The genes rubA and rubB for alkane degradation in Acinetobacter sp. strain ADP1 are in an operon with estB, encoding an esterase, and oxyR

Abstract

Alkanes are oxidized in Acinetobacter sp. strain ADP1 by a three-component alkane monooxygenase, composed of alkane hydroxylase, rubredoxin, and rubredoxin reductase. rubA and rubB encode rubredoxin and a NAD(P)H-dependent rubredoxin reductase. We demonstrate here that single base pair substitutions in rubA or rubB lead to defects in alkane degradation, showing that both genes are essential for alkane utilization. Differences in the degradation capacity for hexadecane and dodecane in these mutants are discussed. Two genes, estB and oxyR, are located downstream of rubB, but are not necessary for alkane degradation. estB encodes a functional esterase. oxyR encodes a LysR-type transcriptional regulator, conferring resistance to hydrogen peroxide. rubA, rubB, estB, and oxyR constitute an operon, which is constitutively transcribed from a sigma70 promoter, and an estB-oxyR containing message is also transcribed from an internal promoter.

FIG. 1

Schematic drawing of the relevant DNA from Acinetobacter sp. strain ADP1 cloned on pWH891. Numbers indicate the kilobase pair scale of the sequence in the EMBL database (accession no. Z46863). Inverted repeats are indicated as stem-loop structures. In the lower part, the chromosomal characteristics of the mutants with inserted lacZ-Km^r cassettes (simplified as lacZ) are shown. Alk, phenotype on alkane (+, growth; −, no growth); estB, esterase; oxyR, peroxide response regulator; lysS, lysyl tRNA synthetase; ppk, polyphosphate kinase; rubA, rubredoxin; rubB, rubredoxin reductase.

FIG. 2

Part of a multiple alignment of OxyR sequences and the deduced consensus sequence (Cons) of the OxyR box. Amino acids are shown in the one-letter code. The conserved cysteine residues forming reversible disulfide bonds are boxed. Ac, Acinetobacter sp. strain ADP1; Eca, E. carotovora; Ec, E. coli; Hi, H. influenzae; Ma, M. avium; Ml, M. leprae; Mm, M. marinum; φ, hydrophobic amino acids; −, acidic amino acids; X, any amino acid.

FIG. 3

Mapping of the 5′ start site of the rubA mRNA by primer extension. (Top) Autoradiograph. RNA was prepared from cells growing exponentially in LB or minimal medium (MM). Dodecane (DD) and hexadecane (HD) were added at 0.6% (vol/vol). Succinate (Suc) was added at 20 mM. Lanes: 1, ADP1 in LB medium; 2, ADP1 in LB medium plus DD; 3, ADP1 in LB medium plus HD; 4, ADP1 in MM plus Suc; 5, ADP1 in MM plus Suc plus DD; 6, ADP1 in MM plus Suc plus HD; 7, ADP1 in MM plus HD; 8, ADP1 transformed with pWH891 in LB medium; 9, WH380 in LB medium; 10, WH386 in LB medium. Lanes A, C, G, and T show the sequencing products obtained with the same primer. The sequence is shown on the left side. Arrows indicate the start sites of transcription (P₁ and P₂). (Bottom) Sequence interpretation. The sequence of the coding strand upstream of rubA is shown. The numbers of nucleotide position are given on the left according to Fig. 1. The start codon of rubA is underlined, and the four N-terminal amino acids are shown in the one-letter code. The start sites of transcription (P₁ and P₂) are indicated by downward arrows, and the sequences with the highest similarity to E. coli ς⁷⁰ promoter sequences (−10 and −35) are boxed.

FIG. 4

Detection of oxyR transcripts. (Top) Northern blot hybridized with an oxyR-specific probe. Eleven micrograms of total RNA was run on each lane of a 1% agarose gel. RNA was prepared from the following cells growing exponentially in LB medium: ADP1 (lane 1), WH380 (lane 2), WH362 (lane 3), WH382 (lane 4), WH384 (lane 5), and WH386 (lane 6). On the left, the positions of RNA molecular size marker bands are given in kilobases. On the right, the RNA bands discussed in the text are marked. (Bottom) Interpretation. For explanation of the genomic situation depicted at the top see Fig. 1. In the bottom part, RNA species detected in Northern blot analyses are indicated as bars, with their sizes given in the right and left margins in kilobases. The rubA-specific 0.3-kb RNA was detected in a 2% agarose gel (data not shown). P_1,2 and P₃, respectively, indicate the promoters P₁ and P₂ determined by primer extension and promoter P₃ proposed on the Northern blot shown in the top panel.

FIG. 5

EstB activity in E. coli. (Top) Mapping of the DNA conferring esterase activity. For explanation of the genomic situation depicted in the upper part, see Fig. 1. Plasmids with successive deletions were derived from pWH891SK6i and pWHSK6 and analyzed by sequencing. The plasmids were transformed into E. coli, and transformants were analyzed for esterase activity after 7 days at 37°C on indicator plates (−, no halo detectable; + and ++, intensity of halo formation). The fragments present on the deletion plasmids are indicated as bars, with the name of the plasmids on the right indicating the number of base pairs that have been deleted. (Bottom) Acyl chain length specificity of EstB. Activity was measured with crude extracts of E. coli cells expressing estB from pAKA22. No activity was found with pNP esters with chain lengths of 14, 16, and 18 carbons or in the vector controls.