Sigma factor RpoS controls alkylresorcinol synthesis through ArpR, a LysR-type regulatory protein, during encystment of Azotobacter vinelandii

Abstract

Azotobacter vinelandii is a bacterium which undergoes a differentiation process leading to the formation of metabolically dormant cysts. During the encystment process, A. vinelandii produces alkylresorcinol lipids (ARs) that replace the membrane phospholipids and are also components of the layers covering the cyst. The synthesis of ARs in A. vinelandii has been shown to occur by the activity of enzymes encoded by the arsABCD operon, which is expressed only during the differentiation process. Also, the production of ARs has been shown to be dependent on the stationary-phase sigma factor RpoS, which is also implicated in the control of the synthesis of other cyst components (i.e., alginate and poly-β-hydroxybutyrate). In this study, we identified ArpR, a LysR-type transcriptional regulator expressed only during encystment that positively regulates arsABCD transcription. We show that this activation is dependent on acetoacetyl-coenzyme A (acetoacetyl-CoA), which might provide a metabolic signal for encystment. We also show that RpoS regulates arsABCD expression through the control of arpR transcription.

Fig 1

Inactivation of arpR impairs alkylresorcinol production. Staining (A) and quantification (B) are shown for alkylresorcinols produced by A. vinelandii SW136 (WT) and mutant strains OV8 (arsA::Tn5), SW7 (arpR::Tn5), SW9 (rpoS::Sp), and SW7 complemented with a plasmid expressing ArpR. The cells were grown on petri dishes containing encystment induction medium (BBOH). Data for the quantification of alkylresorcinols are averages for two independent experiments.

Fig 2

ArpR and RpoS positively regulate the expression of arsA. (A) Expression of arsA of strain SW136 (WT) and its isogenic mutants SW7 (arpR::Tn5) and SW9 (rpoS::Sp) as measured by qRT-PCR. The data are presented as fold changes of arsA mRNA levels of the SW7 and SW9 mutant strains relative to that of the parental strain (SW136). (B) Expression of arsA, quantified as β-glucuronidase activities, in strains YRR30 (WT derivative), YRR32 (SW7 derivative) and YRR34 (SW9 derivative), containing a chromosomal arsA::gusA transcriptional fusion. One unit of β-glucuronidase activity corresponds to 1 nmol of substrate (X-Gluc) hydrolyzed min⁻¹ mg protein⁻¹. Determinations for both panels were made from bacterial cultures grown for 36 h in liquid BBOH medium at 30°C. Error bars represent standard deviations.

Fig 3

Inactivation of arpR or rpoS negatively affects arsA expression from a single promoter. (A) Primer extension mapping of arsA transcription initiation 36 h after encystment induction in A. vinelandii SW136 (lane 1) and mutant derivatives carrying the arpR::Tn5 mutation (lane 2) or rpoS::Sp (lane 3). (B) Nucleotide sequence of the upstream regulatory region of arsA. The transcription start site mapped by primer extension is shown in bold and marked by a black triangle. The −35 and −10 consensus sequences for the P1 promoter are shown in bold and underlined. The arsA translation initiation codon is shown in bold and marked by an arrow. The analysis was performed using the primer 5′-GAGGATCGACGAACGGAC-3′ and 50 μg of RNA isolated from cells grown for 36 h in liquid BBOH medium.

Fig 4

ArpR is the activator of arsA transcription. (A) Growth of E. coli DH5α carrying plasmids pBAD-ArpR, expressing ArpR from an arabinose-inducible promoter, and pACYC/PKK-ArsA, containing the arsA regulatory region fused to the chloramphenicol acetyltransferase reporter gene (cat). The experiment was done on LB medium plates supplemented with chloramphenicol (Cm) in the presence or absence of 0.2% arabinose. (B) Expression of the arsAp-cat transcriptional fusion in E. coli DH5α transformed with the same plasmids as those in panel A, measured as CAT activity. The experiment was done in liquid LB medium at 37°C. The activity was determined after 3, 6, and 12 h of growth in the presence or absence of arabinose. The CAT specific activity was quantified as μmol of substrate (chloramphenicol) hydrolyzed min⁻¹ mg protein⁻¹. Error bars represent the standard deviations. (C) Western blot analysis showing heterologous expression of His-tagged ArpR from plasmid pBAD-ArpR in the presence of arabinose, but not in its absence, even after 12 h of growth. Monoclonal anti-His antibodies were used, and the samples were taken from parallel cultures grown under the same conditions used for activity determinations.

Fig 5

ArpR specifically binds the regulatory arsA region. Electrophoretic mobility shift assays were performed to analyze ArpR binding to the regulatory region of arsA. (A) Labeled DNA fragments (10 nM) containing the regulatory region of arsA were incubated with increasing concentrations of MBP-ArpR (0 to 1,000 nM). ArpR binding to arsA was further analyzed by competitive EMSA. As a negative control, a fragment containing the regulatory region of rpoS was included in the DNA binding reaction mix. The labeled DNA fragment containing the regulatory region of arsA was mixed with 1,000 nM MBP-ArpR in the presence or absence of a 100-fold excess of unlabeled specific (arsA) or nonspecific (rpoS) competitor. (B) EMSA with MBP as a negative control. The DNA-protein complexes were resolved in a nondenaturing 6% polyacrylamide gel.

Fig 6

ArpR restores the synthesis of ARs in the rpoS mutant strain during encystment but not in vegetative cells. (A) Staining of ARs produced by the A. vinelandii SW7 and SW9 mutant strains grown on agar-fortified medium under vegetative growth or encystment-inducing conditions. Strains were transformed with plasmid pBBR-ArpR, carrying a constitutively expressed ArpR gene. As a negative control, the empty pBBRIMCS-2 plasmid was transformed into the SW136 and SW7 strains. (B) Quantification of ARs produced by the complemented strains under the encystment conditions shown in panel A. (C) Western blot analysis showing expression of His-tagged ArpR from plasmid pBAD-ArpR in strains SW7 and SW9. Samples were taken from vegetative and encysting cultures. Monoclonal anti-His antibodies were used.

Fig 7

ArpR is expressed under encystment-inducing conditions. (A) RT-PCR assay to monitor arpR expression in vegetative and encysting cells of A. vinelandii SW136 and its rpoS mutant SW9. Total RNA was extracted from cells grown at 30°C for 12, 24, or 36 h under vegetative growth conditions (liquid BS medium) or with encystment induction (liquid BBOH medium). cDNA was synthesized as reported previously (25). The expression of gyrA was used as a control of constitutive expression. Both the arpR and gyrA fragments were amplified by PCR with Taq polymerase (Altaenzyme). The PCR products were resolved in a 1% agarose gel. (B) Expression of arpR during growth in liquid BS or BBOH medium, measured as β-glucuronidase activities of an arpR::gusA transcriptional fusion in strain YRR50. One unit of β-glucuronidase activity corresponds to 1 nmol of substrate (X-Gluc) hydrolyzed min⁻¹ mg protein⁻¹. Average values ± standard deviations are shown.

Fig 8

Acetoacetyl-CoA increases the affinity of ArpR for the regulatory region of arsA. EMSA was performed using an arsA DNA fragment in the absence or presence of different amounts of acetoacetyl-CoA. The labeled DNA fragment containing the regulatory region of arsA was incubated with increasing concentrations of MBP-ArpR (0, 0.1, and 8.0 μM) and 0, 1, 4, 8, or 12 mM acetoacetyl-CoA. As a negative control, a 225-bp fragment containing the regulatory region of rpoS was included in each DNA binding reaction mix. (B) EMSA with MBP as a negative control.

Fig 9

The presence of acetoacetyl-CoA under vegetative growth conditions induces the expression of the arsA and arpR genes and the synthesis of ARs. (A) Staining of ARs produced by A. vinelandii SW136 and the arpR and rpoS mutants SW7 and SW9, carrying plasmid pBBRIMCS-2 or pBBR-ArpR. (B) Production levels of ARs of A. vinelandii SW136 at different times. (C) arsA expression measured as the β-glucuronidase activity of strain YRR30, which contains an arsA::gusA reporter fusion. (D) arpR expression measured as the β-glucuronidase activity of strain YRR50, which contains an arpR::gusA reporter fusion. For the experiments in all panels, the strains were grown on agar-fortified BS medium in the absence (no inducer) or presence of 5 mM acetoacetyl-CoA. One unit of β-glucuronidase activity corresponds to 1 nmol of substrate (X-Gluc) hydrolyzed min⁻¹ mg protein⁻¹. Data are averages for two independent experiments. Error bars represent standard deviations.