Role for ferredoxin:NAD(P)H oxidoreductase (FprA) in sulfate assimilation and siderophore biosynthesis in Pseudomonads

Abstract

Pyridine-2,6-bis(thiocarboxylate) (PDTC), produced by certain pseudomonads, is a sulfur-containing siderophore that binds iron, as well as a wide range of transition metals, and it affects the net hydrolysis of the environmental contaminant carbon tetrachloride. The pathway of PDTC biosynthesis has not been defined. Here, we performed a transposon screen of Pseudomonas putida DSM 3601 to identify genes necessary for PDTC production (Pdt phenotype). Transposon insertions within genes for sulfate assimilation (cysD, cysNC, and cysG [cobA2]) dominated the collection of Pdt mutations. In addition, two insertions were within the gene for the LysR-type transcriptional activator FinR (PP1637). Phenotypic characterization indicated that finR mutants were cysteine bradytrophs. The Pdt phenotype of finR mutants could be complemented by the known target of FinR regulation, fprA (encoding ferredoxin:NADP(+) oxidoreductase), or by Escherichia coli cysJI (encoding sulfite reductase). These data indicate that fprA is necessary for effective sulfate assimilation by P. putida and that the effect of finR mutation on PDTC production was due to deficient expression of fprA and sulfite reduction. fprA expression in both P. putida and P. aeruginosa was found to be regulated by FinR, but in a manner dependent upon reduced sulfur sources, implicating FinR in sulfur regulatory physiology. The genes and phenotypes identified in this study indicated a strong dependence upon intracellular reduced sulfur/cysteine for PDTC biosynthesis and that pseudomonads utilize sulfite reduction enzymology distinct from that of E. coli and possibly similar to that of chloroplasts and other proteobacteria.

Fig 1

Predicted sulfur transfer steps in PDTC biosynthesis. The three steps depicted are (i) cysteine desulfurylase activity attributed to PdtF, an autosulfurylation forming persulfide-containing, modified PdtF; (ii) acyl activation; and (iii) transulfurylation activities, giving 6-(monothiocarboxylic acid)-picolinic acid. A second cycle of activation/transulfurylation of that product would give PDTC. Gene products (PdtF, PdtJ, PdtG, and PdtH) catalyzing the respective steps are denoted by boxes within or below each reaction.

Fig 2

Genome segments and transposon insertions characterized. (A) Class I mutants; (B) class II mutants and P. putida and P. aeruginosa genomic segments analyzed involving the finR-fprA locus. Flag symbols indicate positions and orientations of mini-TnXylEKm insertions in respective mutants, with strain designations listed above. Half arrows above genes indicate the positions of PCR primers used to amplify genome segments for cloning/complementation.

Fig 3

P. putida DSM 3601 fprA expression is dependent on sulfur source, as well as finR expression. Northern analysis of strains grown on M9 succinate medium with cystine (C) or sulfate (S) as sole sulfur sources. Strain genotypes are: DSM 3601, wild type; BK8, ΔpvdS (ΔpfrI) (pyoverdine-negative); SO3B9, finR::mini-Tn5; TA690, ΔfinR::Gm^r (gentamicin resistance cassette oriented toward fprA); TA691, ΔfinR::Gm^r (gentamicin resistance cassette oriented opposing fprA). Upper portion, ethidium-stained gel. Lower portion, Northern blot hybridized with fprA probe. 4.5 μg of RNA was loaded per lane.

Fig 4

Growth of finR mutants with sulfate or cystine as sole sulfur sources. (A) M9 succinate medium with 1 mM sulfate as sulfur source; (B) M9 succinate medium with 0.5 mM cystine as sulfur source. Symbols: ○, DSM 3601 (WT); ◆, BK8 (Pvd⁻ parental); ▲, TA690 (ΔfinR::Gm^r-forward); ■, TA691 (ΔfinR::Gm^r-reverse); ●, SO3B9 (finR::mini-Tn5KmxylE). The data are means of triplicate cultures. Error bars represent standard deviations.

Fig 5

Reduced source of sulfur suppresses paraquat-induced fprA expression by P. putida DSM 3601. Northern analysis of control cultures grown on M9 succinate medium with sulfate as the sole sulfur source (S) or sulfate plus l-cysteine (Cys), l-cystine (L-C), or d-cystine (D-C). Paraquat-treated cultures (PQ) were exposed to 1 mM paraquat for 20 min with the respective sulfur supplements prior to RNA extraction. Upper portion, ethidium-stained gel. Lower portion, Northern blot hybridized with fprA_Pp probe. A total of 6 μg of RNA was loaded per lane.

Fig 6

P. aeruginosa shows finR-dependent fprA expression which is responsive to reduced sulfur sources. (A) Northern analysis of cultures of wild type (MPA01) or finR deletion mutant (Δ3398) grown with sulfate as the sole sulfur source (S), or sulfate plus l-cystine (C). Paraquat-treated cultures (PQ) were exposed to 1 mM paraquat for 10 or 30 min with the respective sulfur supplements prior to RNA extraction. Upper portion, ethidium-stained gel; lower portion, Northern blot hybridized with an fprA_Pa probe. A total of 10 μg of RNA was loaded per lane. (B) Complementation analysis of the finR deletion strain Δ3398. Cultures were grown with sulfate as the sole sulfur source. The expression of a chromosomal luxCDABE operon fused to the PfprA_Pa promoter was monitored by luminescence. Left, growth; right, luminescence versus cell density (RLU, relative luminescence units). Symbols: ◆, MPA01; ▲, Δ3398; □, Δ3398/pMF54 (vector control); ■, 3398/pMF418 (plasmid-borne finR_Pa); dashed line, MPA01::Tn7lux control (no promoter). (C) Expression of P_fprA-lux reporter by wild-type P. aeruginosa during growth with various sole sulfur sources. Symbols: ◆, sulfate; □, sulfite; ▲, cystine; ●, cysteine. (D) Expression of P_fprA-lux reporter by wild type and ΔfinR mutant strains in response to paraquat and/or cysteine. Exponential-phase cells grown on minimal medium with sulfate as sulfur source were diluted into the same medium with or without additions of 1 mM paraquat or 0.5 mM cysteine. Left, wild type (MPA01); right, ΔfinR. Symbols: □, no addition; ■, 1 mM paraquat; △, cysteine; ▲, cysteine plus 1 mM paraquat.

Fig 7

Pathway of sulfate assimilation/cysteine biosynthesis in enterics versus pseudomonads. Intermediates of cysteine biosynthesis are shown in bold. Gene products with relevant enzymatic activities (CysDN, CysC, etc.) are given below or beside the respective steps in boxes. Abbreviations: APS, adenosine phosphosulfate; PAPS, phosphoadenosine phosphosulfate; OAS, O-acetylserine. The negative allosteric regulation of CysE activity by intracellular cysteine, and the requirement for N-acetylserine (a product of spontaneous rearrangement of OAS) to induce sulfate assimilatory gene expression (via CysB), affords coordination between serine activation and sulfate activation/reduction activities in response to metabolic demand for cysteine (46).