Transcription Factors CysB and SfnR Constitute the Hierarchical Regulatory System for the Sulfate Starvation Response inPseudomonas putida

Abstract

Pseudomonas putida DS1 is able to utilize dimethyl sulfone as a sulfur source. Expression of the sfnFG operon responsible for dimethyl sulfone oxygenation is directly regulated by a 54 -dependent transcriptional activator, SfnR, which is encoded within the sfnECR operon. We investigated the transcription mechanism for the sulfate starvation-induced expression of these sfn operons. Using an in vivo transcription assay and in vitro DNAbinding experiments, we revealed that SfnR negatively regulates the expression of sfnECR by binding to the downstream region of the transcription start point. Additionally, we demonstrated that a LysR-type transcriptional regulator, CysB, directly activates the expression of sfnECR by binding to its upstream region. CysB is a master regulator that controls the sulfate starvation response of the sfn operons, as is the case for the sulfonate utilization genes of Escherichia coli, although CysB DS1 appeared to differ from that of E. coli CysB in terms of the effect of O-acetylserine on DNA-binding ability. Furthermore, we investigated what effector molecules repress the expression of sfnFG and sfnECR in vivo by using the disruptants of the sulfate assimilatory genes cysNC and cysI. The measurements of mRNA levels of the sfn operons in these gene disruptants suggested that the expression of sfnFG is repressed by sulfate itself while the expression of sfnECR is repressed by the downstream metabolites in the sulfate assimilatory pathway, such as sulfide and cysteine. These results indicate that SfnR plays a role independent of CysB in the sulfate starvation-induced expression of the sfn operons.Sulfur, an essential element for bacterial growth, is assimilated from a range of sources. The preferred sulfur source for bacteria is inorganic sulfate, but in soil environments, sulfate may not be freely available because inorganic sulfate constitutes less than 5% of the total sulfur (1). The remainder is largely in the form of sulfate esters and carbon-bound sulfur such as peptides/amino acids and sulfonates (1). Volatile organic sulfur compounds, for example, dimethyl sulfide (DMS) and methanethiol, are also widely distributed in the environment because they are emitted from oceans (2), freshwater sediments (28, 29), and soils (26, 31). Thus, several soil bacterial species have developed a number of systems that allow the use of these organosulfur compounds.Metabolic use of these organosulfur compounds, especially alkanesulfonates, taurine, and alkanesulfate ester, has been extensively investigated in Escherichia coli and Pseudomonas spp. (reviewed in reference 19). Bacterial organosulfur assimilation is mediated by the sulfate starvation response, which involves a set of proteins synthesized by several species of bacteria when the supply of their preferred sulfur sources, such as inorganic sulfate, cysteine, and thiosulfate, is limited. These proteins are known as sulfate starvation-induced (SSI) proteins and include enzymes and transport systems for scavenging sulfur from organic compounds (19...

CysB Negatively Affects the Transcription of pqsR and Pseudomonas Quinolone Signal Production in Pseudomonas aeruginosa

“…However, because CysB appeared to bind to the pqsR-P4 fragment with low affinity, it is unclear whether this interaction is relevant in vivo or is simply due to nonspecific interactions between CysB and DNA sequences close to the pqsR transcriptional start sites. Aside from a preference for A-T-rich DNA sequences, CysB binding sites lack a strong consensus sequence (59,61), and we were unable to clearly identify specific sequences in the pqsR or cysH promoter that might be recognized by CysB. Nevertheless, these data provide evidence that both cysH and pqsR are directly regulated by CysB in P. aeruginosa.…”

“…6). Furthermore, previous studies showed that the in vitro DNA binding activity of recombinant CysB proteins from P. aeruginosa and P. putida was not affected by NAS or OAS (44,59). So while CysB seems to fulfill the same regulatory functions in P. aeruginosa, it appears to respond to different signals than those in enteric bacteria.…”

“…NAS is derived from the cysteine precursor O-acetylserine (OAS), which accumulates when there is not enough sulfur available for cysteine biosynthesis, and acts as a coinducer with CysB to stimulate sulfate uptake and cysteine production (40,50). Similarly, in P. aeruginosa and Pseudomonas putida, CysB appears to act as a master regulator of sulfur assimilation pathways although its exact regulatory functions are not clearly defined (58,59). So to examine the influence of cysteine availability, we compared the expression of pqsR=-lacZ and cysH=-lacZ fusions in response to various sulfur sources as well as OAS and NAS.…”

Bacterial transport of sulfate, molybdate, and related oxyanions

“…In this case, the inducer N-acetylserine inhibits binding of CysB to the cysB promoter and partially reverses inhibition of transcription initiation caused by CysB; this means that CysB has the ability to respond to acetylserine with either an increase or a decrease in affinity for different DNA sequences (Ostrowski and Kredich 1991). In contrast, in B. cenocepacia and Pseudomonas putida the function of CysB appears to be independent of the presence of O-acetylserine, indicating that CysB action may vary with respect to the recognition of an inducer (IwanickaNowicka et al 2007;Kouzuma et al 2008).…”

The cmaR gene of Corynebacterium ammoniagenes performs a novel regulatory role in the metabolism of sulfur-containing amino acids

“…The data indicate that the mechanism by which the CmaR protein exerts its regulatory effect is via the control of the expression of the cmaR gene, rather than by modulating the activity of CmaR through effectors, as is the case for CysR. In Burkholderia cenocepacia and P. putida, the binding of the activator to the cys genes does not require effector molecules (Iwanicka-Nowicka et al, 2007;Kouzuma et al, 2008).…”