Nucleotide Sequence and Expression of a Plasmid-encoded Chromate Resistance Determinant from Akaligenes eutrophus*

The nucleotide sequence of the 2.6-kilobase pair (kb) EcoRI fragment encoding chromate resistance (Chr’) on plasmid pMOL28 in Alcaligenes eutrophus was de- termined. Three open reading frames were assigned to three polypeptides which were expressed from this determinant in Escherichia coli under the control of a phage T7 transcription promoter. When the roles of the polypeptides and open reading frames were ana- lyzed with deletion derivatives of the 2.6-kilobase fragment, the membrane-bound ChrA (401 amino acids) and ChrB (196 amino acids) polypeptides were essential for inducible chromate resistance and re- duced accumulation of chromate, while the third open reading frame was not needed.

This report describes the nucleotide sequence of the 2.6-kb EcoRI fragment encoding the chromate resistance determinant and evaluates by deletion analysis the roles of the open reading frames in chromate resistance.

Bacterial
Strains and Plasmids-Those used in this study are listed in Table I and Fig. 1. Chromate resistance and reduced accumulation of SICrOq-were tested as described (Nies and Silver, 198913). Chromate uptake could be derepressed by cultivation under sulfate starvation (30 PM sulfate; Nies and Silver, 1989c) as occurs with Pseudomonas aeruginosa (Ohtake et al., 1987). However, to avoid interferences between chromosomally encoded chromate uptake, which may be only partially derepressed, and the plasmid-encoded reduced accumulation of chromate, which may be altered in mutants, all strains were grown in the presence of 3 mM sulfate.

Molecular
Genetics-These techniques were performed as described (Nies et al., 1987;Maniatis et al., 1982). For construction of Ml3 derivatives containing the chromate resistance determinant, the 2.6kb EcoRI fragment was cloned from plasmid pECD300 (Nies et al., 1989a) into phage mTMOl0 (Misra, 1987) in both orientations. Nested deletions were generated using Bal31 exonuclease and the complete DNA sequence was determined (Misra, 1987) from both strands.
The polypeptides expressed from the chromate resistance determinant were analyzed as previously described (Tabor and Richardson, 1985;Nies et al., 1989a (Table  II, Fig. 4).
The 14.5-kDa polypeptide was present when a fragment containing ORF3 was cloned into plasmid pT7-5 and expressed in E. coli (Fig. 4). When a fragment containing ORF3 was cloned into plasmid pT7-6 (plasmids pECD322 and pECD323) and expressed in E. coli, a 12-kDa polypeptide was visible on autoradiograms instead of the 14.5-kDa polypeptide (Fig. 4). ORF3 is not terminated within the 2.6-kb EcoRI fragment (Fig. 2), and only half of the 14.5-or 12-kDa polypeptide is encoded by the 2.6-kb fragment. The other half is encoded by plasmids pT7-5 or pT7-6, respectively.
The polypeptide migrating with a mobility corresponding to 31.5 kDa was assigned to the chrA gene (Table II) which encodes a polypeptide with a predicted size of 401 amino acid residues ( Fig. 2; 43-kDa predicted size). The differences between the sizes predicted from the sequence and the sizes estimated from the mobility on sodium dodecyl sulfate-polyacrylamide gel may be due to the unusual behavior of membrane proteins on polyacrylamide gels (Ferro-Luzzi Ames, 1974).
In contrast to the chrA and the partial ORF3 translation product, the chrB gene product was expressed much more weakly in E. coli. The 21-kDa polypeptide was clearly visible on autoradiograms after expression of plasmids pECD311 and pECD318 in E. coli (Fig. 4). A less radioactive 21-kDa polypeptide and some possible degradation products thereof were    The arrow gives the direction of transcription of the chr determinant. The sizes of the predicted gene products in amino acids (aa) are indicated. E, EcoRI; P, P&I; X, XmaI sites.
visible after expression of plasmid pECD321 (Fig. 4). Because plasmid pECD321 contained the first open reading frame only up to position 628 a part of the particular Xl-kDa polypeptide expressed by plasmid pECD321 might be encoded by plasmid pT7-5. The 21-kDa polypeptide was not visible after expression of plasmids pECD322, pECD323 and pECD327 (Fig. 4). After expression of plasmids pECD317, pECD319, and pECD320 (which all contained the complete first open reading frame), the 21-kDa polypeptide was barely visible, about as strongly as expected for a background polypeptide (Fig. 4). Therefore, the assignment of the 21-kDa polypeptide to the first open reading frame of the chr operon is not as solid as the assignment of the 31.5 and the 14.5kDa polypeptides to chrA and ORF3, respectively.
Function of the chr Gene Products-Derivatives of the 2.6kb EcoRI fragment carrying various deletions were subcloned into plasmids pECD159 or pVDZ'2 ( Fig. 1). Plasmid pECD159 itself is not able to replicate in A. eutrophus but (like plasmid pSUP202) is maintained as a cointegrate with plasmidpDNA121 (Nies et al., 1989a). PlasmidpVDZ'S-based hybrid plasmids were transferred into A. eutrophus strain AE104 and plasmid pECD159-based hybrid plasmids into strain AEl04(pDNAl21).
Plasmids pDNA221 and pDNA220, which contain the 2.6kb EcoRI fragment cloned in both orientations in plasmid pVDZ'2, both conferred the same degree of chromate resistance as plasmid pDNA206 (Table III). Plasmids pECD334 and pECD335, which contain the 2.6-kb EcoRI fragment cloned in both orientations in plasmid pECD159, both conferred (if cointegrated into plasmid pDNA121) the same degree of chromate resistance as plasmid pECD300 (Table III). Therefore, chromate resistance appears to be transcribed from its own promoter present on the 2.6-kb EcoRI fragment rather than from the lac promoter present in plasmids pVDZ'2 or pECD159.
Any deletion of the chrA gene (plasmids pDNA222, pDNA223, pDNA225, pECD331, and pECD329) led to a chromate sensitive phenotype (Table III). Therefore, the chrA gene is essential for expression of Chr' in A. eutrophus.
The 1.5-kb XmaI-EcoRI subfragment (encoding ChrA and Chromate Resistance in A. eutrophus the ORF3 product) of the 2.6-kb EcoRI fragment was cloned into plasmid pVDZ'2 in the opposite orientation to the luc promoter (plasmid pDNA224; Fig. 1) and into plasmid pSUP202 leading to plasmid pECD325. Both constructs led to a chromate sensitive phenotype when transferred into A. eutrophus strain AE104 or AE104(pDNA121), respectively. Therefore, transcription of the chr operon must start downstream of the single XmaI site of the 2.6-kb EcoRI fragment (Fig. 3) and/or the chrB gene product is required for expression of chromate resistance in A. eutrophus. In plasmid pECD330 chrA and ORF3, but not chrB, are cloned in plasmid pECD159 under the control of the lac promoter which is active and not regulated in A. eutrophus (Nies et al., 1989d). Plasmid pECD330 did not confer chromate resistance to strain AE104(pDNA121) (Table III). Therefore, the chrB gene product is necessary for expression of chromate resistance in A. eutrophus.

DISCUSSION
The chromate resistance determinant of A. eutrophus plasmid pMOL28 can be compared with that from P. aeruginosa plasmid pUM505 (Ohtake et al., 1987;Cervantes and Ohtake, 1988;Cervantes et al., 1989). Both chromate resistance systems result in reduced cellular accumulation of chromate (Ohtake et al., 1987;Nies and Silver, 1989b). However, Southern DNA/DNA hybridization experiments did not show significant DNA sequence homology (Cervantes and Ohtake, 1988). The constitutive cloned chromate resistance determinant of plasmid pUM505 consists of two open reading frames (Cervantes et al., 1989), whereas the inducible determinant of plasmid pMOL28 has three open reading frames. The proposed translation products of the two chrA genes show a strong homology with 29% amino acids identical. The relationship seems uniformly distributed for the full length of the polypeptides (Fig. 6). However, the identical residues and their locations do not give rise to a useful hypothesis concerning the function of these hydrophobic, presumedly membrane transport proteins. When the DNA sequences of the two open reading frames were aligned, there was only 55% nucleotide identity, which explains the negative Southern blot DNA/ DNA hybridization results. The ORFs located after the chrA genes of plasmids pMOL28 and pUM505 are not needed for chromate resistance (Table III; Cervantes et al., 1989). However, the proposed translation products of these reading frames have closely homologous amino-terminal sequences (35 of the first 41 amino acids are identical). No significant homology occurs after isoleucine4i (data not shown). What the function of these ORFs might be and why an unnecessary ORF should be more conserved than an essential gene (chrA) is puzzling.
From the comparable physiological results (resistance due to lowered levels of chromate accumulation), the significant amino acid homology and similar hydropathy profiles (data not shown) between the proposed chrA gene products, it can be assumed that the basic mechanism of plasmid-determined chromate resistance is similar in Alcaligenes and Pseudomonas with the chrA gene product functioning as a chromate efflux pump. The two systems differ in the function of the proposed chrB gene product which is absent in the cloned constitutive resistance determinant of plasmid pUM505 but essential for the inducible chromate resistance of plasmid pMOL28. Expression of chrA without chrB in Alcaligenes lead to hyperuptake of chromate (Fig. 5) and to hypersensitivity to chromate. Thus, in Alcaligenes the chrB gene product may participate in the hypothetical chromate efflux system together with the chrA gene product.
Since chromate resistance encoded by the cloned 2.6-kb EcoRI fragment is inducible and ORF3 is not needed for this induction, the chrA and/or the chrB gene product might also serve as a regulatory protein. Alternatively, the bacterial chromosome might encode the regulatory function. Compared with the putative ChrA protein, the putative ChrB protein is more likely a regulatory protein because it contains less nonpolar amino acid residues (47% compared with 62%), does not show any potential membrane-spanning segments in the hydropathy plot (not shown), and is absent in the constitutively expressed cloned chromate resistance determinant from Pseudomonas (Cervantes et al., 1989). Thus, the putative ChrB protein may play a dual role as regulatory protein and part of the hypothetical efflux system in the chromate resistance system from Alcaligenes.