Regulation of the sulfate starvation response in Pseudomonas aeruginosa: role of cysteine biosynthetic intermediates

Abstract

Pseudomonas aemginosa PA01 grew in defined synthetic medium with any of a broad variety of single sulfur sources, including sulfate, cysteine, thiocyanate, alkanesulfonates, organosulfate esters and methionine, but not with aromatic sulfonates, thiophenols or organothiocyanates or isothiocyanates. During growth with any of these compounds except sulfate, cysteine or thiocyanate, a set of I 0 sulfate starvation-induced (SSI) proteins was strongly upregulated, as observed by two-dimensional protein electrophoresis of total cell extracts. A comparable level of up-regulation was found for the hydrolytic enzyme arylsulfatase, which has previously been used as a marker enzyme for the sulfate starvation response. One of the SSI proteins was identified by N-terminal sequencing as a high-affinity periplasmic sulfate-binding protein, and another was related to thiol-specif ic antioxidants, but the N-terminal sequences of the other SSI proteins revealed no similarity to N-termini of proteins of known function, and they probably represent uncharacterized enzymes involved in sulfur scavenging when preferred sulfur sources are absent. T o study the role that cysteine biosynthetic intermediates play in the synthesis of these proteins in wiwo, we isolated mini-Tn5 transposon mutants of P. aeruginosa with insertions in the cysN and cysl genes, which encode subunits of ATP-sulfurylase and sulf ite reductase, respectively. These two genes were cloned and sequenced. cysl showed high similarity to the cognate gene in Escherichia coli, whereas cysN encoded a 693 kDa protein with two domains corresponding to the E. coli CysN and CysC proteins. Sulfate no longer repressed synthesis of the SSI proteins in cysN mutants, but repression was restored by sulfite; in the cysl mutant, sulfate, sulfite and sulfide all led to repression of SSI protein synthesis. This suggests that there are at least t w o independent corepressors of the sulfate starvation response in this species.Keywords : Pseudornonas aeruginosa, sulfate starvation, cysteine biosynthesis INTRODUCTIONCysteine biosynthesis by the sulfate assimilation pathway has been well-characterized in both plants and micro-organisms (Kredich, 1996 ; Leustek, 1996). It Abbreviations: 551, sulfate starvation-induced; SSIS, sulfate starvationinduced sti mu Ion.The GenBank accession numbers for the sequences reported in this paper are AF026066 (cysl) and AF035608 (cysDN).proceeds by activation of inorganic sulfate, followed by reduction of the intermediate phosphoadenosyl phosphosulfate to sulfite and hence to sulfide, and transfer of this molecule onto an organic backbone. In limnic and enteric environments, sulfate is present in excess of microbial requirements, but in aerobic soils sulfur is available almost exclusively ( > 95 % ) in organically bound form, and inorganic sulfate is rare (Autry & Fitzgerald, 1990 al., 1986). This cycling is assumed to be due to microbial activity in the soil, or to microbially released soil enzymes, but the mechanisms involved in sulfur soil cycling and in th...

Construction of improved plasmid vectors for promoter characterization in Pseudomonas aeruginosa and other Gram-negative bacteria

“…This con¢rmed that although the P. aeruginosa cys promoters were recognized by the E. coli transcription machinery, correct repression by sulfate (mediated in E. coli by the CysB protein) did not take place. This corresponds with previous observations that there are signi¢cant di¡erences in regulation of cysteine biosynthesis between these two species [4]. The quantitative use of pME4507 and pME4510 as promoter-probe vectors was evaluated with two di¡erent approaches.…”

“…L-Galactosidase activity in the absence of a promoter sequence was found to be extremely low (Table 1), and pure white colonies were observed after growth on plates containing X-Gal/IPTG. The use of the two vectors for qualitative promoter testing was assessed by cloning PCR fragments carrying the promoter regions of two cysteine biosynthesis genes from P. aeruginosa, cysI (a 457-bp fragment including 173 bp upstream of the cysI translation start site) and cysD (a 789-bp fragment containing 701 bp upstream of the cysD translation start site) [4], into the SmaI site of pME4507, and transforming these constructs into E. coli or P. aeruginosa. These promoters were chosen for testing because genes of sulfur metabolism are often expressed at low levels, and are subject to repression in the presence of inorganic sulfate.…”

“…The strains were routinely grown aerobically in Luria^Bertani medium at 37³C. When a de¢ned medium was required, P. aeruginosa was grown on a sulfate-free succinate-salts medium, with sulfur sources added as described in the text [4]. Antibiotics were added to the growth medium at the following concentrations (per ml): ampicillin, 100 Wg for E. coli; tetracycline, 25 Wg for E. coli and 125 Wg for P. aeruginosa; gentamycin, 15 Wg for E. coli and 200 Wg for P. aeruginosa; streptomycin (500 Wg) and carbenicillin (300 Wg) for P. aeruginosa.…”

Sulfur-limitation-regulated proteins in Bacillus subtilis: a two-dimensional gel electrophoresis study

“…Random lacZ fusions and 2D gel electrophoresis revealed a specific increase in the synthesis of several proteins, such as oxygenases (TauD, SsuD), high-affinity uptake systems (ATP-binding cassette -ABC -transport systems, sulfate-and cystine-binding proteins), alkyl hydroperoxide reductase (AhpC), O-acetylserine lyase A (CysK), and of several unidentified proteins (Kertesz et al, 1993 ;Quadroni et al, 1996 ;van der Ploeg et al, 1996. Several proteins induced under sulfatestarvation conditions have also been identified in Pseudomonas aeruginosa (Hummerjohann et al, 1998 ;Kertesz et al, 1999 ;Quadroni et al, 1999).…”

Bioavailability of water-polluting sulfonoaromatic compounds

“…The detail of the regulation of these desulfonative enzymes is still emerging (Gallardo et al 1997;Hummerjohann et al 1998;Kertesz et al 1994a), but preliminary evidence for this regulation in nature is clear-cut (Mazel and MarlieÁ re 1989). The combination of desulfonation and the generalisation that compounds without the sulfonate group are much more likely to be generally bioavailable (Wellens 1990) suggests that, in the long term, waste streams and leachates containing sulfonates may become more amenable to biotreatment.…”