areCBA is an operon in Acinetobacter sp. strain ADP1 and Is controlled by AreR, a sigma(54)-dependent regulator

Abstract

The areCBA genes in Acinetobacter sp. strain ADP1, determining growth on benzyl alkanoates, are shown to be transcribed as a single operon and regulated by areR, which encodes a regulatory protein of the NtrC/XylR family. Assays of the Are enzymes and of two insertions of lacZ as a reporter gene have shown that the operon is induced by benzyl acetate, benzyl alcohol, and benzaldehyde, as well as 2- and 4-hydroxybenzyl acetates and benzyl propionate and butyrate. Two adjacent sites of transcriptional initiation were 97 and 96 bp upstream of the start codon for areC, near a sigma(54)-dependent -12, -24 promoter. Inactivation of areR and rpoN (for RNA polymerase sigma(54)) drastically reduced growth rates on the Are substrates and induction of the operon.

FIG. 1

Physical map of the DNA adjacent to the ben genes in ADP1. Physical map of the areCBA genes and their locations relative to one end of the supraoperonic ben-cat cluster. The various inserts of the plasmids produced from cloning genomic DNA into vectors are specified in Table 1. Plasmids named in boldface contain inserts that were cloned directly from genomic DNA. All other plasmids were produced by PCR from genomic DNA or by subcloning from plasmids containing genomic DNA. Sites at the termini of the inserts marked with an asterisk were incorporated via PCR primers. The Km cassette or lacZ-Km cassette insertions are not to scale. The abbreviations for the restriction sites are Bc, BclI; Bg, BglII; E, EcoRI; H, HindIII; N, NsiI; P, PstI; S, SacI; X, XbaI.

FIG. 2

Proposed pathway for catabolism of benzyl alkanoates by Acinetobacter sp. strain ADP1.

FIG. 3

Primer extension of mRNA from Acinetobacter sp. strain ADP1. The experiment was performed with total RNA and a primer which overlaps the putative start codon of areC. Lanes A, C, G, and T show the respective products from M13mp18 that were sequenced using the M13 forward primer to size the extension products. The bases on the left are from the corresponding M13 sequence. Lanes E, F, and H represent the signal obtained from the experiment using 10 μg of total RNA from cells of ADP1 grown with benzyl acetate, benzyl alcohol, or succinate, respectively, as the sole carbon sources. The locations corresponding to both transcriptional starts are designated by the arrow and are positions 112 and 111 from the 5′ end of the universal primer. The corresponding bases on the ADP1 sequence are shown in Fig. 4.

FIG. 4

Regulatory sequences upstream of areC. The start codon of areC is indicated, and the putative ribosome binding site (Shine-Dalgarno) of areC is indicated above the sequence (RBS). The nucleotides corresponding to both primer extension signals (Fig. 3) are indicated by the double-headed arrow with the strongest numbered as the +1 site. The putative −12, −24 promoter elements are indicated. Arrows below the sequence mark an inverted repeat that is hypothesized to act as the binding site for the regulator protein. Dashed arrows above the sequence mark a direct repeat.

FIG. 5

Agarose gel electrophoresis of RT-PCR products amplified by primers from ADP1 grown on benzyl acetate and benzyl alcohol. The positions of the primers for spanning the areAB intergenic region (BAr, BAf) and the areCB intragenic region (CBr, CBf) are shown relative to the gene organization of areCBA. The values for molecular size markers (in base pairs) in lanes S (HyperLadder I; Bioline, London, United Kingdom) are indicated on the right side of the gel. Lanes: 1, areCB, benzyl acetate-grown cells (expected size, 953 bp); 2, areCB, benzyl acetate-grown cells digested with SacI (640 and 313 bp); 3, areCB, benzyl alcohol-grown cells (expected size, 953 bp); 4, areCB, benzyl alcohol-grown cells digested with SacI (640 and 313 bp); 5, areBA, benzyl acetate-grown cells (expected size, 946 bp); 6, areBA, benzyl acetate-grown cells digested with AccI (631 and 315 bp); 7, areBA, benzyl alcohol-grown cells (expected size, 946 bp); 8, areBA, benzyl alcohol-grown cells digested with AccI (631 and 315 bp). No detectable products were obtained in control reactions with each pair of primers, from which RT had been omitted, or in reactions carried out on succinate-grown cells (data not shown).