruption of genes encoding RHDO large subunits.
To examine whether the etbA
genes are really involved in biphenyl/PCB degradation, etbA1
were independently disrupted by insertional inactivation using homologous recombination as described in Materials and Methods. The bphA1
gene, which was proved to be involved in biphenyl/PCB degradation in RHA1 (13
), was also disrupted by the same method. The etbA1
, and bphA1
insertion mutants were designated HDT1, HDB1, and HDA1, respectively.
The degradation activity toward 4-CB of each insertion mutant was investigated by GC-MS. Autoclaved cells were used as a negative control. The degradation activities of HDB1 and HDA1 were diminished, and the reduction of 4-CB by either of these insertion mutants was half that by RHA1 after 30 min (Fig. ). The activity of HDT1 was only slightly lower than that of RHA1 (Fig. ). Taking into account the polar effect exerted on the activities of genes downstream from the inactivated gene, these results suggested that the etbA1, ebdA1, and bphA1 genes and/or the genes downstream from them are involved in biphenyl/PCB degradation.
FIG. 3. 4-Chlorobiphenyl degradation activities of RHA1 and RHDO large-subunit mutants. The results for single-insertion mutants (A) and double-insertion mutants (B) are presented. The remaining amount of 4-chlorobiphenyl was determined by gas chromatography-mass (more ...)
To examine whether the reduction of 4-CB degradation activity was caused by the inactivated gene, a wild-type gene was introduced into the mutant to complement each mutation. A plasmid, pK4TEB1, containing ebdA1 and its promoter region was introduced into the etbA1 mutant, HDT1, and the ebdA1 mutant, HDB1, by electroporation. pK4TBA21, containing bphA1 and its promoter region, was introduced into the bphA1 mutant, HDA1. The degradation activity of each recombinant was then examined. However, none of the 4-CB degradation activities of the insertion mutants were restored (Fig. ). In the case of HDA1, the introduction of pK4TBA21 resulted in a further reduction of degradation activity. Excluding the case of HDA1, these results suggested that the expression of RHDO subunit genes located downstream from etbA1 and ebdA1 was suppressed by a polar effect. In the case of HDA1, containing the bphA1 plasmid, the impaired high-level expression of a terminal dioxygenase subunit appears to have resulted in the formation of an inactive heterologous complex between BphA1 and EtbA2/EbdA2.
Double disruption of three RHDO large-subunit genes.
Because the insertion mutants mentioned above did not provide sufficient evidence that RHDO terminal oxygenase component genes play a role in biphenyl/PCB degradation in RHA1, we performed double-disruption experiments. The double-insertion etbA1 bphA1 and ebdA1 bphA1 mutants were constructed by using the bphA1 mutant, HDA1, as the parent strain and were designated HDAT1 and HDAB1, respectively. The double-insertion etbA1 ebdA1 mutant, designated HDBT1, was constructed from the ebdA1 mutant, HDB1.
After examining the ability of the single-disruption mutants to degrade 4-CB, we investigated the 4-CB degradation activity of each double-insertion mutant. The degradation activity of HDAT1 was lower than that of its parent strain, HDA1, and was one-quarter that of RHA1 (Fig. ). This degradation activity was estimated to depend solely on the ebdA1A2-encoded terminal oxygenase component (EbdA1A2 component), suggesting that the EbdA1A2 component plays a part in biphenyl/PCB degradation in RHA1. Because HDAT1 was constructed with the etbA1 inactivation of HDA1, the reduction of degradation activity appears to have originated from the inactivation of etbA1. Thus, the terminal oxygenase component encoded by etbA1A2 (EtbA1A2 component) appears to play an important role in biphenyl/PCB degradation in RHA1. On the other hand, the 4-CB degradation activity of HDAB1 was approximately equal to that of the negative control, irrespective of the presence of intact etbA1A2 (Fig. ). For HDAB1, it was suggested that the expression of both bphA3 and ebdA3 was inhibited by the polar effect caused by the respective insertions into bphA1 and ebdA1, respectively. Because of the absence of the electron transfer component, the EtbA1A2 component, which is intact in HDAB1, appears to have lost its activity. This notion was supported by the introduction of plasmid pK4TEB3, containing ebdA3, into HDAB1. The degradation activity of HDAB1 was restored by the introduction of pK4TEB3 to a level approximately equal to that of HDAT1 (Fig. ). These results again indicated that the EtbA1A2 component is involved in biphenyl/PCB degradation in RHA1. For HDAB1, we conjectured that not only the bphA3 gene but also the adjacent bphA4 gene is inhibited by the polar effect. However, the introduction of the sole ebdA3 gene restored the activity of HDAB1. These results suggested that another ferredoxin reductase gene, probably etbA4, plays a role in biphenyl/PCB degradation.
The degradation activity of HDBT1 was estimated to depend solely on bphA1A2, which confirmed that the bphA1A2-encoded terminal oxygenase component is functional in biphenyl/PCB degradation in RHA1. HDBT1 exhibited almost the same activity as its parent strain, HDB1 (Fig. ), although intact etbA1A2 existed in HDB1. These results may suggest that the sole electron transfer components in HDB1 encoded by bphA3 and bphA4 are not sufficient to account for the obvious activity of the etbA1A2-encoded dioxygenase.
Disruption of two RHDO ferredoxin genes and a ferredoxin reductase gene.
As previously mentioned, ebdA3 and etbA4, which encode a ferredoxin and a ferredoxin reductase, respectively, are thought to be involved in biphenyl/PCB degradation in RHA1. To confirm this involvement, we disrupted these genes by the same method used for the disruption of the RHDO large subunits. The bphA3 gene was also disrupted by the same method. The ebdA3, etbA4, and bphA3 insertion mutants were designated HDB3, HDT4, and HDA3, respectively. We also made repeated attempts to construct a corresponding insertion mutant of bphA4, but these failed.
The ability of each insertion mutant to degrade 4-CB was investigated by GC-MS. The degradation activities of the ferredoxin gene mutants were diminished, with HDB3 and HDA3 realizing one-third and one-half of the 4-CB reduction achieved by RHA1, respectively (Fig. ). To examine whether the reduction of 4-CB degradation activity was caused by the inactivated gene, a wild-type gene was inserted next to the bphA1 promoter in vector pK4tsr, which was introduced into the mutant to complement each mutation. Plasmid pK4TEB3, containing ebdA3, and pK4TBA3, containing bphA3, were independently introduced into HDB3 and HDA3, respectively, by electroporation. The 4-CB degradation activities of both HDB3 and HDA3 were restored by the introduction of either ebdA3 or bphA3 (Fig. ). These results indicated that both ferredoxin genes, bphA3 and ebdA3, are involved in biphenyl/PCB degradation. A double-insertion ebdA3 bphA3 mutant was also constructed by using HDA3 as the parent strain and was designated HDAB33. The 4-CB degradation activity of HDAB33 was approximately equal to that of the negative control (Fig. ), suggesting that EbdA3 and BphA3 are the only dominant ferredoxin components involved in biphenyl/PCB degradation in RHA1.
FIG. 4. 4-Chlorobiphenyl degradation activities of mutant strains with insertion mutations in RHDO electron transfer components. The results for HDB3 (A), HDA3 (B), and HDT4 (C), with insertion mutations in ebdA3, bphA3, and etbA4, respectively, are presented. (more ...)
The degradation activity of the ferredoxin reductase mutant HDT4 was diminished to about half that of RHA1 (Fig. ). To complement the etbA4 gene deficiency, pK4TBA4-2, containing etbA4 and its promoter region, was introduced into HDT4 by electroporation. The 4-CB degradation activity of HDT4 was restored by introduction of the etbA4 gene (Fig. ). These results indicated that a ferredoxin reductase gene of RHDO, etbA4, is involved in biphenyl/PCB degradation in RHA1.
Transformation of PCB congeners by insertion mutants.
It is known that the large subunits of terminal oxygenase components are major determinants of the substrate preference of the RHDO. In consideration of the low sequence similarity between EtbA1A2/EbdA1A2 and BphA1A2, it was expected that the substrate preferences would differ between EtbA/EbdA and BphA dioxygenase species. Therefore, the substrate preferences of EtbA/EbdA and BphA for PCB congeners were investigated by using the insertion mutants. The bphA1
mutant, HDA1, and the etbA1 ebdA1
mutant, HDBT1, were employed as EtbA/EbdA- and BphA-expressing strains, respectively. A mixture of five ortho
-substituted PCB congeners that consisted of 2,2′-, 2,3-, 2,5,2′-, 2,5,2′,5′-, and 2,4,5,2′,5′-chlorobiphenyl (solution A) and a mixture of five para
-substituted PCB congeners that consisted of 4,4′-, 2,4,2′,4′-, 2,4,3′,4′-, 3,4,3′,4′-, and 2,4,5,2′,4′,5′-chlorobiphenyl (solution B) were used as substrates. The results are shown in Fig. . Although the degradation activity of HDA1 was lower than that of RHA1, the substrate preference of EtbA/EbdA-expressing HDA1 was almost identical to that of RHA1. On the other hand, BphA-expressing HDBT1 showed extremely low degradation activities toward 2,4,2′,4′-, 2,4,3′,4′-, 2,5,2′,5′-, and 2,4,5,2′,5′-chlorobiphenyls. The results for HDBT1 were almost correspondent with those obtained by using Rhodococcus erythropolis
IAM1399 expressing bphA1A2A3A4
). These results indicated that EtbA/EbdA and BphA have different substrate preferences for PCB congeners and suggested that EtbA/EbdA plays an important role in the degradation of highly chlorinated biphenyls. BphA appears to play a role in the degradation of PCBs to some extent, because HDBT1 showed good activities toward 2,2′-, 2,3-, and 2,5,2′-chlorobiphenyls in addition to 4-CB (Fig. shows the data for 4-CB).
FIG. 5. Degradation of PCB congeners by RHA1 (black bars), by the bphA1 mutant, HDA1 (gray bars), and by the etbA1 ebdA1 double knockout, HDBT1 (white bars). Solution A consisted of five mainly ortho-substituted PCB congeners, 2,2′-dichlorobiphenyl, 2,3-dichlorobiphenyl, (more ...)
Multiple isozyme genes for biphenyl/PCB degradation in Rhodococcus
strains have been reported (9
). However, the potential involvement of these isozyme genes in degradation has not been investigated. To our knowledge, this is the first report to demonstrate the cooperative involvement of multiple RHDO isozymes encoded by the bphA
, and ebdA
genes in a single PCB-degrading bacterium. These genes were indicated to be under the control of the bphST
regulatory system, which has a broad substrate spectrum for transcription induction (18
). Thus, these isozymes might be simultaneously involved in the catabolism of substrates other than biphenyl.