Second acyl homoserine lactone production system in the extreme acidophile Acidithiobacillus ferrooxidans

Abstract

The acidophilic proteobacterium Acidithiobacillus ferrooxidans is involved in the industrial biorecovery of copper. It is found in acidic environments in biofilms and is important in the biogeochemical cycling of metals and nutrients. Its genome contains a cluster of four genes, glyQ, glysS, gph, and act, that are predicted to encode the alpha and beta subunits of glycine tRNA synthetase, a phosphatase, and an acyltransferase, respectively (GenBank accession no. DQ149607). act, cloned and expressed in Escherichia coli, produces acyl homoserine lactones (AHLs) principally of chain length C14 according to gas chromatography and mass spectrometry measurements. The AHLs have biological activity as shown by in vivo studies using the reporter strain Sinorhizobium meliloti Rm41 SinI-. Reverse transcription-PCR (RT-PCR) experiments indicate that the four genes are expressed as a single transcript, demonstrating that they constitute an operon. According to semiquantitative RT-PCR results, act is expressed more highly when A. ferrooxidans is grown in medium containing iron than when it is grown in medium containing sulfur. Since AHLs are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression, this result suggests that A. ferrooxidans has two quorum-sensing systems, one based on Act, as described herein, and the other based on a Lux-like quorum-sensing system, reported previously. The latter system was shown to be upregulated in A. ferrooxidans grown in sulfur medium, suggesting that the two quorum-sensing systems respond to different environmental signals that may be related to their abilities to colonize and use different solid sulfur- and iron-containing minerals.

FIG. 1.

Organization and coexpression of the act locus of A. ferrooxidans and differential expression of act. (A) Organization of the act locus. Shaded arrows indicate the direction of transcription. Small black arrows indicate the position and orientation (5′ to 3′) of primers used in RT-PCR or PCR studies. A predicted sigma-70-like promoter (−35 and −10 regions separated by 18 bp), a possible ribosome binding site (RBS; underlined), and the proposed ATG initiation codon (bold) are indicated. hyp, conserved hypothetical gene of unknown function; rimK, predicted ribosomal protein S6 modification protein. (B) Coexpression of the act locus. Results of RT-PCR (using RNA as a substrate [columns a]) and PCR experiments (using genomic DNA as s substrate [columns b]) are shown. The nucleotide sequence of the act locus has been deposited in GenBank under accession number DQ149607. (C) Differential expression of act when cells were grown in either iron-containing (Fe²⁺) or sulfur-containing (S⁰) medium as determined by semiquantitative RT-PCR. The numbers refer to the numbers of PCR cycles performed. The expression of the constitutively expressed recA is included as a control.

FIG. 2.

Demonstration of the biological activity of A. ferrooxidans Act. (A) E. coli containing pAf-act is capable of inducing the expression of β-galactosidase (dark spot in streak b) in a reporter strain of S. meliloti, SinI⁻. On the other hand, E. coli containing the vector plasmid but lacking act does not express β-galactosidase (streak a). The positive-control S. meliloti Rm41 wild type, which produces AHLs of various chain lengths from C₁₂ to C₁₆ (streak c), induces the expression of β-galactosidase in S. meliloti SinI⁻ (streak c). (B) Same as panel A except that the reporter strain used is A. tumefaciens NT1, which responds mainly to shorter substituted AHLs and AHL chain lengths of C₄ to C₁₄.

FIG. 3.

Identification by MS of an AHL in the extracellular supernatant derived from a culture of E. coli JM109 containing pAf-act. (A) Structure of a standard unsubstituted AHL of chain length C₁₄. MW, molecular weight; HSL, homoserine lactone. (B and C) Mass spectra of a supernatant extract from E. coli JM109 pAf-act (B) and a synthetic standard of unsubstituted C₁₄-AHL (C) (the scale to the right of the line break on the x axis has been amplified five times to show the m/z at 311). The characteristic mass-to-ion charge ratios (m/z) of the AHL are indicated.