Observations of the regulation of production of this surfactant in various culture conditions suggest a role for this surfactant primarily in fully hydrated environments. Its coordinated expression with flagella suggests an intimate role between surfactant production and flagellar motility, but the identification of many other regulatory elements reveals a complicated mechanism of regulation. Examinations of the interaction of this surfactant with the bacterial cell, its flagella, and with the surfaces that this bacterium colonizes should illuminate its role in the epiphytic lifestyle of P. syringae. Biosurfactants, or biologically-produced surface active agents, are a broad group of natural amphiphilic compounds that are capable of lowering the interfacial tension between two phases. Although many different types of natural products have been identified to function as biosurfactants, one of the most intriguing classes of these compounds is the lipopeptides. Lipopeptides have a peptide head group attached to a lipid tail, and the peptide moiety is unique in that it is synthesized non-ribosomally; ie, it is not translated from an mRNA. Rather, lipopeptides are generally synthesized by non-ribosomal peptide synthetases , large enzyme complexes that catalyze the sequential assembly of a small peptide, as well as direct the addition of a carbon tail. Additionally, these assembly units can specify the incorporation of unusual amino acids,ebb and flow bench modifications, and/or cyclization. Because of this flexibility and ability to create unique peptides, NRPSs have found widespread use in bacteria and fungi for the synthesis of a variety of compounds including antibiotics, siderophores, pigments and many others.
NRPSs specific for lipopeptide synthesis contain an initial condensation domain that catalyzes tail addition to the first amino acid of the peptide, and thus can be readily identified bioinformatically in genomes. The most familiar example of a lipopeptide is that of surfactin produced by Bacillus subtilis. This cyclic lipopeptide is composed of seven amino acids linked to a 12- to 16-carbon tail; the seven amino acids being somewhat variable in composition due to the low fidelity of many NRPSs. Although generally linked to biofilm formation and motility, surfactin has recently been revealed to act as an autoinducer signal, wherein surfactin production is sensed by non-surfactin producing cells, inducing them to produce an extracellular matrix. Other Bacillus lipopeptide families include the iturins and fengycins, while Pseudomonads have been found to produce an even broader range of lipopeptides. Two of the most extensively studied Pseudomonas lipopeptides are syringomycin and syringopeptin produced by Pseudomonas syringae, which have been noted for their membrane-disrupting and resultant phytotoxic properties. These cyclic lipopeptides contain 9 and 22 to 25 amino acids, respectively, and contribute to the virulence of this microorganism. Recently, production of syringafactin, an 8-amino acid linear lipopeptide, was also described in P. syringae DC3000 and B728a. With the continued identification of new lipopeptides and the sequencing of their genetic loci, an interesting pattern has emerged; many of the NRPSs for lipopeptide production in Pseudomonads possess divergently transcribed LuxR-type regulators both upstream and often also immediately downstream, of the biosynthetic cluster. When they have been characterized, disruptions in the upstream and sometimes downstream regulator results in blockage of lipopeptide production. These LuxR-type regulators have a characteristic C-terminal helix-turn-helix DNA-binding region, but form a distinct family separate from other characterized LuxR-type regulators.
Classic LuxR homologs have an autoinducer-binding domain, while other LuxR-type response regulators have receiver domains typical of two-component systems.However, the NRPS-associated LuxR-type regulators have neither domain, and thus form their own group of regulators. SalA and SyrF are the best characterized of these LuxR-type NRPS regulators; they control and are located in close proximity to the NRPS loci for syringomycin and syringopeptin in P. syringae B301D. They have been shown to dimerize, and that a dimerized SalA binds the promoter region upstream of syrF, while dimerized SyrF binds to the promoter region of syringomycin. Thus, similar to V. fischeri LuxR, they become active after forming a multimeric complex. However, while LuxR must first bind an autoinducer to dimerize and become an activate transcription factor , it is unclear what if any factors contribute to the activation of the NRPS associated LuxR-type regulators. In addition to SalA and SyrF, a third LuxR-type regulator, SyrG, also exhibits partial control over syringomycin synthesis in P. syringae, although it operates independent of SalA and SyrF. Furthermore, P. syringae B728a possesses two additional regulators of this type , which flank the syringafactin biosynthetic cluster on both sides. SyfR, the regulator physically upstream of the cluster, was previously demonstrated to be required for syringafactin production in P. syringae DC3000. However, the LuxR homolog downstream of the syringafactin biosynthetis cluster had no effect on syringafactin production when deleted, and remains unnamed. No further characterization of SyfR has appeared. Although it is clear that these LuxR-type regulators often control lipopeptide synthesis in Pseudomonads, there has been little investigation of how environmental signals feed into this regulation. Some plant signals have been shown to induce lipopeptide production in plant associated Pseudomonads, supporting their proposed roles in virulence. Additionally, lipopeptides are regulated in a manner dependent on quorum sensing and cell density in a few Bacillus and Pseudomonas species.
We recently found that expression of syringafactin in P. syringae is dependent on contact of cells with surfaces. The current study was undertaken to investigate the role of SyfR in such contact-dependent syringafactin production in strain B728a. We will show that SyfR controls more than syringafactin production,4x8ft rolling benches and is involved in a complex web of cross regulation between other LuxR-type regulators and other lipopeptides in P. syringae. Neither of the truncated SyfR constructs restored syringafactin production in a syfR– mutant. Either SyfR164-257 apparently did not include the correct regions of the DNA-binding domain, or SyfR has a different structural organization than LuxR. We also introduced these truncated SyfR variants into the wild-type strain to test for dominant negative interference which would indicate that SyfR forms multimers similar to SalA and SyrF. While the wild type strain expressing SyfR164-257 retained full syringfactin production, the wild-type strain constitutively expressing SyfR1-198 produced only the same size small surfactant halo as a syfR– mutant strain. Additionally, when we introduced the pPsyfA-gfp reporter fusion into these strains, we observed a similar pattern of GFP fluorescence as production of syringafactin in these strains; overexpression of the SyfR binding domain has a repressive effect on syfA transcription. This supports the hypothesis that this regulator forms a multimeric complex in order to induce syringafactin transcription. We tested the hypothesis that SyfR might be involved in conveying the preferential production of the surfactant syringafactin when cells were cultured on agar plates compared to broth cultures. Initially, we determined if constitutive expression of SyfR is sufficient to induce high levels of syringafactin production in broth culture. We grew the wild-type strain, a syfR– mutant, and a wild-type strain that over-produced SyfR by expressing syfR constitutively on the plasmid p519n–syfR, in both plate and broth conditions, and tested for surfactant production by the drop collapse method. Similar to the oil spray assay depicted in Figure 1, the water drop collapse assay indicated that relatively large quantities of syringafactin were produced in both the wild type and wild-type harboring plasmid p519n-syfR strains on agar plates, while a syfR– mutant was deficient in surfactant production. In contrast, while syringafactin production was low in a wild-type strain when these strains were grown in shaken broth cultures, constitutive expression of SyfR induced sufficient syringafactin production to enable drop collapse under these culture conditions. This suggested that low levels of SyfR might be responsible for the low levels of syringafactin production seen in broth cultures. We thus hypothesized that the surface regulation of syringafactin is at least in part mediated by SyfR.We determined if the apparently low levels of SyfR in broth culture stemmed from low levels of syfR transcription. To test this model we constructed a bioreporter in which a gfp reporter gene was expressed under the control of the promoter of syfR in plasmid pPsyfR-gfp. When a wild type strain carrying pPsyfR-gfp was grown in broth media, apparent syfR transcription was about 3-fold lower than when grown on agar plates.
As a control, similar levels of GFP fluorescence were observed in a strain constitutively expressing the gfp reporter gene in these two culture conditions. It should be noted that rates of syfA transcription itself were more than 10-fold higher in cells cultured on agar plates compared to broth. We attribute the larger effect of broth culture on syfA expression than on expression of its regulator syfR as a consequence of the strong concentration dependence of oligomerization of SyfR that would be expected to contribute to its activation. We investigated the possibility that syfR is subject to autoregulation in P. syringae since LuxR induces its own expression at least 2- to 3-fold compared to that in luxR– mutant strains. Similarly, constitutive SalA expression results in a 2- to 3-fold upregulation of salA. It is noteworthy that this range of autoregulation is of the same magnitude as the differences in syfR transcription observed between broth and plate cultures. Therefore, we investigated the transcription of syfR in the absence of functional SyfR protein. Surprisingly, we observed equally low GFP fluorescence of a syfR– mutant strain harboring pPsyfR-gfp cultured on both agar plates and in broth media. This finding suggested two important points. First, it suggests that SyfR is autoregulated, and is necessary for the induction of its own transcription above a low baseline level. Second, it suggests that the surface regulation of both syringafactin production and SyfR abundance are conferred by a post-transcriptional process that affects SyfR levels or activity. Thus, we hypothesize that broth culture conditions reduce the magnitude of SyfR autoregulation, either through degradation of the syfR transcript or SyfR itself, or by alteration of SyfR. Further biochemical experimentation will be necessary to determine the mode of this control. If broth conditions foster the hypothesized destruction or modification of SyfR, then we might expect that constitutive production of SyfR would nonetheless result in lower promoter induction of syfR and syfA in broth cultures compared to growth on agar plates. We earlier observed that constitutive expression of SyfR enabled syringafactin production even in broth culture, but we did not examine syfA expression per se. Apparent syfA expression in broth culture, as estimated with the plasmid pPsyfA-gfp introduced into a strain constitutively expressing SyfR, was slightly below that observed on agar plates , which might lend support to our hypothesis. However, the promoter activity of syfR in a strain with constitutive expression of SyfR wasslightly higher in broth cultures than in cells recovered from agar plates. We have no explanation for why syfA expression was lower in broth cultures than on agar plates while syfR was higher. Further biochemical work might help elucidate any additional factors that contribute to syfA regulation. Nonetheless, the observation that constitutive expression of SyfR results in a further up-regulation of syfR further supports our claim that SyfR is autoregulated. A test of the self-sufficiency of the autoinduction process of syfR would be to demonstrate that SyfR is sufficient for syfA induction in another bacterial taxa that might lack ancillary components found only in P. syringae. Introduction of luxR from V. fischeri along with its regulated bioluminescence-encoding operon resulted in expression of of bioluminescence in E. coli. We sequentially transformed E. coli strain DH5α with both p519n-syfR and pPsyfA-gfp.This indicates either that additional transcription factors are necessary for syfA transcription, that processing or some unknown activation of SyfR cannot occur in E. coli, that these components were not efficiency transcribed in this E. coli host, or that SyfR does not directly regulate syfA. Additional investigation to distinguish these possibilities is warranted. We investigated the possibility that SyfR functions downstream from other global regulators in P. syringae. There have been multiple reports that the GacA/GacS two-component regulatory system controls lipopeptide production. In P. syringae, it has been further demonstrated that GacA/S controls lipopeptide production through its regulation of SalA. We hypothesized that Gac might also control syringafactin production, and thus tested surfactant production in a ∆gacS deletion mutant using the atomized oil assay as well as determining the expression of various genes involved in syringafactin production using transcription reporters.