Fig 1.
Production of ARs by different A. vinelandii mutants.
(A) Staining with Fast Blue B of the ARs produced by colonies of A. vinelandii strain SW136 (Wild type), and its mutant derivatives inactivated in the genes gacA, rsmZ1, rsmA (strains SW5, SW13 and SW11) and the double mutant gacA-rsmA (SW15). The colonies were grown on Burk-Butanol (encystment induction medium) for 5 days prior to staining. (B). Quantification of the ARs produced by the mutants under the same conditions. The data represent the mean of triplicates and the error bars represent the standard deviations. Asterisks indicate statistical significance (unpaired Student's t-test) in the comparison of each mutant versus the wild type strain, *P < 0.05.
Fig 2.
Effect of different gene inactivations on arsA gene expression measured by RT-qPCR.
The level of the arsA transcripts was measured under encystment inducing conditions and it was normalized according to the level of the gyrA mRNA. The data are presented as fold change of arsA mRNA levels in mutants with gacA, rsmZ1, rsmA, and gacA-rsmA gene inactivations (strains SW5, SW13, SW11 and SW15), relative to those of the wild type strain (SW136). Determinations were made from bacterial cultures grown for 36 h in liquid BBOH medium at 30°C. The data represent the mean of triplicates and the error bars represent standard deviations. Asterisks indicate statistical significance (unpaired Student's t-test) in the comparison of each mutant versus the wild type strain, *P < 0.05.
Fig 3.
Effect of different gene inactivations on arpR gene expression measured by RT-qPCR.
The level of the arpR transcripts was measured under encystment inducing conditions and it was normalized according to the level of the gyrA mRNA. The data are presented as fold change of arpR mRNA levels in mutants with gacA, rsmZ1, rsmA, and gacA-rsmA gene inactivations (strains SW5, SW13, SW11 and SW15), relative to those of the wild type strain (SW136). Determinations were made from bacterial cultures grown for 36 h in liquid BBOH medium at 30°C. The data represent the mean of triplicates and the error bars represent standard deviations. Asterisks indicate statistical significance (unpaired Student's t-test) in the comparison of each mutant versus the wild type strain, *P < 0.05.
Fig 4.
Effect of gacA, rsmZ1, rsmA gene inactivations on the expression of the ARs biosynthetic gene arsA and its regulatory gene arpR measured using chromosomal transcriptional and translational gene fusions.
Expression levels of arsA (A) and arpR (B) were quantified as ß-glucuronidase activity of strains carrying arsA::gusA and arpR::gusA transcriptional (black bars) and translational (grey bars) gene fusions. The strains used for arsA gene expression determinations were YRR30, YRR36, YRR40 and YRR38 (Wild type and its gacA, rsmZ1 and rsmA mutant derivatives containing an arsA::gusA transcriptional gene fusion respectively); and YRR31, YRR33, YRR41, YRR39 (Wild type and its gacA, rsmZ1 and rsmA mutant derivatives containing an arsA::gusA translational gene fusion). For arpR, the strains were YRR50, YRR54, YRR58, and YRR56 (Wild type and its gacA, rsmZ1 and rsmA mutant derivatives containing an arpR::gusA transcriptional gene fusion respectively); YRR51, YRR53, YRR59 and YRR57 (Wild type and its gacA, rsmZ1 and rsmA mutant derivatives containing the arpR::gusA translational gene fusion). One unit of ß-glucuronidase corresponds to 1 nmol of substrate (X-Gluc) hydrolyzed min-1 mg Protein-1. Determinations in both panels were made from bacterial cultures grown for 36 h in liquid BBOH medium at 30°C. Error bars represent standard deviations. Asterisks indicate statistical significance (unpaired Student's t-test) in the comparison of each mutant versus the corresponding wild type strain with the same gene fusion, *P < 0.05.
Fig 5.
Binding of the RsmA protein to the mRNA of arpR.
RNA gel mobility shift assay to analyze RsmA binding to the regulatory region of the arpR mRNA. (A) Labelled RNA fragments (10 nM) containing the regulatory region of arpR were incubated with increasing concentrations of His6-RsmA (0–720 nM). (B) The binding of RsmA to the arpR RNA was further analyzed by competitive EMSA. As a negative control, a fragment containing the regulatory region of the sodA RNA was included in the RNA binding reaction. The labelled RNA fragment containing the regulatory region of arpR was mixed with 720 nM of His6-RsmA in the presence or absence of up to 200-fold excess of unlabelled specific (arpR) or non-specific (sodA) competitor RNAs. (C) RNA gel mobility shift assay of an RNA fragment (10nM) containing the regulatory region of the sodA RNA (negative control) with the His6-RsmA protein (0 and 720 nM). The RNA-protein complexes are indicated (RsmZ1-RsmA, B; and RsmZ1 free, F) and were resolved in non-denaturing 6% polyacrylamide gels.
Fig 6.
Effect of ArpR constitutive expression and acetoacetyl-CoA coinducer addition on ARs production of the mutant strains.
(A) Staining with Fast Blue B of alkylresorcinols produced by colonies of A. vinelandii SW 136 wild type strain and its mutant derivatives gacA, rsmZ1, rsmA and gacA-rsmA. All strains were transformed with plasmid pBBR-ArpR, carrying a constitutively expressed arpR gene. The colonies were grown on Petri dishes under encystment inducing conditions for 5 days. (B) Quantification of the ARs produced by the same strains also expressing ArpR from plasmid pBBR-ArpR, under the same conditions shown in (A) Asterisks indicate statistical significance (unpaired Student's t-test) in the comparison of ARs content of each mutant versus the wild type strain, *P < 0.05. (C) Effect of the addition to the medium of acetoacetyl-CoA, the coinducer of ArpR, on ARs production visualized by staining with Fast Blue B. The colonies were grown on Petri dishes under vegetative conditions for 5 days in the absence or presence of 5 μM acetoacetyl-CoA. The stains were also transformed with plasmid pBBR-ArpR.
Fig 7.
Proposed model for the control of ARs synthesis by the Gac-Rsm system in A. vinelandii SW136.
The regulatory protein GacA controls ARs synthesis by two different pathways, one dependent on the Rsm system, that regulates the expression of the transcriptional activator ArpR, and a second pathway that positively controls expression of the ars biosynthetic genes independently of these regulators. Dashed lines indicate unknown intermediates or unknown mechanism of regulation.