SfnR2 Regulates Dimethyl Sulfide-Related Utilization in Pseudomonas aeruginosa PAO1

Abstract

Dimethyl sulfide (DMS) is a volatile sulfur compound produced mainly from the degradation of dimethylsulfoniopropionate (DMSP) in marine environments. DMS undergoes oxidation to form dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO 2 ), and methanesulfonate (MSA), all of which occur in terrestrial environments and are accessible for consumption by various microorganisms. The purpose of the present study was to determine how the enhancer-binding proteins SfnR1 and SfnR2 contribute to the utilization of DMS and its derivatives in Pseudomonas aeruginosa PAO1. First, results from cell growth experiments showed that deletion of either sfnR2 or sfnG, a gene encoding a DMSO 2 -monooxygenase, significantly inhibits the ability of P. aeruginosa PAO1 to use DMSP, DMS, DMSO, and DMSO 2 as sulfur sources. Deletion of the sfnR1 or msuEDC genes, which encode a MSA desulfurization pathway, did not abolish the growth of P. aeruginosa PAO1 on any sulfur compound tested. Second, data collected from ␤-galactosidase assays revealed that the msuEDC-sfnR1 operon and the sfnG gene are induced in response to sulfur limitation or nonpreferred sulfur sources, such as DMSP, DMS, and DMSO, etc. Importantly, SfnR2 (and not SfnR1) is essential for this induction. Expression of sfnR2 is induced under sulfur limitation but independently of SfnR1 or SfnR2. Finally, the results of this study suggest that the main function of SfnR2 is to direct the initial activation of the msuEDC-sfnR1 operon in response to sulfur limitation or nonpreferred sulfur sources. Once expressed, SfnR1 contributes to the expression of msuEDC-sfnR1, sfnG, and other target genes involved in DMS-related metabolism in P. aeruginosa PAO1. IMPORTANCE Dimethyl sulfide (DMS) is an important environmental source of sulfur, carbon, and/or energy for microorganisms. For various bacteria, including Pseudomonas, Xanthomonas, and Azotobacter, DMS utilization is thought to be controlled by the transcriptional regulator SfnR. Adding more complexity, some bacteria, such as Acinetobacter baumannii, Enterobacter cloacae, and Pseudomonas aeruginosa, possess two, nonidentical SfnR proteins. In this study, we demonstrate that SfnR2 and not SfnR1 is the principal regulator of DMS metabolism in P. aeruginosa PAO1. Results suggest that SfnR1 has a supportive but nonessential role in the positive regulation of genes required for DMS utilization. This study not only enhances our understanding of SfnR regulation but, importantly, also provides a framework for addressing gene regulation through dual SfnR proteins in other bacteria.

Identification of a complete dibenzothiophene biodesulfurization operon and its regulator by functional metagenomics

“…DszR and the SfnR sequences annotated in GenBank appear to be N-dependent activators of the group IV (Bush and Dixon, 2012), which lack the N-terminal regulatory domain, and appear to be constitutively active, as recently shown for SfnR2 in vivo (Lundgren et al, 2019), and for DszR in vitro ( Supplementary Fig. S3) and in vivo (Fig.…”

“…An ssuEADCBF operon, whose first gene codes for an FMN-reductase, has been reported as essential for dimethyl sulfide utilization in P. putida strain DS1, and the function of SsuE in this pathway has been compared to that of DszD in DBT desulfurization (Endoh et al, 2003b). SfnR has also been reported as an activator essential for utilization of dimethyl sulfone in P. putida DS1 (Endoh et al, 2003a) and methane sulfonate in P. aeruginosa PAO1 (Lundgren et al, 2019) as sulfur sources, and involved in transcription activation of sfnFG (in DS1) and msuED (in PAO1), coding both for an FMN-reductase and an FMNdependent monooxygenase in turn involved in dimethyl sulfone (Endoh et al, 2005) or metane sulfonate desulfonation (Kertesz et al, 1999), respectively. Therefore, an attractive hypothesis is that the DBT desulfurization pathway reported here has recruited not only the FMN-reductase from an aliphatic sulfonate biodesulfurization pathway but also a regulated N-dependent promoter and its activator.…”

MifS, a DctB family histidine kinase, is a specific regulator of α-ketoglutarate response in Pseudomonas aeruginosa PAO1

“…The metabolic versatility of P. aeruginosa is dependent on its ability to precisely detect specific metabolites in its environment and regulate gene expression accordingly [3, 15, 45–49]. Dicarboxylates are an important group of metabolites for P.…”

Utilization of L-glutamate as a preferred or sole nutrient in Pseudomonas aeruginosa PAO1 depends on genes encoding for the enhancer-binding protein AauR, the sigma factor RpoN and the transporter complex AatJQMP

“…Bacteria were grown at 37 °C to an OD 600 of ~ 0.2 and subsequently challenged with the addition of a final concentration of 20 mM of substrate: L-alanine, succinate, α-ketoglutarate, L-arginine, L-histidine, L-glutamate, L-aspartate, L-glutamine and L-asparagine. LacZ activity was measured at 1.0 and 3.0 h post addition of substrate using a microplate reader protocol as previously described [ 45 ]. Briefly, in a 96-well flat-bottom polystyrene plate (Falcon), 5.0 μL of cell culture was mixed with 45 μL of permeabilization solution (100 mM Na 2 HPO 4 , 80 μg mL − 1 CTAB, 40 μg mL − 1 deoxycholate, 2.0 mM MgSO 4 , 5.4 μL mL − 1 β-mercaptoethanol).…”