Abstract
In Bacillus subtilis, the extracytoplasmic function σ factor σM regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σM, and the absence of YhdL leads to runaway expression of the σM regulon and cell death. Here, we report that this lethality can be suppressed by gain-of-function mutations in spoIIIJ, which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 SpoIIIJ contains a single amino acid substitution in the substrate-binding channel (Q140K), and this allele suppresses the lethality of high SigM. Analysis of a library of YidC variants reveals that increased charge (+2 or +3) in the substrate-binding channel can compensate for high expression of the σM regulon. Derepression of the σM regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidCQ140K substitution. We further show that the fitness defect caused by high σM activity is exacerbated in the absence of SecDF protein translocase or σM-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific σM-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the σM regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.
Author Summary Bacteria frequently produce antibiotics that inhibit the growth of competitors, and many naturally occurring antibiotics target cell wall synthesis. In Bacillus subtilis, the alternative σ factor σM is induced by cell wall antibiotics, and upregulates genes for peptidoglycan and cell envelope synthesis. However, dysregulation of the σM regulon, resulting from loss of the YhdL anti-σM protein, is lethal. We here identify charge variants of the SpoIIIJ(YidC) membrane protein insertase that suppress the lethal effects of high σM activity. Further analyses reveal that induction of the σM regulon leads to high level expression of membrane proteins that trigger envelope stress, and this stress is countered by specific genes in the σM regulon.
Introduction
The ability of cells to adapt to changing conditions relies in large part on the expression of specific stress responses controlled by transcription regulators. The extracytoplasmic function (ECF) subfamily of σ factors are frequently involved in bacterial responses to stresses affecting the cell envelope [1, 2]. In Bacillus subtilis, there are seven ECF σ factors with the σM regulon playing a particularly important role in modulating pathways involved in peptidoglycan synthesis, cell envelope stress responses, and intrinsic resistance to antibiotics [3]. Cells lacking σM (sigM null mutants) grow normally in unstressed conditions, but have greatly increased sensitivity to high salt and to cell wall active antibiotics, including ß-lactams and moenomycin [4–6].
Like many other ECF σ factors, the activity of σM is regulated by two membrane-localized anti-σ factors encoded as part of the sigM operon, sigM-yhdL-yhdK [7, 8]. The major anti-σ factor is YhdL, a transmembrane protein that directly binds σM [9]. However, full YhdL activity requires a second transmembrane protein YhdK. Although a lack of σM is well tolerated under unstressed conditions, the lack of the anti-σM factors leads to a runaway activation of the autoregulated sigM operon, and overexpression of the σM regulon [10]. A null mutation of yhdK leads to an ∼100-fold elevation of σM activity, morphological abnormalities, and slow growth [10]. A null mutation in yhdL is lethal, but suppressors arise readily that have inactivated sigM and grow normally in unstressed conditions [4, 10]. These findings imply that high level expression of σM is toxic to cells, but the basis for this toxicity has not been defined.
Previously, we explored the basis for σM toxicity by selecting for suppression of yhdL lethality in a sigM merodiploid strain to reduce the frequency of suppressors that had inactivated σM. These studies led to the recovery of mutations in rpoB and rpoC, encoding the ß and ß’ subunits of RNA polymerase, that led to a reduction of σM activity sufficient to restore viability [10]. These mutations, which acted selectively on σM, affected a region of core RNA polymerase involved in σ factor binding. In the course of these studies we also demonstrated that the toxicity from high σM could be alleviated by mutation of the autoregulatory promoter for the sigM operon, or by overexpression of the housekeeping σ factor, σA [10]. These results suggest that the lack of a functional anti-σM factor (yhdL null mutant) leads to runaway activation of the sigM operon and a high level of σM activity that is incompatible with growth. However, it is unclear whether σM toxicity results from a decrease in activity of the essential housekeeping σA, overexpression of one or more σM-regulated genes, or both.
To further explore the impact of overexpression of specific σM-regulated genes on cell physiology we generated a library of strains in which specific σM-dependent promoters (PM) are inactivated by point mutations. This approach, which removes σM-dependent activation while leaving other promoters and regulatory inputs intact, is important since many σM-regulated genes have multiple promoters and encode essential genes, including several involved in peptidoglycan synthesis and cell division [3]. In the course of developing this library we received a previously described strain with a mutation inactivating the PM of rodA, encoding a SEDS family transglycosylase important for peptidoglycan synthesis [6]. We unexpectedly discovered that in this strain yhdL could be inactivated, and the same was true for the parent strain (B. subtilis PY79). These serendipitous observations led us to hypothesize that B. subtilis PY79 differs from other B. subtilis 168 strains in its ability to tolerate high level expression of the σM regulon.
Here, we report that the ability of B. subtilis PY79 to tolerate σM regulon overexpression results from a single amino acid substitution in the spoIIIJ gene, which encodes the major YidC membrane insertase in B. subtilis [11, 12]. This finding motivated a detailed structure-function analysis of YidC, which led to the discovery of mutations that increase the positive charge within the hydrophilic, substrate-binding channel of YidC from +1 (wild-type) to +2 or +3 (in specific combinations) increase tolerance to overexpression of σM-regulated membrane proteins. Moreover, high level activity of σM leads to induction of genes associated with secretion stress, oxidative stress, and DNA damage responses, and the σM regulon itself includes functions that help compensate for stresses associated with membrane protein overexpression.
Results
A single amino acid change in SpoIIIJ necessary and sufficient for tolerance of high σM
The anti-σ factors YhdL and YhdK regulate σM, and the absence of yhdL is lethal in B. subtilis strain 168 due to toxic levels of σM [4, 10]. Since σM controls a large regulon, including many essential genes involved in cell wall synthesis [1, 3], we sought to construct a library of strains in which specific σM-dependent promoters are inactivated by point mutations. One such promoter precedes rodA, which encodes a peptidoglycan transglycosylase [6]. We thus acquired a ΔPM-rodA strain (BAM1077[6]), and tested whether yhdL is essential in that strain background, with its parent wild type strain PY79 as a control. Surprisingly, we found that yhdL is not essential in either PY79 strain. A yhdL mutant in PY79 exhibits reduced colony size compared to WT and high σM activity as indicated by the blue color on LB plates containing X-Gal (Fig. 1a). This mutant is relatively stable, with the occasional appearance of suppressors that have a large white colony morphology (likely containing mutations in sigM). In contrast, introduction of a yhdL null mutation into other B. subtilis 168 strains results in tiny, pinpoint colonies that cannot be re-streaked, consistent with prior work [4, 10].
To identify the genetic differences in PY79 that confer tolerance of high σM, we compared the genome between B. subtilis strain 168 and PY79. There are over a hundred single nucleotide polymorphisms (SNPs) between the 168 reference sequence and PY79, as well as four large deletions in the genome of PY79 (including the SPβ prophage), causing a reduction of 180 kb for the PY79 genome compared with 168 [13, 14]. To identify differences that might correlate with tolerance of high σM activity, we compared the sequences of genes encoding RNA polymerase subunits, and genes in the σM and Spx regulons (Spx is a transcription regulator that is regulated by σM). However, no differences were noted in these 103 genes between the PY79 and 168 reference genomes (Table S1).
Next, we used an unbiased, forward genetics approach to identity the mutation in PY79 that suppresses the lethality of a yhdL null mutation. We used genomic DNA from a PY79 yhdL::kan strain to transform 168 to kanamycin resistance, reasoning that the only viable transformants will likely also acquire the suppressing mutation. Because each competent cell of B. subtilis contains about 50 binding sites for DNA uptake, a competent cell can import multiple fragments of DNA during transformation in a process known as congression [15]. When a 168 strain containing a PM-lacZ reporter was transformed with chromosomal DNA from the viable PY79 yhdL::kan strain and selected on an LB plate supplemented with kanamycin and X-gal, we recovered numerous tiny blue colonies that did not grow when re-streaked onto fresh plates (consistent with the essentiality of YhdL in the 168 background), a few large white colonies (likely sigM mutants), and intermediate sized blue colonies (Fig. 1B). The intermediate blue colonies grew to a similar size as a yhdL null mutant in PY79, consistent with acquisition of both the yhdL::kan allele and a second locus that suppresses the toxicity of high σM. Whole genome sequencing was performed on these transformants and the reads were mapped to the reference genome of 168. Out of 15 sequenced 168 transformants, 14 contained the same SNP imported from PY79 that generates a missense mutation in spoIIIJ (encoding SpoIIIJQ140K) (Fig. S1A, Table S2). We therefore hypothesized that the spoIIIJQ140K allele was the suppressor needed for cells to tolerate the yhdL mutation.
SpoIIIJ belongs to the YidC membrane protein insertase family and is responsible for inserting membrane proteins into the lipid bilayer, independently or in association with the Sec secretion system [16–18]. E. coli encodes one essential homolog of YidC, while some bacteria such as B. subtilis encode two homologs, SpoIIIJ(YidC) and YidC2 [18, 19]. The gene encoding YidC was named spoIIIJ because mutations at this locus lead to a block at stage III of sporulation [19, 20]. However, spoIIIJ is constitutively expressed and functional in vegetative cells. The expression of the paralog YidC2 is regulated by an upstream gene mifM, which monitors the total membrane protein insertase activity and only allows expression of YidC2 when MifM is not efficiently inserted into the membrane [21]. Both SpoIIIJ and YidC2 can fulfill the essential function of YidC insertase, with SpoIIIJ essential for sporulation [22] and YidC2 important for the development of competence (Fig. S2A) [23]. Interestingly, an alignment of YidC homologs revealed that the Gln140 residue is highly conserved among bacteria, and only B. subtilis PY79 SpoIIIJ contains Lys at this position (Fig. 1C).
To test if this SpoIIIJ Gln to Lys variant (SpoIIIJQ140K) is necessary and sufficient for tolerance of high σM, we introduced the spoIIIJQ140K mutation at the native locus of strain 168 using CRISPR, and found that yhdL was no longer essential (Fig. 1D, Fig. S1B). Conversely, changing the Lys140 into Gln in PY79 abolished the ability of PY79 to tolerate loss of yhdL (Fig. S1B), suggesting that the SpoIIIJQ140K is necessary and sufficient for tolerance of a yhdL deletion mutation. To test if the spoIIIJK140 allele is dominant over the spoIIIJQ140 allele, we constructed merodiploid strains expressing both alleles of SpoIIIJ (using a vector with xylose-inducible promoter PxylA that integrates into ganA and when induced produces about 70% amount of the native protein level (Fig. 2B)). Strains with either a PxylA-spoIIIJK140 in the 168 strain, or PxylA-spoIIIJQ140 in PY79 strain background could still tolerate the loss of yhdL (Fig. S1B). This dominance suggests that SpoIIIJQ140K leads to a gain of function that enables cells to tolerate high σM activity. Phase contrast microscopy revealed that a 168 SpoIIIJQ140K yhdL mutant had a similar but slightly more elongated cell morphology compared with a PY79 yhdL mutant (Fig. 1E), confirming the major role of SpoIIIJQ140K in tolerance of a yhdL null mutation.
As a general strategy to monitor cell fitness, we compared the impact of spoIIIJ alleles on morphology and colony size in the PY79 and 168 backgrounds mutant for yhdK. YhdK functions together with YhdL as an anti-σ complex, but unlike yhdL a yhdK null mutant is tolerated in 168 strains, although it does lead to an ∼100-fold increase in σM regulon expression and severe growth defects [10]. Five independent trials with a 168 yhdK mutant, known to generate small colonies, revealed up to a ∼10% change in average colony area. This variation likely results from small differences in the plating conditions. Although these small differences were in some cases judged to be statistically significant (based on P value in a t test, two tails, assuming unequal variances) (Fig. S1C), this reflects the high sample number in each measurement (100-1000 colonies per measurement). Considering this level of variation between genetically identical strains, we only regard as significant those changes of >10% in colony size. Using this assay, we found that the small colony size, as well as the filamentous cell morphology of the 168 yhdK mutant, can be largely rescued by the spoIIIJQ140K allele (Fig. 1F, G). Conversely, a spoIIIJK140Q mutation in PY79 yhdK converted the large colonies of the parent strain into the small round morphology of the 168 yhdK strain, and the cells exhibited increased filamentation (Fig. 1F, G). Deletion of spoIIIJ in a PY79 yhdK mutant mimicked a SpoIIIJK140Q mutation (Fig. 1G), likely because the cells now rely on the other YidC paralog, YidC2, which contains a glutamine residue in the equivalent position (Fig. 1C) [19]. Overall, our results show that SpoIIIJQ140K mutation is necessary and sufficient for B. subtilis to tolerate high σM activity caused by the absence of the anti-σ factor YhdL or its partner protein YhdK.
Overexpression of SpoIIIJ increases tolerance of high σM activity
We hypothesized that the SpoIIIJQ140K protein may simply be more active or abundant in cells than the native protein. To test if increasing insertase activity is sufficient to alleviate toxicity associated with high σM, we overexpressed the wild-type 168 SpoIIIJQ140 protein using a strong IPTG inducible promoter, Pspac(hy) [24]. Induction of the Pspac(hy)-spoIIIJQ140 allele led to a three-fold increase in the amount of SpoIIIJ protein compared with WT (Fig. 2B), and increased the colony size of a yhdK mutant by 68% (Fig. 2A). This increase is less than the effect of the spoIIIJQ140K allele at the native locus (which increased yhdK colony size by 134%), and consistently it only marginally increased the growth of a yhdL depletion strain under depletion condition (Fig. S2C). The increase in the fitness of the yhdK mutant supports the hypothesis that higher insertase activity is beneficial for cells with elevated σM activity, but overexpression alone does not phenocopy the effect of the altered function allele.
We next tested whether overexpression of YidC2, the other YidC homolog in Bacillus, could benefit cells with high σM expression. Interestingly, when yidC2 was overexpressed from the Pspac(hy) promoter, the growth defect of either the yhdK mutant or the yhdL depletion strain was exacerbated (Fig. 2A, S2C). Furthermore, when the equivalent glutamine residue of SpoIIIJQ140 was mutated to lysine, the YidC2Q148K mutant protein was toxic when overexpressed in a yhdK mutant (Fig. S2B). Thus, YidC2 is unable to compensate for SpoIIIJ in alleviating stress associated with high σM activity, even when the corresponding Gln to Lys substitution is present. Similarly, overexpression of E. coli YidC did not provide any benefit to a yhdK or yhdL mutant (Fig. 2A). However, when the equivalent Gln to Lys substitution was present, overexpression of the E. coli YidCQ429K mutant was modestly beneficial, and colony size of the yhdK mutant increased by 58% (Fig. 2A). These results suggest that different YidC homologs vary in their substrate preferences, and the Gln to Lys mutation may enhance the ability of the B. subtilis SpoIIIJ and E. coli YidC insertases to facilitate membrane insertion of at least some σM-dependent proteins.
SpoIIIJQ140K increases the positive charge inside the substrate binding groove
The structure of YidC2 from Bacillus halodurans revealed a positively charged hydrophilic groove formed by five transmembrane segments [16]. A positively charged residue in the substrate-binding groove is essential for the function of the insertase, as a R73A substitution in B. subtilis SpoIIIJ completely abolished the essential function of SpoIIIJ in vivo, while an R73K substitution retained function [16]. Although the positive charge is essential, the R73 residue is not, as the positive charge can be provided by mutation of any of six other residues inside the hydrophilic groove to Arg. These six positions include Ile72 and Ile76 in transmembrane region 1 (TM1), Gln140 and Leu144 in TM2, and Trp228 and Gly231 in TM5 [25] (Fig. 3A, S3B). Since both Arg and Lys are positively charged, we hypothesized that the key feature of the PY79 SpoIIIJK140 protein is the +2 charge inside its substrate binding chamber. To test this hypothesis, we engineered a SpoIIIJR73AQ140K double substitution protein with a net charge of +1, where R73 is functionally replaced by K140. Expression of this protein is sufficient to support viability, as judged in a strain with depletion of yidC2 (Fig. 3B), but is not sufficient to allow depletion of yhdL (Fig. 1D). This supports the idea that the key effect of the Q140K substitution is to increase the positive charge in the substrate binding groove.
Increased charge inside the substrate binding groove is key for tolerance of high σM activity
Since it is the increased positive charge from +1 to +2, rather than the Q140K substitution per se, that rescues cells from high σM toxicity, we next set out to test if other combinations of positively charged residues can also rescue cells from high σM activity. To this end, we generated a library of SpoIIIJ variants with five of the six positions mentioned above substituted (or not) with Arg, as shown previously to support function in the absence of Arg73 [25], and Gln140 substituted (or not) with Lys, as seen in PY79. In addition, we mutated Arg73 to Ala (or not) (Fig. 3A). This leads to 27=128 possible charge combinations, ranging from 0 to a maximum of +7, with most containing a nominal positive charge of +2 to +5 in the substrate binding groove (Fig. S3A, S3B). Note that for the sake of simplicity, we assume that the K140 residue is positively charged, since the epsilon-amino group of free Lys has a pKa of ∼10.5, but this will likely vary depending on the local charge environment.
To identify SpoIIIJ variants that can function to support growth of a yhdL depletion strain, we transformed a spoIIIJ null yhdL depletion strain and selected for transformants that grew in the absence of yhdL induction. After sequencing and validation, we identified 103 spoIIIJ mutants that supported growth in the absence of yhdL induction (Table S3). Among them, 88 mutants contained a nominal double positive charge in 13 unique combinations (Fig. S3A). Five of these 13 combinations include K140, including the combination present in the PY79 SpoIIIJ protein: R73 K140. The remaining 15 mutants contained a nominal triple positive charge, with four unique combinations. Interestingly, all four of these variants contained K140, and in each case K140 was present together with a pair Arg residues that also supported growth with Q140. It is possible that the presence of nearby Arg residues lowers the pKa of K140, and the protonation state of this residue may also vary depending on the nature of the bound substrate. Among the 17 unique combinations of double and triple positive charges, none has more than one positive charge in TM1, whereas TM2 and TM5 can each harbor double positive charges (Fig. S3A, Table S3). We conclude that all 17 functional SpoIIIJ variants have an effective charge of between +2 and +3 in the substrate binding channel. This strongly suggests that a modest increase of charge inside the substrate binding chamber facilitates the insertion of σM-regulated proteins overproduced under YhdL depletion conditions, whereas a further increase may be detrimental to the activity or the stability of the insertase.
Each of these 17 SpoIIIJ variants can alleviate the stress imposed by high σM activity and support growth of the YhdL depletion strain (Fig. S3C), and increase the colony size of the yhdK mutant by up to 74% (Fig. 3C). We also found 16 of these 17 variants can support growth of a YidC2 depletion strain (Fig. S3D). Only the SpoIIIJ R72 K140 R144 variant was unable to support cell growth under these conditions, suggesting that it is compromised in the ability to insert proteins essential for cell growth, despite its ability to modestly increase colony size of the yhdK mutant (31%).
SpoIIIJ also inserts MifM into the membrane, which serves as a sensor of SpoIIIJ function to regulate expression of yidC2 [21, 26]. We used a yidC2’-lacZ translational fusion reporter to measure the ability of each SpoIIIJ variant to insert MifM. A WT 168 strain exhibited very low level of yidC2’-lacZ activity in the presence of native spoIIIJ expression (∼42 Miller Units (MU), Fig. 3D), and deletion of spoIIIJ increased the reporter activity by about 3-fold (∼132 MU, Fig. 3D). Complementation of a spoIIIJ null mutant with the 168 version of spoIIIJ (single positive charge at R73) at the thrC locus reduced the reporter activity to ∼101 MU (Fig. 3D). The lack of complete complementation may be caused by the location of the gene, as the native locus is close to the origin of the chromosome and thus has higher copy number than thrC in fast growing cells. Indeed, under our growth condition (late exponential phase in LB at 37°C), less SpoIIIJ protein was detected in the thrC::spoIIIJ complementation strain than the WT (Fig. 3D).
Using this strain background, we found that most of the SpoIIIJ variants with double positive charge appear to have higher MifM insertion ability as they exhibited lower yidC2’-lacZ activity than the WT protein. Interestingly, the three mutants with double positive charge that have the largest colony size in a yhdK mutant background (R73 K140, K140 R144, and K140 R228), also showed the lowest yidC2’-lacZ activity, consistent with the hypothesis that they have higher insertase activity for both MifM and for proteins that contribute to toxicity in strains with high σM-activity. Conversely, one mutant with a nominal triple positive charge (R76 K140 R228) exhibited a strong increase in colony size in the yhdK mutant background (Fig. 3C), but appeared to have low MifM insertion activity (high yidC2’-lacZ expression; Fig. 3D). Overall these results suggest that increasing the positive charge inside the substrate binding groove affects the efficiency of inserting membrane proteins, with several variants that appear to enhance the insertion efficiency for both MifM and σM-regulated proteins, while also retaining the ability to insert essential membrane proteins.
High σM activity causes a cascade of stresses that can be partially compensated by its regulon
SpoIIIJ functions as a membrane insertase, by either independently inserting some single pass membrane proteins or by functioning as part of the Sec translocon to facilitate the folding of the translocated proteins [18, 27]. The finding that SpoIIIJQ140K can suppress growth defects caused by high σM leads us to reason that the toxicity of high σM is caused by overexpression of membrane proteins that overwhelm the secretion system, and that SpoIIIJQ140K suppresses this stress by functioning as a more efficient insertase/foldase or chaperon. In B. subtilis, membrane secretion stress is sensed by the CssRS (control of secretion stress regulator/sensor) two component system. In the presence of membrane secretion stress, caused either by overproduction of secreted proteins or high temperature, CssR upregulates expression of membrane proteases HtrA (high temperature requirement A) and HtrB to facilitate the re-folding or degradation of misfolded proteins [28, 29].
To test whether high σM causes secretion stress, we constructed a PhtrA-lux reporter to monitor induction of the CssR-regulon. Indeed, PhtrA activity was increased about ten-fold in a yhdK mutant, and this induction can be reduced by the SpoIIIJQ140K allele by about 70% (Fig. 4A). To evaluate the role of the CssRS system in alleviating the membrane protein overproduction stress under high σM condition, we constructed mutants missing components of the CssR regulon and used yhdK mutant colony size as a measure of fitness. Surprisingly, we found that neither CssR-regulated membrane protease (HtrA and HtrB) is important for fitness of the yhdK mutant as judged by the effect of their deletion on colony size. Deletion of the response regulator CssR abolished the induction of the regulon [29], and has no effect on the fitness of yhdK mutant. In contrast to a previous report [29], the absence of CssS increased transcription of PhtrA-lux reporter by ∼80-fold. This suggests that CssS may act both as a sensor kinase to activate CssR and the regulon under induction conditions, and also as a phosphatase to deactivate CssR in the absence of induction signals, as also noted for sensor kinases in other two component systems [30].
Deletion of cssS reduced yhdK mutant colony size by over 60% (Fig. 4B), and the reduction of colony size is reversed in a cssS htrB double mutant, suggesting that high HtrB activity is detrimental for the yhdK mutant. Consistently, when htrB but not htrA was overexpressed using the Pspac(hy) promoter, the colony size of yhdK mutant was reduced by about 50% (Fig. 4B). Deletion of these genes in the WT 168 background did not have much effect on the colony size (Fig. S4A). These results suggest that the yhdK mutant is sensitive to the overproduction of membrane proteases. Single deletion of other proteases, including SipT, SipS, HtpX, PrsW and GlpG in a yhdK null mutant was also not detrimental, and in some cases led to a small beneficial effect in colony size (Fig S4B), again indicating that membrane protease activity is not beneficial for the yhdK mutant. We speculate that under high σM conditions, the overproduced proteins may cause a backlog of membrane proteins that require a longer time to correctly insert and fold in the membrane. Some of these proteins may be essential for cell growth and are vulnerable to degradation by membrane proteases.
Misfolding of membrane proteins, as well as jamming the Sec translocon by hybrid proteins, leads to the generation of reactive oxygen species (ROS) and ultimately DNA damage [31, 32]. To test whether high σM also triggers a similar cascade of stresses, we performed quantitative RT-PCR to measure induction of representative stress genes. Indeed, the high σM activity in the yhdK null mutant was correlated with strong induction of genes involved in secretion stress (secG, cssR), oxidative stress (katA, sodA), and DNA repair (lexA, dinB and recA) (Fig 4C). The induction of these stress response genes was largely suppressed in the presence of the spoIIIJQ140K allele, suggesting that their induction is a downstream effect resulting from inefficient insertion of membrane proteins (Fig 4C).
Interestingly, while many genes in the σM regulon are involved in the synthesis and maintenance of the cell wall, there are also genes involved in regulation of redox balance (spx and the Spx regulon), DNA repair and recombination (for example, radA, radC and recU), and ppGpp synthesis and stringent response (sasA) [3]. There is also a candidate σM promoter associated with the secDF operon [3]. This suggests that a subset of genes in the σM regulon are involved in compensating for stresses associated with upregulation of σM. Indeed, deletion of secDF, sasA or the PM of spx dramatically reduced the colony size of the yhdK mutant (Fig. 4D), while there was very little effect noted in the WT background (Fig. S4C). Thus, these genes seem to play an important role in cell fitness specifically under conditions of high σM expression. Deleting single genes inside the Spx regulon in the yhdK mutant did not lead to noticeable reduction of colony size (Fig. S4D), suggesting functional redundancy within the Spx regulon.
Among the 69 genes currently assigned to the σM regulon according to SubtiWiki [33], 38 code for membrane-associated or secreted proteins (Table S4). To demonstrate the burden these membrane proteins may cause when overexpressed under high σM condition, we mutated the σM-dependent promoters (PM) for genes important for the elongasome and the divisome and tested the consequence on fitness measured by colony size of the yhdK mutant. We focused on a PM inside maf gene that transcribes radC-mreB-mreC-mreD-minC-minD operon, a PM upstream of rodA gene, and a PM inside murG that contributes to transcription of the murB-divIB-ylxW-ylxX-sbp-ftsA-ftsZ operon (Fig 4E). Mutation of these three promoters individually or in combination had little effect on the colony size of the WT strain (Fig. S4E), since these genes are also expressed from σA-dependent promoters. In a yhdK mutant, however, mutation of PM(maf) and PM (murG) led to an additive increase of colony size, while mutation of PM(rodA) led to a small detrimental effect (Fig. 4F). We conclude that overexpression of genes downstream of PM(maf) and PM(murG) may overwhelm the membrane-protein insertion pathway, and thereby contribute to a net negative effect on cell fitness. Consistent with this idea, in cells expressing SpoIIIJQ140K, the beneficial effect of mutating PM(maf) and PM(murG) was largely abolished (Fig. 4G).
Discussion
The YidC membrane protein insertase is the bacterial representative of the YidC/Oxa1/Alb3 protein family of evolutionary conserved integral membrane proteins [18, 27]. In E. coli, YidC is required for the assembly of one or more essential protein complexes that reside in the inner membrane, including subunits of the energy generating F1FO ATPase and NADH dehydrogenase I [34]. Although YidC can function in concert with the SecYEG translocon as a foldase/chaperone, YidC can also function independently for the insertion of small membrane proteins with one or two transmembrane segments [35].
Bacillus subtilis encodes two YidC paralogs, SpoIIIJ and YidC2 (formerly YqjG). Either protein can support growth of B. subtilis, but a double mutant is inviable. Under most conditions SpoIIIJ is the functional YidC homolog and is responsible for insertion of transmembrane proteins, including the F1FO ATPase [19]. MifM, a membrane protein encoded upstream of yidC2, is also a substrate of SpoIIIJ and serves as a sensor for SpoIIIJ activity [21]. In the absence of SpoIIIJ activity, the translational pause caused by the membrane insertion of MifM is abolished and YidC2 is translationally activated [26, 36]. Although either SpoIIIJ or YidC2 can support growth, they appear to differ in the efficacy of inserting specific proteins: cells lacking SpoIIIJ are impaired in sporulation [20], whereas those lacking YidC2 have decreased competence [23]. The relationship between YidC protein sequence and the selection of specific client proteins is poorly understood. In cells depleted for both SpoIIIJ and YidC2 there was a substantial upregulation in expression of the Clp protease system and the LiaIH membrane-stress proteins that are regulated by the LiaRS two-component system [23]. This suggests that impairment of membrane protein insertion leads to an accumulation of misfolded proteins and disruption of the cell membrane.
One critical feature required for YidC function, as first visualized in the structure of the B. halodurans YidC protein [16], is a hydrophilic substrate-binding channel postulated to interact transiently with transmembrane segments of nascent integral membrane proteins. In YidC proteins the first transmembrane region (TM1) contains a conserved Arg residue that is essential for function in many, but not all, YidC orthologs [37]. This positively charged residue is postulated to form a salt-bridge with single-pass transmembrane client proteins with acidic residues in their amino-terminal region [16]. Remarkably, the function of this conserved Arg residues (R73 in Bacillus SpoIIIJ) can be replaced by Arg residues introduced at any of six other positions in transmembrane segments of YidC [25].
In this work, we have described an unusual variant of SpoIIIJ found in B. subtilis strain PY79 which contains the conserved R73 residue and additionally a second positively charged residue (K140) at a position that can functionally replace R73. This variant SpoIIIJ protein (SpoIIIJQ140K) is necessary and sufficient for B. subtilis to survive high level expression of the σM regulon. By screening of a library of SpoIIIJ variants with different charges, we revealed that all SpoIIIJ proteins that enable cells to tolerate loss of yhdL contain at least two, and in some cases three, positively charged residues in this channel. We postulated that these SpoIIIJ variants may be more capable of accommodating the increased expression of membrane proteins under σM control. Support for this hypothesis derives from experiments in which the deletion of individual σM-dependent promoters that control operons encoding multiple integral membrane proteins was found to improve fitness of strains with high σM activity. Thus we reason that the relevant feature of SpoIIIJQ140K is the presence of increased positive charge in the substrate-binding channel, which probably results in an increased insertase/foldase activity for specific σM-regulated membrane proteins.
Our results have clarified the nature of the lethality associated with a yhdL deletion that unleashes high level σM activity. In strains with elevated σM activity there is an increased flux of proteins targeted to the inner membrane and a subset of these may be inefficiently inserted by the native SpoIIIJ protein. This can result in a jamming of YidC-dependent protein translocation and a disruption of membrane function. The downstream sequelae associated with this disruption includes misfolding of proteins and induction of the secretion stress response (CssR), as well as genes associated with oxidative stress and DNA damage. These types of stresses may explain, in part, the inclusion of appropriate compensatory functions (including Spx, some DNA repair functions, and SecDF) as part of the σM regulon. It is presently unclear why the spoIIIJQ140K allele arose in the PY79 strain, or what conditions may have led to its selection. However, this strain was derived from strains treated with chemical mutagens to cure the endogenous prophage SPß [14] and this or other selection conditions may have contributed to emergence of this mutation. Further studies will be required to better understand how variations in YidC structure can fine-tune the substrate selectivity of this essential membrane protein insertase, and the stresses that arise when this system is challenged by the induction of highly expressed membrane proteins.
Material and Methods
Strains, plasmids and growth condition
All strains used in this work are listed in Table S5, and all DNA primers are listed in Table S6. Bacteria were routinely grown in liquid lysogeny broth (LB) with vigorous shaking, or on plates (1.5% agar; Difco) at 37 °C unless otherwise stated. LB medium contains 10 g tryptone, 5 g yeast extract, and 5 g NaCl per liter. Plasmids were constructed using standard methods [38], and amplified in E. coli DH5α or TG1 before transforming into B. subtilis. For selection of transformants, 100 μg ml-1 ampicillin or 30 μg ml-1 kanamycin was used for E. coli. Antibiotics used for selection of B. subtilis transformants include: kanamycin 15 μg ml-1, spectinomycin 100 μg ml-1, macrolide-lincosamide-streptogramin B (MLS, contains 1 μg ml-1 erythromycin and 25 μg ml-1 lincomycin), and chloramphenicol 10 μg ml-1. For spot dilution assays, cells were first grown in liquid culture at 37 °C with shaking to mid-exponential phase (OD600∼0.3-0.4), washed twice in LB medium without inducer, then serial diluted in LB medium without inducer. 10 μl of each diluted culture was then spotted onto plates and allowed to dry before incubation at 37 °C for 12-24 hours.
Genetic techniques
Chromosomal and plasmid DNA transformation was performed as previously stated [39]. The pPL82 plasmid-based Pspac(hy) overexpression constructs were sequencing confirmed before linearized and integrated into the amyE locus [24]. The pAX01 plasmid-based PxylA overexpression constructs were sequencing confirmed before linearized and integrated into the ganA locus [40]. The ganA::PxylA-yhdL-cat and thrC::PM-spoVG-lacZ-spec constructs were made with LFH PCR to avoid antibiotic marker conflicts [10]. Markerless in-frame deletion mutants were constructed from BKE or BKK strains as described [41]. Briefly, BKE or BKK strains were acquired from the Bacillus Genetics Stock Center (http://www.bgsc.org), chromosomal DNA was extracted, and the mutation containing an ermR (for BKE strains) or kanR (for BKK strains) cassette was transformed into our WT 168 strain. The antibiotic cassette was subsequently removed by introduction of the Cre recombinase carried on plasmid pDR244, which was later cured by growing at the non-permissive temperature of 42 °C. Gene deletions were confirmed by PCR screening using flanking primers. Unless otherwise described, all PCR products were generated using B. subtilis 168 strain chromosomal DNA as template. DNA fragments used for gene over-expression were verified by sequencing. Null mutant constructions were verified by PCR.
Mutations to selectively inactivate σM-dependent promoters were generated by either promoter deletion (rodA) or by inactivating point mutations (murG, maf, spx). To inactivate PM(rodA), a 91 bp region containing the PM (located upstream of a σA -dependent promoter) was deleted using CRISPR. The resulting deletion had a junction sequence of: CACATTATCGC/TTTCGTGTAGC. The point mutations inactivating murG and maf both changed the −10 region sequence from consensus, CGTC, to TGTT. The PM* mutation inactivating the promoter regulating the yjbC-spx operon is a 3 bp substitution changing the −10 region from consensus, CGTC, to AAGT, as previously described [42].
Mutations of spoIIIJ at native locus, as well as ΔPM-rodA were constructed using a clustered regularly interspaced short palindromic repeats (CRISPR)-based mutagenesis method as previously described [10]. Briefly, possible protospacer adjacent motifs (PAM) site, which is NGG for Streptococcus pyogenes Cas9, was identified and off-target sites were checked against B. subtilis genome using BLAST. If no off-target site was identified, the PAM site was chosen and 20 bps upstream of the site were used as sgRNA and cloned into vector pJOE8999 [43]. The repair template was generated by joining two or more PCR products, with intended mutation (with additional mutation to abolish gRNA recognition if necessary) introduced by PCR primers, and cloned into the pJOE8999-sgRNA vector. DNA sequence for amino acid substitution was chosen according to the preferred codons of B. subtilis [44]. The pJOE8999 derivative containing both the sgRNA and repair template was then cloned into competent cells of E. coli strain TG1 to produce concatemer plasmids, which were transformed into B. subtilis at 30°C. Transformants were then grown at 42°C to cure the plasmid, and intended mutations were confirmed by sequencing. ΔPM-rodA was constructed with repair template amplified using primers 7426, 7427, 7428 and 7429, and the gRNA constructed using 7430 and 7431. SpoIIIJQ140K mutation in 168 was constructed with repair template amplified using primers 7868, 7869, 7870 and 7871, and the gRNA constructed using 7866 and 7867. SpoIIIJK140Q mutation in PY79 was constructed with repair template amplified using primers 7866, 8247, 8246 and 7871, and the gRNA constructed using 8248 and 8249. SpoIIIJR73A mutation was constructed with repair template amplified using strain 168 genomic DNA as template with primers 7868, 8280, 8281 and 7871, and the gRNA constructed using 8278 and 8279. SpoIIIJR73AQ140K mutation was constructed with the same primer sets for SpoIIIJR73A mutation, expect the repair template was amplified using strain PY79 genomic DNA. All constructs were sequencing confirmed.
SpoIIIJ library of different positive charges were constructed using degenerate primers and LFH PCR. Four DNA fragments were amplified and joined using LFH PCR. The fragments include one with part of gene hom and thrC (amplified with primers 8766 and 8767), one with the spoIIIJ gene from 168 (amplified with primers 8768 and 8681, containing the native promoter and ribosomal binding site), one with a specR cassette (amplified with primers 8682 and 8769), and one with part of thrC and full length of thrB (amplified with primers 8770 and 8771). The joined PCR product was first transformed into strain 168 to generate HB23976. Then using genomic DNA of HB23976 as template, four new DNA fragments were amplified using degenerate primer pairs 8766 and 8689, 8686 and 8690, 8687 and 8691, and 8688 and 8771. These fragments were joined together using LFH PCR and the joined PCR product (more than 10 μg DNA) was expected to contain all 128 possible combinations of SpoIIIJ charge from 0 to +7. The PCR product library was used to transform strain 168, and more than ten-thousand transformants (from 20 plates, each contains more than 500 colonies) were pooled together to extract genomic DNA to form a virtually equivalent DNA library of the spoIIIJ variants. This genomic DNA library provides high transformation efficiency and was used to transform a yhdL depletion strain (HB23953) and transformants were selected on LB plate supplemented with X-gal but no xylose for yhdL induction. More than 200 transformants were re-streaked onto fresh LB plate with X-gal but no xylose to confirm robust growth under high SigM condition, and 103 of them were Sanger sequenced for the thrC-spoIIIJ-spec region to identify the spoIIIJ variants.
Whole genome re-sequencing and sequence analysis
Chromosomal DNA of suppressor strains was extracted using Qiagen DNeasy Blood & Tissue Kit. DNA was then sent to Cornell University Institute of Biotechnology for sequencing using Illumina HiSeq2500 with Single-end 100 bp reads. Sequencing results were analyzed using CLC workbench version 8.5.1 and mapped to the genome of strain 168 (reference accession number NC_000964.3). Note that our working stock of B. subtilis 168 has 21 SNPs compared to the cited reference sequence, and these common SNPs were not considered, and only newly introduced SNPs from the PY79 strain were tabulated (Table S2). Unmapped reads were de novo assembled and contigs larger than 1 kb were BLASTed against genome of strain PY79 (reference accession number NC_022898.1). Single nucleotide variants (SNVs) were detected using default settings, and gene deletions larger than 300 bps were identified by manually scanning regions of low coverage.
Colony size measurement
Colony size was measured using Fiji Image J [45]. Briefly, bacterial cells were grown in liquid LB medium at 37°C with vigorous shaking to mid-exponential phase (OD600∼0.3-0.4), then serial diluted to desired concentrations. Diluted cells were plated onto fresh LB plates (15 ml medium per plate, the diameter of the plate is 10 cm and the height 15 mm, VWR, US, Catalog number 25384-342), and multiple dilutions were used. Plates were incubated at 37 °C for 24 hours. Plates containing less than 100 separate single colonies were used for size measurement, because this number of colonies per plate ensures sufficient sample size and does not cause reduced colony size due to crowdedness and nutrient limitation. Pictures of plates were taken with a ruler as a length reference, and colony size was measured using Fiji Image J per software’s instruction. For each strain, at least 100 colonies were measured, and box and whisker plots were used.
Luciferase reporter construction and measurement
Luciferase reporter construction and measurement was performed as previous described [46]. The luciferase reporters were constructed by inserting the tested promoters into the multicloning sites of pBS3Clux [47]. The promoter PhtrA was amplified using primers 8403 and 8404. For luciferase measurements, 1 μl of exponentially growing cells were inoculated into 99 μl of fresh medium in a 96 well plate, incubated at 37 °C with shaking using a SpectraMax i3x plate reader, and OD600 and luminescence were measured every 12 min. The data was analyzed using SoftMax Pro 7.0 software. Promoter activity was normalized by dividing the relative light units (RLU) by OD600.
Phase contrast microscopy
Cells were grown in liquid LB medium to exponential phase (OD600 ∼0.4) and loaded on saline (0.90% NaCl, w/v) agarose pads (0.8% final concentration) on a glass slide. Phase contrast images were taken using a Leica DMi8 microscope equipped with a 100x immersion objective and Leica Application Suite X software.
Western blot
Western blot was performed as described previously [42]. Briefly, cells were grown in 5 ml LB medium in a 20 ml test tube a 37 °C with vigorous shaking. Inducer IPTG or xylose were added when required by the construct to induce SpoIIIJ. After reaching exponential phase (OD600∼0.3-0.4), 1 ml cells were pelleted by centrifugation at 4°C, resuspended in 100 μl pre-chilled buffer (containing 25 μl 4X Laemmli Sample buffer (Bio-Rad, USA), 10 μl 1M DTT, 65 μl H2O) and kept on ice. Cells were then lysed and crude cell lysate was loaded to a 4-20% SDS-PAGE stain-free gel for electrophoresis. The gel was visualized using ImageLab with stain-free gel protocol. Proteins were then transferred onto a PVDF membrane using the TransBlot Turbo Transfer System (Bio-Rad, USA), and immune blotting was performed using anti-SpoIIIJ antiserum. The blot was visualized using the Clarity Western ECL substrate (Bio-Rad) and ImageLab software. Band intensity was calculated using the ImageLab software and normalized using total protein amount according to SDS-PAGE gel image.
β-galactosidase assay for yidC2’-lacZ
Bacterial cells containing the yidC2’-lacZ translational fusion were grown to late exponential phase (OD600 0.6-0.8) in 96-well plates with 200 μl LB medium per well at 37 °C with vigorous shaking. Cells were pelleted by centrifugation, resuspended in Z buffer supplemented with DTT (Dithiothreitol, 400 nM final concentration), and lysed by lysozyme. OD600 was measured before lysozyme treatment. After lysis, ONPG (ortho-nitrophenyl-β-galactoside) was added and OD420 and OD550 were measured every 2 minutes. Product accumulation was calculated using formula product=1000×[OD420-(1.75×OD550)] and plotted against time. The slope of the linear part of the product accumulation curve was calculated using Excel and Miller Unit (MU) was calculated using formula MU=Slope/OD600/V, where V is the volume of cells used for the reaction (200 μl).
RNA extraction and qRT-PCR
Cells were grown to mid-exponential phase (OD600 ∼0.4) and RNA was extracted using RNeasy Mini Kit (Qiagen). The extracted was then treated with DNase I (Invitrogen) and the quality of RNA was checked with electrophoresis. RNA was then reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green (BioRad) and the topA gene was used for reference of data normalization.
Author Contributions
Conceptualization: Heng Zhao, John D. Helmann
Funding acquisition: John D. Helmann.
Investigation: Heng Zhao, Anita Sachla
Project administration: John D. Helmann.
Writing - original draft: Heng Zhao, John D. Helmann.
Writing - review & editing: Heng Zhao, Anita Sachla, John D. Helmann.
Acknowledgements:
We thank Dr. Shinobu Chiba for providing the yidC2’-lacZ translational fusion reporter and antiserum for SpoIIIJ, Dr. David Rudner and Alexander Meeske for strain BAM1077, Dr. Vaidehi Patel and Wen-Wen Zhou for providing the PM* mutations for the murG and maf operons, and Dr. Tobias Doerr for valuable discussions and sharing equipment. We also thank Dr. Pete Chandrangsu and Ahmed Gaballa for help with experimental design and interpretation, and Daniel Roistacher for helping with experiments.
Footnotes
Co-author emails: hz285{at}cornell.edu, ajs588{at}cornell.edu