Coregulation of the cyclic lipopeptides orfamide and sessilin in the biocontrol strain Pseudomonas sp. CMR12a

Abstract Cyclic lipopeptides (CLPs) are synthesized by nonribosomal peptide synthetases (NRPS), which are often flanked by LuxR‐type transcriptional regulators. Pseudomonas sp. CMR12a, an effective biocontrol strain, produces two different classes of CLPs namely sessilins and orfamides. The orfamide biosynthesis gene cluster is flanked up‐ and downstream by LuxR‐type regulatory genes designated ofaR1 and ofaR2, respectively, whereas the sessilin biosynthesis gene cluster has one LuxR‐type regulatory gene which is situated upstream of the cluster and is designated sesR. Our study investigated the role of these three regulators in the biosynthesis of orfamides and sessilins. Phylogenetic analyses positioned OfaR1 and OfaR2 with LuxR regulatory proteins of similar orfamide‐producing Pseudomonas strains and the SesR with that of the tolaasin producer, Pseudomonas tolaasii. LC‐ESI‐MS analyses revealed that sessilins and orfamides are coproduced and that production starts in the late exponential phase. However, sessilins are secreted earlier and in large amounts, while orfamides are predominantly retained in the cell. Deletion mutants in ofaR1 and ofaR2 lost the capacity to produce both orfamides and sessilins, whereas the sesR mutant showed no clear phenotype. Additionally, RT‐PCR analysis showed that in the sessilin cluster, a mutation in either ofaR1 or ofaR2 led to weaker transcripts of the biosynthesis genes, sesABC, and putative transporter genes, macA1B1. In the orfamide cluster, mainly the biosynthesis genes ofaBC were affected, while the first biosynthesis gene ofaA and putative macA2B2 transport genes were still transcribed. A mutation in either ofaR1, ofaR2, or sesR genes did not abolish the transcription of any of the other two.

The LuxR superfamily consists of transcriptional regulators that contain a DNA-binding helix-turn-helix (HTH) motif in the C-terminal region (Fuqua, Winans, & Greenberg, 1996). In this superfamily, four subfamilies can be distinguished based on domain architecture and the mechanism of regulatory activation. LuxR-like proteins SalA, SyrF, and SyrG are a part of the fourth subfamily, which is characterized by the lack of any defined N-terminal domain. These proteins have been associated with the regulation of the CLPs syringomycin and syringopeptin in Pseudomonas syringae pv. syringae, a plant pathogenic bacterium (Vaughn & Gross, 2016). In various other Pseudomonas species and strains, regulatory genes encoding similar LuxR-like proteins are positioned up-and downstream of the CLP biosynthesis genes (De Bruijn & Raaijmakers, 2009a). Within several CLP families, the regulation of CLP biosynthesis has been attributed to LuxR-type regulators including PsoR (putisolvin) in P. putida (Dubern et al., 2008), ViscA and ViscBC XtlR (xantholysin) in P. putida BW11M1 , and PcoR and RfiA (corpeptin) in P. corrugata CFBP 5454 (Strano et al., 2015).
In several Pseudomonas strains, the principal regulator of CLP biosynthesis is the GacA/GacS two-component system since a mutation in one of both encoding genes leads to a loss in CLP production (De Bruijn & Raaijmakers, 2009a). The GacA/GacS system is known to activate small RNAs that bind to and sequester translational repressor proteins, which block the ribosomal binding sites in the mRNA of Gac-regulated genes. Two small RNAs (sRNAs) and two repressor proteins, RsmA and RsmE, have been linked to the regulation of entolysin (Vallet-Gely et al., 2010) and massetolide A biosynthesis (Song, Voort, et al., 2015). In the massetolide producer P. fluorescens SS101, these repressor proteins most likely block translation of the LuxR-type transcriptional regulator, MassAR (Song, Voort, et al., 2015), by binding to a specific site called the GacA box. This site comprises a nontranslated leader sequence upstream of the AUG codon on the messenger RNA.
In several CLP-producing Pseudomonas strains, a GacA box is present upstream the LuxR regulators flanking the CLP biosynthesis gene cluster suggesting that other CLP-producing Pseudomonas strains may show a similar regulation of lipopeptide biosynthesis (Song, Voort, et al., 2015).
In CMR12a, sessilin biosynthesis is governed by three linked NRPS genes namely sesA, sesB, and sesC ( Figure 1a) (D'aes et al., 2014). These genes are flanked upstream by a nodT-like gene designated sesT, and downstream by macA1 and macB1 genes, which are probably involved in sessilin secretion. MacA and MacB are part of a tripartite secretion system involving an inner membrane protein (MacB), a periplasmic adaptor protein (MacA), and an outer membrane protein (NodT). Similar to sessilin, orfamide biosynthesis is governed by three linked NRPS genes namely ofaA, ofaB, and ofaC (Figure 1b) (D'aes et al., 2014). MacA-and macB-like genes putatively involved in orfamide secretion are located downstream of ofaC. Intriguingly, there is no nodT-like gene in the orfamide gene cluster of Pseudomonas sp.
CMR12a, while this gene is present in the orfamide gene clusters of P.
protegens isolates (Ma, Geudens, et al., 2016). In addition, a LuxR-type regulatory gene, ofaR1, is located upstream of the orfamide biosynthesis cluster and a second one, ofaR2, is situated downstream of the macA2B2 genes, whereas a single LuxR-type regulatory gene, sesR, is located upstream of the sessilin biosynthesis cluster next to the sesT gene (D'aes et al., 2014).
In this study, we hypothesized that in Pseudomonas sp. CMR12a, OfaR1 and OfaR2 regulate the biosynthesis of orfamides, whereas SesR is vital for sessilin biosynthesis. To test our hypothesis, sitedirected mutagenesis of the corresponding genes was conducted followed by biochemical and transcriptional analyses.

| Bacterial strains and culture conditions
Bacterial strains, plasmids, and primers used in this study are listed in Table 1. Pseudomonas sp. CMR12a was cultured on Luria-Bertani (LB) agar plates or in liquid LB broth at 28°C. All molecular techniques were performed using standard protocols (Sambrook, Frithsch, & Maniatis, 1989). Escherichia coli strains were grown on LB agar plates or LB broth amended with appropriate antibiotics. Saccharomyces cerevisiae InvSc1 was cultivated on yeast extract-peptone-dextrose (YPD) (Shanks, Caiazza, Hinsa, Toutain, & O'Toole, 2006). Escherichia coli strain WM3064 was used as a host for the plasmids used in sitedirected mutagenesis.

| Analysis of CLP production
For LC-ESI-MS analyses, bacterial strains were grown at 28°C in sixwell plates with 2.5 ml LB broth per well. Cultures were maintained for variable time periods after which 1 ml of each was centrifuged at 18,900g for 4 min. Filter-sterilized supernatants were subjected to reverse-phase LC-ESI-MS as described by D'aes et al. (2014). Cells obtained after the centrifugation step were washed once with sterile distilled water resuspended in 1 ml of acetonitrile solution (50%) after which sonication was carried out for 30 s. Following centrifugation, the cell supernatant was filter sterilized and subjected to LC-ESI-MS analysis. Data generated from supernatant and cell analyses were processed to either extract chromatograms or obtain the relative production of sessilins and orfamides using the MassLynx V4.1 software.

| Site-directed mutagenesis
Site-directed mutagenesis of the ofaR1, ofaR2, and sesR genes was performed based on methods described previously (D'aes et al., 2014). To construct each mutant, a fragment of the corresponding LuxR biosynthesis gene was deleted by allelic replacement with vector pMQ30 (Shanks et al., 2006). Primers used for polymerase chain reaction (PCR) and plasmids are described in Table 1. To obtain a deletion plasmid, two coding regions of each LuxR gene were amplified by PCR and these products were cloned next to each other by homologous recombination in S. cerevisiae InvSc1. This plasmid was mobilized into CMR12a by conjugation with E. coli WM3064 and selection on gentamycin. Subsequently, transconjugants that had lost the plasmid during the second crossover event were selected on LB with 10% sucrose after which gene deletion was confirmed by PCR and sequencing (LGC Genomics, Germany).

| Construction of pME6032-based vectors for complementation
A fragment containing the luxR gene was obtained by PCR with specific primers (Table 1). These PCR products were subsequently cloned in the expression vector pME6032 comprising the pTac promoter.
The plasmids obtained, pME6032-OfaR1, pME6032-OfaR2, and pME6032-SesR were transformed into E. coli WM3064 via heat shock after which transformed colonies were selected on LB agar plates supplemented with tetracycline 50 μg/ml. Correct integration of fragments F I G U R E 1 Schematic representation of sessilin (a) and orfamide (b) gene clusters of Pseudomonas sp. CMR12a. On both clusters, RT-PCR amplicon positions are lettered A to X. SesT (NodT-like outer membrane lipoprotein); SesR: LuxR-type transcriptional regulator; SesD: SyrD-like ABC transporter protein; OfaR1: LuxR-type transcriptional regulator upstream of the orfamide gene cluster; OfaR2: LuxR-type transcriptional regulator downstream of the orfamide gene cluster; MacA: periplasmic membrane protein; MacB: inner membrane protein. MacA1 and MacB1: associated with the sessilin gene cluster; MacA2 and MacB2: associated with the orfamide gene cluster. MacA1/MacA2 and MacB1/MacB2 share 78% and 80% identity, respectively was verified by PCR analysis, restriction analysis of isolated plasmids, and sequencing. These three pME6032-based E. coli WM3064 vectors were transformed into the corresponding Pseudomonas sp. CMR12a LuxR mutants by conjugation. Transformed cells were selected on LB supplemented with 100 μg/ml tetracycline and the presence of pME6032-OfaR1, pME6032-OfaR2, or pME6032-SesR was confirmed by PCR analysis using primers specific for pME6032 and the corresponding luxR gene.

| White line-in-agar and swarming motility assays
The protegens Pf-5) in the middle of the plates was made from three drops

| RNA extraction and reverse transcription-PCR (RT-PCR)
Bacterial cells were grown in still cultures using a six-well plate con- cDNA with RNA equivalent of 100-200 ng was subjected to PCR with specific primers listed in Table S1. The thermal profile used consisted of an initial denaturation step at 95°C for 2 min, followed by 30 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 1 min.
The primer pairs were used to amplify cDNA obtained from transcripts corresponding to genes of the sessilin and orfamide biosynthesis gene clusters and their flanking genes including the sesT, sesR, ofaR1, ofaR2, ofaABC, sesABC, and the macAB genes. Transcripts covering adjacent gene pairs of the aforementioned genes were also amplified.

| Bioinformatic analyses
LuxR-like protein sequences for Pseudomonas sp. CMR12a were obtained from the nucleotide sequences of the sessilin and orfamide biosynthesis gene clusters with GenBank accession numbers JQ309920 and JQ309921, respectively. Other amino acid sequences used for phylogenetic analyses were collected from the National Centre for Biotechnology Information (NCBI) website. Characteristics of strains and protein sequences used in the phylogenetic analyses of LuxR proteins are presented in Table S2.
Sequence alignments were made using Muscle (Edgar, 2004)  Furthermore, bioinformatic tools were employed to check for the presence of Rsm binding sites upstream of the ofaR1, ofaR2, and sesR genes. The query search was conducted using the conserved motif 5′-A /U CANGGANG U /A-3′, where N denotes any nucleotide (Song, Voort, et al., 2015). Subsequently, similar nontranslated leader sequences flanking the LuxR transcriptional regulators of several CLPproducing Pseudomonas strains were aligned with the three LuxR regulators of Pseudomonas sp. CMR12a.

| Growth and production of sessilins and orfamides by CMR12a in shaken and still LB broth cultures
To quantify the production of sessilins and orfamides by CMR12a in shaking (150 rpm

| Functional analysis of luxR-type regulatory genes in sessilins and orfamides biosynthesis
LC-ESI-MS analysis revealed the complete abolishment of orfamide and sessilin production in the ofaR1 and ofaR2 mutants (Figure 3a).
However, the mutant in the sesR gene, located upstream of the sessilin biosynthesis cluster, still produced sessilins and orfamides.
Additionally, quantitative measurements (relative peak area/ OD 620 ) of the two CLPs did not reveal any difference between CMR12a and CMR12a-∆sesR (data not shown). Restored sessilin and orfamide production was observed in the complemented ofaR1 mutant, but not in the complemented ofaR2 mutant (Figure 3a).  CMR12a-∆ofaR2, and the complemented ofaR2 mutants no longer secrete sessilins, since they did not give the white line-in-agar interaction when challenged with the orfamide producer, P. protegens Pf-5. The white line-in-agar phenotype was observed, however, for CMR12a, CMR12a-∆sesR, and the complemented ofaR1 mutant strains (Figure 3b).   Furthermore, a mutation in either of the three LuxR-type genes of CMR12a did not appear to abolish the transcription of the other ( Figure 4A and B). OfaR1 and ofaR2 mutants appeared to show a weaker transcription of the sesD (syrD-like) gene, whereas the sesR mutant showed similar results with CMR12a ( Figure 4c).

| Phylogenetic analyses of LuxR-type regulatory proteins associated with CLP gene clusters
Phylogenetic analyses of the CLP cluster-associated LuxR-type proteins of CMR12a together with that of other Pseudomonas strains, showed several distinct clusters ( Figure 5) as follows: OfaR1 and SesR proteins clustered together with other LuxR-type regulators located upstream of CLP biosynthesis genes. Similarly, OfaR2 clustered with LuxR-type regulators located downstream of the CLP biosynthesis genes. Specifically, SesR clustered with other LuxR-type regulators within the tolaasin group, while OfaR1 and OfaR2 clustered with regulators which flank orfamide-coding genes in other Pseudomonas strains including P. protegens Pf-5 . The AHLbinding regulators of CMR12a, CmrR and PhzR, formed a separate cluster together with the LuxR of V. fischeri indicating that they belong to a separate subfamily of regulators ( Figure 5).

| Presence of Rsm binding sites upstream of LuxR transcriptional regulators
Genomic search for putative Rsm binding sites was conducted within the sequences upstream of the three luxR regulatory genes of CMR12a. Conserved GGA motifs upstream of the ATG start codon could be identified. Sequence alignment of these sequences with their homologs in CLP-producing Pseudomonas strains showed the similarity of these regions upstream of sessilins and orfamide biosynthetic gene clusters with those of previously described CLPs (Figure 6).
F I G U R E 4 RT-PCR analyses for the sessilin (a) and orfamide (b) biosynthesis gene clusters and flanking genes in CMR12a and LuxR mutants, (c) sesD (syrD-like) gene associated with the sessilins gene cluster. Bacterial cells analyzed were collected from 24 hr culture of Pseudomonas sp. CMR12a and its LuxR mutants. For each gene within the sessilin and orfamide gene clusters, the same bacterial culture was analyzed in duplicate and representative results are shown for one experiment. Agarose gel results are shown for analysis of single genes together with gene coexpression to distinguish monocistronic and polycistronic transcription. Primers used are listed in Table S1 and the amplicon positions are as indicated in Figures 1a and

| DISCUSSION
Our study revealed that the LuxR-like transcriptional regulators, OfaR1 and OfaR2, which are associated with the orfamide gene cluster not only regulate orfamide biosynthesis but also sessilin biosynthesis, while we could not find a clear function for the LuxR-like regulator, SesR, associated with the sessilin gene cluster.
LC-ESI-MS analysis revealed that orfamide and sessilin production commences concurrently in the late exponential phase, but orfamide is mainly retained inside the cell and secreted much later and in lower amounts than sessilin. We have previously shown that the release of orfamide in the environment is hampered by sessilin and hypothe- has only been demonstrated for plant pathogenic Pseudomonas bacteria. In the bean pathogen P. syringae pv. syringae B728a, three LuxR-like proteins, SalA, Syrf, and SyrG, were shown to control the biosynthesis of the CLPs syringopeptin and syringomycin (Vaughn & Gross, 2016). SalA controls the expression of both syrG and syrF (Lu, Scholz-Schroeder, & Gross, 2002). Furthermore, qRT-PCR analysis of deletion mutants in syrF and syrG showed that both genes require a functional salA gene for activation. In addition, SyrG appears to function as an upstream transcriptional activator of syrF (Vaughn & Gross, 2016 De Bruijn et al., 2007Li et al., 2013;Rokni-Zadeh et al., 2012;Vallet-Gely et al., 2010;Zachow et al., 2015).
During this study, we were unable to complement the CMR12a-∆ofaR2 mutant. Considering the fact that the macB2 gene associated with the orfamide gene cluster gave a weaker transcript than macA2 for CMR12a, it is possible that ofaR2 is transcribed from a longer transcript which spans across part of the macB2 gene. This would result in an antisense overlap that could influence the expression of macB2 by transcription attenuation (Sesto, Wurtzel, Archambaud, Sorek, & Cossart, 2012). This obviously requires further investigation.
In our study, phylogenetic analysis of LuxR-type proteins, positioned up-and downstream of the CLP gene clusters of CMR12a together with previously described CLP-associated LuxR regulators revealed that OfaR1 and SesR clustered together with known LuxR-type regulators located upstream of the CLP biosynthesis genes, whereas OfaR2 clustered with those located downstream. LuxR regulators from strains which produce similar CLPs, for example, orfamide producers P.
protegens Pf-5 and Pseudomonas sp. CMR12a, cluster together. An exception is the LuxR regulator for poaeamide, P. poae RE*1-1-14 which although shares a structural relationship with orfamide (Zachow et al., 2015), clusters with LuxR regulators of CLPs belonging to the viscosin family. The LuxR regulator (WipR) of the WLIP producer-P. reactans LMG 5329, showed a higher homology with LuxR regulators of the viscosin family compared with that of another WLIP producer-P. putida RW10S2 (Rokni-Zadeh et al., 2013). This decreased conservation suggests that the biosynthetic gene cluster of poaeamide might have evolved separately. Our results further indicate that LuxR-type regulators of CMR12a belong to the same subfamily as in other plant beneficial Pseudomonas strains including P. protegens Pf-5, P. fluorescens SS101, and P. fluorescens SBW25, which produce orfamide, massetolide, and viscosin, respectively (De Bruijn & Raaijmakers, 2009a;De Bruijn et al., 2008;. Given that LuxR transcriptional regulators of P. syringae pv. syringae cluster with all LuxR regulators analyzed during this study, our results indicate that similar to this plant pathogenic strain, these other LuxR regulators, including OfaR1, OfaR2, and SesR, belong to the fourth LuxR family which is characterized by the absence of any defined N-terminal domain (Vaughn & Gross, 2016).
During this study, a genomic search and subsequent alignment of sequences upstream of ofaR1, ofaR2, and sesR with their homologs in other lipopeptide biosynthesis genes of Pseudomonas strains, showed that Rsm binding sites were located upstream of all three luxR-like genes of CMR12a. Given the fact that this Rsm binding site, alternatively called the GacA box, was found upstream of multiple CLP biosynthesis genes (Song, Voort, et al., 2015a) in different Pseudomonas strains, our results suggest that the Gac/Rsm-mediated regulation of CLPs might be a general phenomenon in most biocontrol CLPproducing Pseudomonas spp.
In conclusion, this study establishes that sessilin and orfamide production in CMR12a are coregulated by two of the three luxR-type genes namely ofaR1 and ofaR2. Our findings show that either OfaR1 or OfaR2 can regulate the biosynthesis of these two CLPs, while the function of SesR remains unclear.