Abstract
The nucleotide sequence of the 2.6-kilobase pair (kb) EcoRI fragment encoding chromate resistance (Chrr) on plasmid pMOL28 in Alcaligenes eutrophus was determined. 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 analyzed 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 reduced accumulation of chromate, while the third open reading frame was not needed.
Highlights
ChrA (401 amino acids) and ChrB (196 amino acids) polypeptides were essential for inducible chromate resistance and reduced accumulation of chromate, while the third open reading frame was not needed
Cobalt and nickel resistances result from inducible energydependent cation efflux (Sensfuss and Schlegel, 1988; Nies and Silver, 1989b) governed by a resistance determinant located on a 8.5-kb EcoRI-PstI fragment adjoining the 2.6-kb EcoRI fragment on plasmid pMOL28 that determines Chr’ (Nies et al, 1989a); Siddiqui et al, 1989)
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)
Summary
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). For construction of Ml3 derivatives containing the chromate resistance determinant, the 2.6-. Kb EcoRI fragment was cloned from plasmid pECD300 (Nies et al., 1989a) into phage mTMOl0 (Misra, 1987) in both orientations. EcoRI site, (ii) contains a single PstI site, (iii) has a size of about 7.2 kb, (iv) can be mobilized into A. eutrophus, and (v) forms cointegrates with plasmid pDNA121 (Nies et al, 1989a) in A. eutrophus. Isolation of Mutants-DNA fragments with Bul31-generated deletions (which’were used for the DNA sequence analysis) in the 2.6-kb EcoRI fragment were cloned from the corresponding phage mTMOl0 derivatives into plasmids pT7-5, pT7-6, pVDZ’2, and pECD159 using
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