New Results

SwrA as global modulator of the two-component system DegS/U in B. subtilis

Francesca Ermoli, Giulia Vitali, Cinzia Calvio
doi: https://doi.org/10.1101/2021.05.07.443137

ABSTRACT

The two-component system DegS/U of Bacillus subtilis controls more than one hundred genes involved in several different cellular behaviours. Since the consensus sequence recognized by the response regulator DegU has not been clearly defined yet, mutations in either component have been crucial in the identification of the cellular targets of this regulatory system. Over the years, the degU32Hy mutant allele, that was supposed to mimic the activated regulator, has been commonly used to define the impact of this TCS on its regulated genes in domestic strains.

SwrA encodes a small protein essential for swarming motility and for poly-γ-glutamate biosynthesis and is only present in wild strains. Previous work indicated that SwrA is partnering with DegU~P in exerting its role on both phenotypes.

In this work, inserting a degS200Hy mutation in swrA+ and swrA- isogenic strains we demonstrate that SwrA modulates the action of DegU~P on two new phenotypes, subtilisin expression and competence for DNA uptake, with a remarkable effect on transformation. These effects cannot not be appreciated with the DegU32Hy mutant as it does not mirror the wild-type DegU protein in its ability to interact with SwrA.

INTRODUCTION

Two-component systems (TCS) are signal transduction modules common in bacteria and archaea, composed by a sensor histidine kinase and a cognate response regulator. Sensor kinases auto-phosphorylate themselves on a histidine residue in response to specific environmental signals and then transfer the phosphate group to a specific aspartic acid residue of the regulator inducing a structural rearrangement that enables it to modify its DNA binding properties and regulate gene expression. Moreover, sensor kinases can often quench spurious signals by dephosphorylating their cognate regulators (1). The DegS/U TCS, composed by the cytoplasmic DegS kinase and the DegU transcription factor, is involved in the regulation of several important physiological pathways of Bacillus subtilis, among which flagella-mediated motility, degradative enzyme synthesis, genetic competence, and sporulation (2). The extremely wide impact of this TCS has been evidenced through several transcriptional profiling experiments (3–5). A particular class of mutations in either DegS or DegU leads to the hyperproduction of several degradative enzymes, including the aprE-encoded protease subtilisin, and has therefore been named “Hy” (6–8). Besides promoting the synthesis of several degradative enzymes, the pleiotropic Hy mutations also cause the so called Hy phenotype which includes loss of DNA competence, absence of flagella, sporulation in the presence of glucose and elongated cell morphology (6, 9, 10). A Hy phenotype is also observed when two small proteins, DegQ and DegR, are overexpressed. They are both involved in the DegS/U signalling pathway: DegQ stimulates the transfer of the phosphate moiety from DegS to DegU (5), while DegR stabilizes DegU~P by preventing DegS-mediated DegU~P dephosphorylation (11). The overexpression of DegQ naturally occurs in wild B. subtilis strains, thanks to a nucleotide change in the −10 box of its promoter that leads to 10-fold increase in transcription with respect to domestic strains (12, 13); however, the degQHy mutation present in undomesticated strains only generates a mild phenotype, as these strains, differently from degS/UHy mutants, do not copiously produce γ-PGA (see below).

Among the number of originally isolated Hy mutants (6), subsequent studies have heavily relayed on degU32Hy, a particular degU allele which carries an A-to-T transversion at nucleotide 35 of the degU ORF, leading to a His to Leu amino acid change at position 12 (14). In early studies, all DegU32Hy phenotypes matched those obtained with the DegS200Hy mutant, in which the Gly to Glu mutation at position 218 of DegS impairs the phosphatase activity of the sensor kinase, thus leading to the accumulation of DegU~P (15). From the perfectly overlapping phenotypes of the two mutants, DegU32Hy has been since considered as a constitutively active proxy of DegU~P, without any further structural characterization. Only recently such interpretation has been challenged, thanks to the introduction of a new player, SwrA (16).

SwrA is also a small protein, 117 aa, which has been discovered thanks to its fundamental role in swarming motility (17). Its existence remained long undisclosed due to the fact that domestic B. subtilis strains only encode a non-functional 13 aa truncated peptide because of the insertion of an extra adenine in a poly-A tract in swrA ORF causing a frameshift mutation (17–19). This type of mutations can easily flip back to the wild-type form (wt) and vice versa with the frequency of a phase-variation event (17). It is thus frequent to obtain mixed swrA+/- populations even in laboratory strains upon prolonged incubations. When functional, SwrA stimulates flagella production through its activity at the Pfla/che promoter, thereby promoting sigD transcription which also permits efficient cell septation (16, 19–21). Although initially confined to motility regulation, the role of SwrA was also shown to be essential for the expression of the otherwise-silent pgs operon, encoding the enzymes required for the synthesis of the biopolymer poly-γ-glutamic acid (γ-PGA) (22). Interestingly, γ-PGA production not only strictly depends on SwrA but also on a Hy mutation in either DegQ (23) or DegU/S (24). This was the first evidence of a strict connection between SwrA and the DegS/U TCS.

Further genetic evidence of the link between SwrA and the DegS/U was gained by studies on the fla/che operon, in which the direct interaction between DegU~P and SwrA was demonstrated genetically and biochemically. Genetically, it was observed that while degU32Hy and degS200Hy mutants completely suppress Pfla/che expression in laboratory strain lacking SwrA, leading to the classically non-flagellated Hy-phenotype (6, 10), the restoration of a functional swrA allele leads to hyperflagellation in degS200Hy mutants as well as in degS/Uwt backgrounds, although not in degUS32Hy mutants (16). Biochemically, it was shown that, in electro mobility shift assays, the DegU~P-bound fla/che promoter is super-shifted in the presence of SwrA while DegU32Hy is not; moreover, DegU32Hy does not require phosphorylation for DNA binding (16). The physical interaction between DegU and SwrA was also evidenced in other studies (25). Ultimately, the impact of SwrA on motility is to remarkably turn the Pfla/che repressive effect of DegU~P, naturally produced or induced by a degS200Hy mutation, into a transcriptional boost, thus allowing swarming motility (16, 21). This dramatic overturn of DegU~P impact on the motility operon could not be appreciated in domestic strains, because of the absence of SwrA and in wild strains if the degU32Hy allele is used as a proxy of DegU~P (26).

Although the above data suggest that SwrA does not interact with DegU32Hy, this is not always true. Indeed, γ-PGA production is induced by SwrA in the presence of either degU32Hy or degS200Hy (24). However, a deep characterization of the differential impacts of the two Hy alleles on Ppgs has yet to be conducted.

In competence, Hy mutants have been shown to have a negative impact on the overall process in laboratory strains. However, degS/U null mutants were also shown to be non-competent, suggesting the requirement of this TCS in the pathway (9). The current model is the following: DegU~P has a negative impact on comS, while unphosphorylated DegU is required, possibly because it mediates the binding of ComK to its own promoter (27). More recently, the degQHy allele was shown to negatively affect comS and comK expression in both domestic and wild strains (28).

In this work we demonstrate that SwrA heavily impacts not only motility and γ-PGA production but also other DegS/U regulated behaviours; SwrA positively modulates DegS/U activity in competence for transformation and reduces aprE transcription. Moreover, we characterized the differential influence of degU32Hy and degS200Hy mutations on pgs transcription. Finally, we once more demonstrate that the degU32Hy allele encodes a constitutively active mutant protein whose activity dramatically differs from the phosphorylated DegU~P protein. Our results suggest that, as it happened in the past for motility, the use of this mutant may lead to misleading interpretations of the real physiological role of DegS/U TCS in B. subtilis physiology.

RESULTS

SwrA and motility in undomesticated strains

Our previous work showed that SwrA acts by subverting the impact of DegU~P on the fla/che promoter, transforming its action into a positive boost on flagellar gene expression. The functional interaction SwrA-DegU~P only occurs with the wild-type phosphorylated form of the response regulator, while the DegU32Hy mutant protein does not effectively interface with SwrA at this promoter (16). To generalize this effect also to undomesticated strains, either degU32Hy or degS200Hy mutation was introduced in the transformable com/Q12L mutant of the undomesticated NCIB3610 (29). The introduction of the DegU32Hy mutation caused a complete loss of motility, as already shown by Stanley-Wall and collaborators (26). Conversely, the degS200Hy derivative of the undomesticated strain proficiently swam and swarmed, paralleling the results obtained in domestic strain (Fig. S1). The only difference with domestic strains is the presence of a well-defined “lump” of γ-PGA that can be observed in the central part of the degSHy plates in Fig. S1. This characteristic is due to the abundant production of the polymer in DegS200Hy mutants, which is much higher than in the degU32Hy background. Interestingly, γ-PGA production was never visible in degS/Uwt undomesticated strains, although they naturally contain the degQHy mutation. This finding suggests that the degQHy mutation, which does not impact on the protein structure of DegU, is less effective than the degS200Hy mutation in generating DegU~P.

Therefore, we concluded that the powerful overturn of the DegU~P action on motility genes is a general phenomenon occurring not only in laboratory strains, but also in wild, undomesticated strains, even when the phosphorylation of DegU is maximal.

SwrA and competence for DNA uptake

The voluminous literature data reporting the negative effect of Hy mutations on competence were acquired in domestic B. subtilis strains which lack the SwrA protein. To establish whether SwrA has a general role as regulatory factor for DegS/U activity in competence, genetic transformation was analyzed in isogenic mutants differing for the status of the swrA allele as well as for the source of DegU~P: either the intact phosphoprotein obtained in the presence of a degS200Hy or the mutant DegU32Hy protein.

Transformation efficiency was assessed in PB5370 and PB5249, respectively the swrA- and swrA+ versions of the commonly used JH642 domestic strain (30), which do not contain any selectable marker. The swrA- and swrA+ isogenic strains did not show any significant difference in transformation efficiency (Fig. 1), but for a slightly better performance of the swrA+ strain, PB5249, which was thus taken as reference strain for determining the efficiency of the others. Both strains were transformed with the Hy mutation in either degU or degS and the resulting Hy mutants were transformed with a selectable genomic DNA. As shown in Fig. 1, consistently with literature data, in swrA- strains transformation efficiency was abolished by both degUHy and degSHy mutations (efficiency 0.7% and 2.7%, respectively). Even in the presence of a functional swrA allele the degUHy strain did not substantially modify competence, i.e., the swrA+ degU32Hy strain remained non-transformable (0.7% efficiency). However, the presence of SwrA in the degSHy strain was sufficient to restore competence to 36% of efficiency (Fig. 1).

Fig. 1.
Fig. 1. Transformation efficiency.

Competence was evaluated in laboratory strains derived from JH642 (swrA- degQwt trpC1 pheA2), differing for the status of the degS/U and swrA alleles (listed in Table 1), by counting resistant colonies obtained by transformation with selectable genomic DNA. Each experiment is the average of at least three independent replicates; error bars account for the standard error of the mean. The genotype of each strain is given on the x-axis. In each experiment, 100 % efficiency was assumed for the swrA+ strain (PB5249).

Taken together, these data indicate that if SwrA is functional, competence is reduced but no longer abolished by phosphorylation of DegU. Thus, SwrA is able to modulate the activity of DegU~P, partially suppressing its negative effect. This positive action is possible only in the presence of a degSHy mutation, i.e., in the presence of a phosphorylated wild-type DegU protein.

SwrA and subtilisin expression

The restoration of competence in a degSHy strain prompted us to extend our investigation to aprE expression, which is known to be induced by the presence of a Hy mutation in either degS or degU (31). As already described, swrA+ revertants often arise in the swrA- population upon long incubations and might generate confusing results. To avoid the development of such revertants, a swrA null mutant was created together with an isogenic swrA+ strain. To verify whether SwrA has a role also in subtilisin production, the PaprE-GFP reporter, developed by Veening et al. (32), was inserted in swrA+ and ΔswrA JH642-derived strains. Analyses were carried out by imaging flow cytometry, which not only allows to quantify the average single cell fluorescence, but also to dissect the aprE-ON and -OFF populations due to heterogeneity in aprE expression (32, 33). As the expression of the reporter was not detected in these conditions (data not shown), a degU32Hy or degS200Hy allele was introduced in each strain. The analyses of the PaprE-GFP degSHy or degUHy in both swrA+ and ΔswrA were focused on the transition phase (T0), and 5 and 15 h later (T5 and T15). As shown in Fig. 2A, in DegUHy strains, the percentage of aprE-ON cells did not substantially vary in dependence of the presence of SwrA both at T0 and at later time points. Conversely, in DegSHy strains the percentage of aprE-ON cells was highly affected by SwrA. The presence of SwrA led to a substantial decrease in the number of ON cells, particularly at T0 (−70%). Moreover, the percentage of ON cells was similar between DegU32Hy or DegS200Hy mutants in ΔswrA strains, but it was significantly reduced in the swrA+ degS200Hy background.

Fig. 2.
Fig. 2. Expression of PaprE-GFP.

Domestic strains, differing for the status of the degS/UHy alleles and for the presence of a functional swrA gene, were analysed by imaging flow cytometry to evaluate the percentage of GFP-ON/OFF cells and the peak of fluorescence intensity. Cultures were sampled at the transition point (T0), 5 and 15 hours later (T5 and T15), as indicated below each graph, where the swrA status is also indicated. A, C: data collected for the degU32Hy strains; B, D: data collected for degS200Hy strains. The upper panels, A and B, represent the percentage of cells expressing the reporter gene (ON-population). The lower panels, C and D, show the peak of intensity of the ON-population. Values represent the average of at least three independent replicates; error bars account for the standard error of the mean.

Also, the expression level of PaprE, i.e., the single cell fluorescent intensity, did not vary in the presence or absence of SwrA in DegUHy strains; however, in DegSHy mutants the presence of SwrA significantly decreased fluorescence intensity (Fig. 2B). Also in this case, there are no appreciable differences in GFP levels among ΔswrA strains, while in the swrA+ background the degS200Hy allele is not as efficient as degUHy in driving aprE expression. A gallery of images of aprE ON and OFF cells acquired during flow cytometry are provided in Fig. S2.

These data allow to conclude that SwrA modulates the activity of DegU~P also at the aprE promoter. SwrA reduces the efficacy of DegU~P on subtilisin expression. Analogously to what observed in competence, the SwrA-mediated effect only occurs in the presence of a degSHy mutation, i.e., in the presence of a wild-type phosphorylated DegU protein.

DegSHy and DegUHy mutants in pgs expression

The activation of the pgs operon expression is known to depend on the co-presence of at least a degS/UHy allele and SwrA. However, so far, most of the data have been collected using DegUHy mutants, while scant information is given on γ-PGA production in DegSHy strains (24). To fill this gap, a Ppgs-sfGFP reporter construct was devised and inserted in locus in the swrA+ laboratory strain. Since no fluorescence was detected in this strain (data not shown), it was further transformed with either degSHy or degUHy alleles. The Hy strains were grown under vigorous shaking in a glutamate-rich medium that supports γ-PGA production, with periodic sampling over a 48-h prolonged incubation. At relevant time-points, Ppgs expression was quantified by imaging flow cytometry. In the DegUHy mutant, Ppgs appeared to be homogeneously active from the beginning of the analysis (2-h post inoculum, data not shown), with intensity reaching a peak at T-2 (8-h post inoculum). This early peak of maximal intensity was followed by a monomodal decline over the next 40 h (Fig. 3A), with the majority of the population already OFF after T18. Conversely, in the DegSHy strain, Ppgs activation showed a 2-h delay: cells started displaying fluorescence at T0 (10-h post inoculum), with a gradual increase over time. Intensity reached a peak at T14 which was followed by a slower decline of the GFP signal, which remained however appreciable, in most of the cell population, up to the end of the experiment (T38) (Fig. 3B).

Fig. 3.
Fig. 3. Expression profile of Ppgs-GFP.

Strains (A) swrA+ degUHy and (B) swrA+degSHy were grown in E-medium and cells were collected for imaging flow cytometry at different time points. The colour of the plots refers to the collection time (in h relative to the transition point), which is indicated in each graph. A dashed line marks the ON threshold. The intensity values, represented in logarithmic scale on the x-axis, refer to the median pixel intensity of each single event.

From these data it emerges that the impact of SwrA on pgs transcription is dramatic for both Hy mutants: no transcription is observed in swrA- backgrounds (22–24), and data not shown). However, there is a remarkable difference in the expression profile using the two partner proteins; upon interaction with SwrA, the constitutively active mutant protein DegU32Hy immediately exerts its pressure on the pgs promoter but the effect is rapidly relieved. In the degSHy strain, a delay in pgs activation is observed, most likely due to the requirement of the physiological trigger of the signalling pathway. However, once activated, the SwrA-DegU~P stimulus on Ppgs is sustained up to 24 h (T14), although the intensity of the fluorescent signal is reduced with respect to what observed in cells containing DegUHy. These data are in line with our experimental evidence that DegSHy strains produce a much higher amount of γ-PGA (data not shown) and indicate that, although an interaction between SwrA and DegUHy occurs, the effect on transcription is considerably different from that obtained when SwrA interacts with DegU~P.

DISCUSSION

This work extends the array of DegS/U regulated phenotypes in which SwrA plays a pivotal role. The data have been summarized in Table 2. Considering the phenotypes thus far analyzed, SwrA emerges as key modulator of DegS/U on all the promoters tested so far, PaprE, Ppgs (Figs. 2 & 3) and Pfla/che, (16) (Fig. 4). Notably, SwrA also mitigates the negative effect of DegU~P on genetic competence (Fig. 1) and makes degSHy swrA+ strains easily transformable.

Fig. 4.
Fig. 4. SwrA effects on DegS/U regulated genes.

The schematic representations above summarizes the data collected on aprE, pgs and fla/che transcription on the global effects exerted DegU~P alone (left) and with SwrA (right). The size of the curved arrows in front of each gene is indicative of the efficiency of transcription. Symbols are described in the box at the bottom.

The results shown in this work do not confute literature data obtained in swrA- domestic B. subtilis strains (168, JH642, and others) (18). The non-transformability of degUHy as well as degSHy swrA- strains is indeed validated in our experimental settings (Fig. 1). Rather, a piece of literature data appears to support our results. In 1991, Hahn and Dubnau, analyzing the impact of degU32Hy and degS200Hy alleles on PsrfA expression, could not interpret the fact that, differently from DegUHy, DegSHy did not repress srfA transcription (34). It is tempting to imagine that a high percentage of swrA+ revertant cells arose in the DegSHy strain used in the experiment, due to the high frequency of phase variation events (10-4) (17), and in those revertants SwrA was able to mitigate -or supress-the negative effect of DegU~P, turning it into a less negative -or positive-signal.

Presently, the main target of DegU~P in competence has not been clearly identified, because of the coexistence of at least two possible target genes: Pcomk (8, 27, 28) and PsrfA (28, 34). A negative effect of the degQHy allele has been evidenced on both promoters, in domestic and undomesticated strains (swrA- and swrA+, respectively) (28). Since the degQHy mutation increases DegU~P levels (5) without impacting on the DegU structure, its interaction with SwrA is preserved. The way in which the effects of SwrA and DegQ are balanced needs to be further analyzed in well-defined genetic backgrounds.

A second fundamental result that emerges from this work is that the DegU32Hy mutant protein does not behave as the phosphorylated wild-type DegU protein. A proxy for DegU~P is represented by the degS200Hy mutation, which produces high levels of DegU~P without directly modifying the structure of the transcriptional activator DegU. Moreover, from the lack of activation of the pgs promoter in undomesticated strains, which are naturally swrA+ degQHy (data not shown), it can be hypothesized that the level of DegU phosphorylation attained in degQHy cells is lower compared to that gained with the degS200Hy mutation.

From Ppgs analyzes it can be hypothesized that DegU32Hy is able to bind directly to DNA, even before activation of the signalling pathway that would lead to its phosphorylation, i.e., in the non-phosphorylated form (see Fig. 3A). This notion is not novel: Stanely-Wall and collaborators showed that γ-PGA production in a degU32Hy background also occurs in a degS null mutant (35). Also in vitro, Mordini et al. (2013) showed that DegU32Hy binds to DNA independently from the presence of its cognate kinase. Moreover, the interaction of SwrA (physical or genetical) with the mutant DegU32Hy protein is compromised. Either it does not occur at all, as it appears by the lack of differences between the phenotypes of degU32Hy swrA+ and swrA- strains in competence, aprE expression and motility (Figs. 1 & 2 and ref. 16), or it markedly differs from the interaction with DegU~P produced by degS200Hy, as it appears from the differential activation profile of Ppgs in the two mutants. In any case, the physiological role of DegU~P in B. subtilis should be approached using a DegS200Hy mutant. This also suggests that our current view of the impact of the DegS/U on B. subtilis physiology gained through the use of degU32Hy mutants might require some revamping, as it happened for motility.

MATERIALS AND METHODS

Strain construction

All strains used in this study are listed in Table 1.

PB5630, corresponding to strain DK1042 obtained by D. Kearns and co-workers (29) by introducing the com/Q12L mutation in the resident plasmid of the undomesticated NCIB3610, was transformed with pLoxSpec/degSU(Hy) and pLoxSpec/degS200 (24). PB5814 and PB5815, respectively, were obtained after selection for spectinomycin resistance (60 μg/ml).

The clean deletion of the swrA gene was obtained by transforming PB5249 with pCCΔswrA, a non-replicative plasmid that, completely removing the swrA ORF, inserts a kanamycin resistance gene upstream of the swrA promoter to control the expression of the downstream minJ gene. pCCΔswrA plasmid was obtained through the following steps: a PCR fragment comprising the region upstream the swrA gene, containing all the regulatory elements, was amplified from PB5249 genomic DNA with primers UPPromA/E (EcoRI)5’-ccgaattctttgtgcttaaagagattatggatc-3’ and CC_A_rev (XhoI) 5’-aacgctcgagttgtgaacccccattttctttatacagataagcac-3’; the initial part of the following ORF, minJ, was amplified from the same source with primers CC_B_for (XhoI) 5’-accgctcgaggtgtctgttcaatggggaattgaactgttaaaaagc-3’ and CC_C_rev (SmaI) 5’-tcccccggggtttgccagctgctgtccgatcg-3’. The two products were digested with XhoI (restriction sites underlined) and ligated. The 934 bp resulting product was inserted between the EcoRI and SmaI sites of the pJM114-derived pCC1 (21). The plasmid pCCΔswrA, verified by multiple restriction digestion and by sequencing of the relevant portions.

The plasmid pCCΔswrA was used to transform PB5249. PB5606 was obtained by selecting one clone for kanamycin (2 μg/ml) resistance; deletion of the coding sequence of swrA and the integrity of its promoter and minJ were verified by PCR and DNA sequencing.

The PaprE-gfp strains were obtained by in-locus integration of the pGFP-aprE plasmid (a generous gift from Prof. J.W. Veening, ref. 32) into the chromosome of swrA+ and ΔswrA isogenic strains, respectively PB5393 (21) and PB5606 (described above), both carrying a kanamycin resistance gene upstream of the swrA promoter region. The resulting strains were named PB5717 and PB5719, respectively (Table 1). degU32(Hy) and degS200(Hy) alleles, were introduced in PB5717 and PB5719 by transformation with pLoxSpec/degSUHy) and pLoxSpec/degS200 (24) and selection for spectinomycin resistance (60 μg/ml). In the derived strains, PB5720, PB5722, PB5723 and PB5725 (Table 1), the single copy insertion of each construct was assessed.

The Ppgs-SFgfp strains were obtained using a modified pMutin vector (pMATywsC). The construction of the plasmid occurred in multiple steps. First, in the pMutin-GFP vector (ECE149, obtained from the Bacillus genetic stock centre, http://www.bgsc.org/) the gfp gene was substituted by Gibson assembly with a super folder version of the GFP (SFgfp) amplified from pECE323 plasmid (Bacillus genetic stock centre) with primers RXeGFPda321-5’-ggctgcactagtgctcgaattcattatttataaagttcgtccataccgtg-3’ and FXeGFPda321-5’-tcggccggaaggagatatacatatgtcaaaaggagaagaactttttacag-3’ to give pMutinsfGFP. The 5’ portion of ywsC together with the Phyperspank promoter were inserted in the resulting pMutinsfGFP through a tripartite Gibson assembly. The Phyperspank promoter was amplified from plasmid Phyp.R0.sfGFP(sp).LacI_operon (36) using primers PHypFor 5’-agcttccaagaaagatatccctcggatacccttactctgttg-3’ and PHypRev 5’-ggctataatgagtaaccacatgtttgtcctccttattagttaatc-3’; the 5’ portion of ywsC (647 bp) was amplified from PB5249 chromosomal DNA using primers ywsCFor 5’-taactaataaggaggacaaacatgtggttactcattatagcctgtg-3’ and ywsCRev 5’-gtaaaaagttcttctccttttgacagagaagcgttatcagggaatac-3’. In the plasmid obtained, pPhsywsCsfGFP, the spoVG RBS and initial codons were translationally fused to the sfGFP using the partially overlapping oligos oligoFORSpoVG 5’-ccctgataacgcttctctggaattcccgggatccccagcttgttgatacactaatgcttttatatagggaaaaggtggtgaactactatgTCAAAAGGAG-3’ and oligoREVSpoVG 5’-CTCCTTTTGAcatagtagttcaccaccttttccctatataaaagcattagtgtatcaacaagctggggatcccgggaattccagagaagcgttatcaggg-3’, derived from the pJM116 vector (37) The final construct was verified by sequencing and saved as pMATywsC. This plasmid was used to transform PB5249 (swrA+) and PB5370 (swrA-), using erythromycin resistance (5 μg/ml) for selection, resulting in PB5741 and PB5742 strains, respectively. degU32(Hy) and degS200(Hy) alleles were introduced in PB5741 by transformation with pLoxSpec/degSU(Hy) and pLoxSpec/degS200 (24) by spectinomycin resistance (60 μg/ml) selection, giving rise to PB5743 and PB5745, respectively. The single copy insertion of each construct was assessed.

Competence evaluation

Cells were inoculated in LM (LB supplemented with MgSO4, 9μM; tryptophan, 50 μg/mL; phenylalanine, 50 μg/mL) at OD600=0.2 and grown at 37°C shaking. At OD600=1, cells were diluted 1:20 in MD (K2HPO4, 9.8 mg/ml; KH2PO4, 5.52 mg/ml; Na3Citrate·5H2O, 0.92 mg/ml; glucose, 20 mg/ml; tryptophan, 50 μg/ml; phenylalanine, 50 μg/ml; ferric ammonium citrate, 11 μg/ml; K-aspartate, 2.5 mg/ml; MgSO4, 0.36 mg/ml) and grown at 37°C until stationary phase (T0). About 200 ng of chloramphenicol (Cm)-selectable B. subtilis chromosomal DNA was added to 0.5 ml cells which were further incubated for 1.5 h at 37°C with shaking. Transformants were isolated on 5 mg/ml chloramphenicol on several selective plates. Resistant colonies were counted and related to cell density at T0 to calculate the transformation efficiency, taking into account each dilution step before plating. Data shown in Fig. 1 represent the average of three independent experiments.

Gene expression evaluation by flow cytometry

For the analysis of PaprE activity, cells were inoculated in Shaeffer’s sporulation medium (38) at 0.2 OD600 and grown at 37°C under continuous shaking for 20 h. Aliquots were collected every 60’ for OD600 readings; at the transition point (5h), 5 and 15 h later, 10% glycerol (final concentration) was added to culture aliquots for storage at −20°C.

For the analysis of Ppgs activity, cells were inoculated in E-medium (39) at OD600=0.1 and grown at 37°C under continuous shaking for 48h. Aliquots were collected at 2-h intervals for OD600 readings and direct cytofluorimetric analyses. Before analyses, fresh and/or frozen samples were centrifuged for 5 minutes at 14000 xg; cell pellets were re-suspended in D-PBS for flow cytometry (Gibco).

Samples were acquired on an Amnis® ImageStream®X Mk II Imaging Flow Cytometer using the INSPIRE software with the following set up: Channel 02 (GFP fluorescence), Channel 06 (scattering channel); the Brightfield was visualized on Channel 01 and on Channel 05, depending on the GFP expression level, to avoid interference from Channel 02. The 488 nm laser was used at either 50 mW or 100 mW power, according to the GFP expression level, in order to avoid sensor saturation. The flow rate was set to low speed/high sensitivity and images were taken at 60X magnification. For each sample at least 10000 events were acquired.

All data were analyzed using the IDEAS software (version 6.2). In-focus events were gated in a histogram displaying the Gradient RMS_M01_Ch01 on the x-axis. A plot of the Area versus Aspect Ratio Intensity in the Brightfield channel was used to exclude doublets from the analysis. A plot of the Area versus Intensity in the Scattering channel was used to exclude events characterized by high scatter such as beads. To avoid any bias due to cell size in evaluating fluorescence intensity, the GFP level of each cell was calculated through the Median Pixel feature on the fluorescence channel. The threshold value to distinguish the ON population was set at the maximum autofluorescence of a non-fluorescent population used as negative control (OFF). Data presented in Figs. 2 and 3 represent the average of three independent experiments.

SUPPLEMENTARY MATERIALS

Fig. S1.
Fig. S1. Motility of undomesticated DegU/SHy strains.

Swimming (on the left of each panel) and swarming (on the right) motility performances of undomesticated strains (swrA+). A): PB5630 degS/Uwt; B): PB5814 degUHy; C) PB5815 degSHy. The genotype of each strain is also indicated on the right. Strains are listed in Table 1.

Motility plates were prepared as described in Mordini et al., 2013, except for the addition of surfactin, that was omitted in the swarming plates.

Fig. S2.
Fig. S2. Cell images collected during Flow Cytometry.

Representative images of PaprE-GFP containing cells collected from the different intensity bins which are pointed by an arrow on the graphs in the right panels. On the left, identical images as acquired in the fluorescence channel (Ch02) and in the brightfield channel (Ch05).

ACKNOWLEDGEMENTS

This research was supported by the Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (2018–2022) - Dept. of Biology and Biotechnology “L. Spallanzani”, University of Pavia. Authors are grateful to Prof. J. W. Veening for the PaprE plasmid. The contribution of Erlinda Rama, Matteo Cavaletti, Jessica Bollini, Laura Nucci is acknowledged. We also thank Dr A. Azzalin, from the departmental imaging facility, and Dr A. Serra, from Luminex Corporation, for support with the Amnis ImageStream data acquisition and analyses.

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Posted May 08, 2021.
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SwrA as global modulator of the two-component system DegS/U in B. subtilis
Francesca Ermoli, Giulia Vitali, Cinzia Calvio
bioRxiv 2021.05.07.443137; doi: https://doi.org/10.1101/2021.05.07.443137
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