https://orcid.org/0000-0001-8177-3280
Figures
Abstract
The type VI secretion system (T6SS) is a complex secretion system encoded by many Gram-negative bacteria to translocate effector proteins directly into target cells. Due to its high complexity and energy-intensive firing process, regulation of the T6SS is tightly controlled in many organisms. Y. pseudotuberculosis encodes four complete T6SS clusters but lacks genes implicated in T6SS gene regulation in other microorganisms, indicating a distinct control mechanism. Here, we could show that the T6SS4 of Y. pseudotuberculosis is heterogeneously expressed within a population, which is determined by the transcriptional T6SS4 activator RovC. Moreover, the T6SS4 and RovC are embedded in a complex and global regulatory network, including the global post-transcriptional regulator CsrA, the Yersinia modulator A (YmoA), the global protease Lon, and RNases (PNP and RNase III). Post-transcriptional processing of the T6SS4 polycistron and different transcript stability within the operon also achieve a higher regulatory complexity. In summary, our work provides new insights into the sophisticated and complex regulatory network of the T6SS4 of Y. pseudotuberculosis, which clearly differs from regulation in other organisms.
Authors summary
Bacteria use a specialized multi-protein complex called the Type VI secretion system (T6SS) to inject toxic proteins into other cells to compete with target microorganisms or to infect host organisms. While the T6SS has been extensively studied in some model organisms, much less is known about the function and regulation of the four T6SS clusters of the food-borne human pathogen Yersinia pseudotuberculosis. In this study, we found that the T6SS4 of Y. pseudotuberculosis is only expressed in a small subpopulation in vitro. This suggests that its regulation is fundamentally different from what is known in other organisms. We show that a complex regulatory network regulates T6SS4 gene expression, and the T6SS4 transcript is post-transcriptionally processed, resulting in different mRNA levels of the individual T6SS components. These findings contribute to a deeper understanding of how bacteria, especially Y. pseudotuberculosis, regulate complex secretion systems at multiple levels.
Citation: Kerwien A, Körner B, Meyer I, Teschke Y, Köster CS, Salto IP, et al. (2025) T6SS4 is heterogeneously expressed in Yersinia pseudotuberculosis and is a target for transcriptional and post-transcriptional regulation. PLoS Pathog 21(9): e1013356. https://doi.org/10.1371/journal.ppat.1013356
Editor: Igor E. Brodsky, University of Pennsylvania, UNITED STATES OF AMERICA
Received: July 6, 2025; Accepted: September 5, 2025; Published: September 24, 2025
Copyright: © 2025 Kerwien et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files and the RNA-seq data are publicly available from GEO with the identifier(s) GSE249386.
Funding: This work was supported by the University of Münster (AK, BK, IM, YT, CSK, IPS, PD, ASH) and the German Research Foundation (DFG), with grant No. DFG-STO1208/2-1 given to ASH, and grant of the priority programme SPP2002 grant No. DFG DE616/7-2 given to P. Dersch. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Bacteria in complex environmental settings are exposed to various rapidly changing conditions, such as temperature and nutrient availability, competing with other microorganisms or evading the mammalian immune system. Yersinia pseudotuberculosis can be found in soil, water, plants and is a human pathogen that causes various gut-associated symptoms such as diarrhea and enteritis [1–4]. Entry into the human host involves a significant temperature and nutrient composition change. Moreover, the bacteria have to switch from defending a niche against other microorganisms to evading the host’s immune system, which requires a rapid and precise change in gene expression of the respective virulence factors. Due to these distinct lifestyles, it is not surprising that the expression of virulence-associated and fitness-relevant genes of Y. pseudotuberculosis, including its Type III (T3SS) and Type VI secretion systems (T6SS), is tightly regulated by temperature [5,6]. One well-described example is the regulation of the Y. pseudotuberculosis Ysc-Type III secretion system (Ysc-T3SS). The Ysc-T3SS promotes the injection of antiphagocytic Yop (Yersinia outer protein) effector proteins into host cells and plays an important role in defending against the attack of phagocytic cells [7–11]. Expression of the Ysc-T3SS is activated by the transcriptional regulator LcrF (low calcium-response regulator) at 37°C via an RNA-thermometer and is transcriptionally repressed at 25°C by the Yersinia modulator A (YmoA) [12–17]. YmoA acts as a protein thermometer as it undergoes a conformational change and is rapidly degraded by the Lon/ClpP proteases upon a temperature upshift from moderate (25°C) to body temperature (37°C) [14,16].
In contrast to the regulation of the T3SS, much less is known about the regulation of the four T6SS clusters of Y. pseudotuberculosis, differing in their chromosomal arrangement and number of genes [18]. Exclusively encoded in Gram-negative bacteria, T6SSs are large contact-dependent secretion systems of 13 highly conserved core components that directly translocate a broad spectrum of different effector proteins into competing prokaryotic or eukaryotic cells [19–25]. As the assembly and disassembly of the apparatus are very energy-consuming, we expected that the expression of T6SSs of Y. pseudotuberculosis would be tightly regulated in response to environmental signals, as observed in other microorganisms [26–29]. Previous studies on the T6SS4 gene cluster of Y. pseudotuberculosis revealed that its expression is strongly temperature-dependent. It can only be induced at moderate growth temperature (25°C), predominantly during the stationary phase, but not at 37°C in vitro [18,30]. It was also found that expression of T6SS4 is promoted by direct binding of global regulators such as RpoS, OmpR, or RovM, and T6SS4 expression is positively regulated by quorum sensing [18,31–34]. Unlike any other yet known T6SS, Yersinia-T6SS4 gene expression further requires the expression of the specific hexameric transcriptional activator RovC [30]. RovC is encoded in the opposite direction upstream of the T6SS4 cluster and activates T6SS4 gene expression by direct binding within the T6SS4 promoter region [30]. The T6SS4 of Y. pseudotuberculosis is additionally controlled by the global regulator CsrA (carbon storage regulator A) [30]. The CsrA protein belongs to the Csr system, which includes two small non-coding RNAs CsrB and CsrC. They can bind and sequester multiple CsrA proteins, thus preventing CsrA from binding to its target mRNAs. The Csr system is known to be an important post-transcriptional regulatory system that influences the expression of many virulence- and fitness-relevant genes in bacteria [35–38]. In Y. pseudotuberculosis, CsrA was found to play an essential role in T6SS4 expression, as it affects rovC on the transcriptional and post-transcriptional levels [30]. Although the exact mechanism of how CsrA influences RovC synthesis is still unclear, CsrA was shown to repress the rovC transcription indirectly, but also to stabilize rovC mRNA, indicating a complex role in the regulation of RovC and, thus, of T6SS4 [30]. It is further known that the CsrA homologous protein RsmA (regulator of secondary metabolites A) negatively regulates the expression of all three T6SS islands in Pseudomonas aeruginosa, highlights the critical role of the Rsm/Csr system in T6SS regulation [39].
As several studies have shown that the expression of the T6SS4 is repressed at 37°C [18,30,31], it can be assumed that T6SS4 targets (micro-)organisms other than mammals, e.g., competing bacteria in their environmental niches. However, no antibacterial effector that is exclusively translocated by T6SS4 has yet been identified. In contrast, it was hypothesized that Y. pseudotuberculosis uses its T6SS4 to maintain the intracellular ion homeostasis of, e.g., manganese or zinc [40–43]. This suggests a role in the resistance to oxidative stress and could promote a higher virulence in mice [40,42,43]. However, no expression of the T6SS4 was detectable by RNA-sequencing under different virulence-relevant conditions at 37°C and during infection in other studies [6,44–47].
To gain further insight into the role of the T6SS4, its temperature control mechanisms, and function, we analyzed the expression control of the Y. pseudotuberculosis T6SS4 at transcriptional, post-transcriptional, and translational levels. To this end, we used a flow cytometry-based approach to study T6SS4 expression at a single-cell level. We found that T6SS4 cluster expression within a population is very heterogeneous, unlike T6SS gene clusters in Vibrio or Pseudomonas spp [19,22,23,26,48–50]. We could further show that the heterogeneous expression of the transcriptional regulator gene rovC promotes phenotypic heterogeneity. Moreover, heterogeneous rovC and T6SS4 gene expression are impacted by the global protease Lon, the RNases PNPase and RNase III, the RNA-binding protein CsrA, and the transcriptional regulator YmoA. The rapid downregulation of rovC and T6SS4 gene expression upon temperature shift from 25°C to 37°C involves distinct temperature-dependent post-transcriptional modifications of both rovC and T6SS4 mRNA.
Results
Flow cytometry-based method revealed heterogeneous expression of the T6SS4 at different temperatures
In previous studies, T6SS4 expression of Y. pseudotuberculosis was mainly studied in bulk approaches, revealing only an overall up- or downregulation of gene expression in a bacterial population [18,31,34]. In contrast to this general approach to analyze gene expression, we applied flow cytometry and fluorescence microscopy to study T6SS4 expression at a single-cell level. Therefore, we used a strain in which the core gene clpV4 of the T6SS gene cluster 4 (T6SS4) is chromosomally fused to gfp (Fig 1A). We could show that only a small subpopulation (approximately 10–15%) highly expressed clpV4-gfp (T6SS4-ON subpopulation), whereas the majority of the population remained repressed (T6SS4-OFF subpopulation) (Figs 1B and S1). This finding of phenotypic heterogeneity—referring to variable T6SS4 expression within a genetically identical population—differs from what was found in P. aeruginosa and Vibrio cholerae. In these model organisms, T6SS gene cluster expression was reported to be homogeneous among genetically identical cells [22,48,51–53]. To analyze a higher bacterial cell number, a flow cytometry-based method was established by gating for GFP-positive (GFP+, T6SS4-ON) and negative (GFP-, T6SS4-OFF) subpopulations (Figs 1C and S2). Incubation at 25°C resulted in an increasing amount of clpV4-gfp expressing bacteria after 8 h, when the cultures reached the stationary phase. In contrast, clpV4-gfp expression was rapidly downregulated after a temperature shift to 37°C and remained repressed throughout the bacterial growth phase (Fig 1B and 1C). This aligns with previous findings, describing a strong regulatory effect of temperature and growth phase on the T6SS4 expression [18]. It also illustrates that the overall induction of the T6SS4 expression is only triggered in a subset of the population.
(A) Scheme of YPIII T6SS4 cluster. Chromosomal fusion of clpV4 to gfp was used as a reporter to analyze expression of T6SS4 genes. (B) Fluorescence microscopy of wt clpV4-gfp. The bacteria were incubated for 6 h at 25°C or 37°C and imaged on 1% agarose pads. The scale bar represents 10 μm, and representative overlays of the GFP channel and brightfield are shown. (C) Quantification of clpV4-gfp expressing bacteria (GFP+) incubated at 25°C or 37°C. 1 x 105 bacteria were analyzed using flow cytometry. Data depict the mean and standard deviation of three independent experiments. Statistical differences were determined using a Two-Way ANOVA with Šidák correction. **** = p ≤ 0.0001.
The heterogeneous expression of the transcriptional regulator gene rovC causes heterogeneity of T6SS4
Our previous study revealed that expression of T6SS4 genes is activated by the hexameric transcriptional regulator RovC. It was further shown that deletion of rovC completely abolishes T6SS4 expression at 25°C [30]. Therefore, we assumed that rovC might only be expressed in the T6SS4-ON subpopulation. To test this hypothesis, we introduced a low-copy-number plasmid containing the translationally fused promoter region of rovC to mCherry (S2 Fig) into the Y. pseudotuberculosis strains expressing the chromosomally encoded clpV4-gfp fusion. With this dual reporter strain, the expression of rovC-mCherry and clpV4-gfp was analyzed simultaneously. As shown in Fig 2A, the rovC-mCherry fusion was weakly expressed in the majority of the bacteria (mCherrylow population), and these bacteria did not express clpV4-gfp (Fig 2B and 2C). In contrast, 15–20% of the bacteria showed a high expression level of rovC-mCherry and expressed clpV4-gfp. This strongly indicates that the heterogeneous expression of the transcriptional regulator gene rovC causes heterogeneity of T6SS4 expression.
(A) Flow cytometric analysis of a translational rovC promoter fusion to mCherry (pProvC rovC’-‘mCherry) resulted in two subpopulations of mCherrylow and mCherryhigh expression, which differed in their expression intensity (GeoMean ECD-A). Significant differences were determined using an unpaired t-test. (B) Fluorescence microscopy of Y. pseudotuberculosis YPIII clpV4-gfp pProvC rovC’-‘mCherry. Images of brightfield, GFP, and mCherry channel are shown, as well as an overlay of all channels. Representative images from one of three independent experiments are shown. Scale bar indicates 10 μm. (C) Quantitative Venn diagram of mCherry and gfp expressing bacteria, analysed by flow cytometry of wt clpV4-gfp with translational fusion of promoter region of rovC to mCherry (pProvC rovC’-‘mCherry). 1 x 105 bacteria were analysed. Data depict the mean of three independent experiments. Absolute numbers of GFP-expressing bacteria (green), mCherryhigh-expressing bacteria (red), mCherrylow-expressing bacteria (orange), and bacteria expressing both (yellow). ** = p ≤ 0.01, *** = p ≤ 0.001.
Influence of the global post-translational, post-transcriptional, and transcriptional regulators Lon, CsrA, and YmoA on temperature-dependent rovC and T6SS4 gene expression
Next, we aimed to gain insight into the molecular mechanisms of how heterogenous T6SS4 expression is controlled in response to temperature, e.g., which factors contribute to the rapid decrease of the number of T6SS4-ON bacteria in the population upon a temperature shift from 25°C to 37°C. Several global regulators of Yersinia have been shown to control gene expression in a temperature-dependent manner. One example is the global protease Lon [54–59], which was shown to be involved in the temperature-dependent degradation of the virulence regulators YmoA and RovA of Y. pestis and Y. pseudotuberculosis, respectively [60,61]. We first examined whether the Lon protease affects the number of T6SS4-ON bacteria in a temperature-dependent manner. We found that deleting the lon gene substantially increased clpV4-gfp (T6SS4-ON)-expressing bacteria at 25°C compared to the wildtype (Fig 3A). However, the number of the clpV4-gfp (T6SS4-ON)-expressing bacteria decreased rapidly when the culture was shifted from 25°C to 37°C. Based on this result, it is possible that Lon directly targets RovC or influences rovC transcription indirectly (Fig 3B). Western blot analysis demonstrated that the levels of T6SS4 components, such as Hcp4 and ClpV4, as well as the T6SS4 activator RovC are strongly increased in the absence of Lon at 25°C but not at 37°C (Fig 3C and 3D). This indicates that Lon exerts its influence on T6SS4 gene expression via the regulation of RovC. We further tested the impact of the lon gene deletion on the number of mCherryhigh (RovC-ON) bacteria in Y. pseudotuberculosis expressing the ProvCrovC’-‘mCherry reporter (S3 Fig) and the overall amount of the rovC transcript in the bacterial cells (Fig 3E). We found a higher number of RovC-ON bacteria and a significant increase in rovC transcript levels in the lon deletion mutant at 25°C. In contrast, the rovC transcript was downregulated at 37°C independently of the presence of Lon (Fig 3E), indicating that Lon controls the overall level of RovC but is not involved in temperature control.
(A) Quantification of clpV4-gfp expressing bacteria in wt clpV4-gfp (YP412) and ∆lon clpV4-gfp (YP544) at either 25°C or 37°C. 1 x 105 bacteria were analyzed using flow cytometry. Data depict the mean and standard deviation of three independent experiments. Significant differences were determined using a Two-Way ANOVA with Šidák correction. (B) Scheme of potential downregulation at 37°C mediated by Lon-dependent inhibition of the rovC transcription or by Lon-mediated proteolysis of RovC. Created in BioRender. Dersch, P. (2025) https://BioRender.com/8el9map. (C-D) Western blotting of wt clpV4-gfp and ∆lon clpV4-gfp. Protein levels of ClpV4-GFP, RovC, and Hcp4 were detected using antibodies against GFP, RovC, and Hcp4. Detection of RNAP was used as a loading control. Experiments were performed in three independent replicates; one representative Western blot is shown. (E) qRT-PCR was performed of total RNA extracted from wt and ∆lon incubated for 6 h at 25°C or 37°C. Specific primer pairs were used to determine expression levels of rovC, and log2 fold changes to sopB as a non-temperature-regulated reference gene [6] were calculated. Experiments were performed in three independent replicates, and the mean and standard deviations are shown. Significant differences were determined using an unpaired t-test. * = p ≤ 0.05, *** = p ≤ 0.001, **** = p ≤ 0.0001, ns = not significant p > 0.05.
Previous work has shown that the expression of the Csr system components of Yersinia, which are known to repress RovC and thus T6SS4 synthesis [30], is strongly controlled by temperature and carbon/nutrient sources [16]. CsrA regulates RovC synthesis in opposing ways: it represses transcription while promoting post-transcriptional stability [30]. Moreover, a Yersinia-specific histone-like protein YmoA, with homology to the E. coli Hha protein, is known to regulate Yersinia virulence factors, including the master regulator LcrF of the Ysc-T3SS gene cluster in a temperature-dependent manner [12,13]. From a transcriptomic analysis, it is further known that YmoA also influences the expression of several virulence-associated genes, such as vipA4, vipB4, and rovC in Y. pseudotuberculosis, and modulates the expression of the Csr system [12,15,16]. Based on this knowledge, we used csrA and ymoA deletion strains to test their influence on the synthesis of RovC and T6SS4 components at 25°C and 37°C (Fig 4A). A deletion of csrA or ymoA resulted in a significant increase in the number of T6SS4-ON bacteria at 25°C (Fig 4B), whereby the overall expression was significantly higher in the ∆csrA compared to the ∆ymoA mutant strain (Fig 4B and 4C). In addition, both gene deletions also led to a substantial increase in the RovC and Hcp4 levels at 25°C compared to wt (Fig 4D). However, no upregulation of RovC and the T6SS4 components was observed in the mutant strains at 37°C (Fig 4B-D). A qRT-PCR analysis further showed that rovC and T6SS4 gene transcript levels decreased significantly between 25°C and 37°C in both mutants (Figs 4E and S4), indicating that CsrA and YmoA affect RovC and T6SS4 expression but are not involved in temperature control.
(A) Schematic overview of potential regulation pathways, controlling the temperature-dependent synthesis of RovC and T6SS4. Regulation can be mediated by an independent repressor or by affecting transcript or protein stability on a post-transcriptional level. Created in BioRender. Dersch, P. (2025) https://BioRender.com/9cztzkr. (B-C) Quantification of clpV4-gfp expressing bacteria (B) and GFP expression intensity (GeoMean FITC-A) (C) using the wt clpV4-gfp (YP412), ∆csrA clpV4-gfp (YP559), and ∆ymoA clpV4-gfp (YP474) strains incubated overnight at either 25°C or 37°C. 1 x 105 bacteria were analyzed using flow cytometry. Data depict the mean and standard deviation of three independent experiments. Significant differences in B) and C) were determined using a Two-Way ANOVA with Tukey’s correction. (D) Western blotting of samples analyzed in (B). ClpV4-GFP, RovC, and Hcp4 protein levels were detected using antibodies against GFP, RovC, and Hcp4. RNAP was detected as a loading control. Experiments were performed in three independent replicates; one representative Western blot is shown. (E) Total RNA of an overnight culture of wt, ∆csrA (YP53), and ∆ymoA (YP50) was isolated to perform qRT-PCR. Specific primer pairs for rovC were used to determine the expression of rovC. Log2 fold changes were calculated between rovC and sopB as a non-temperature-regulated reference gene [6]. Experiments were performed in three biological replicates, and significant differences were determined using a Two-Way ANOVA with uncorrected Fisher’s LSD. ** = p ≤ 0.01, **** = p ≤ 0.0001, ns = not significant, p > 0.05.
Downregulation of T6SS4 expression at 37°C is promoted by post-transcriptional control of rovC mRNA levels
A previous experiment in this study comparing rovC mRNA levels at different temperatures revealed that the amount of rovC transcripts is significantly reduced at a growth temperature of 37°C compared to 25°C (Figs 3E and 4E). To gain further insight into the mechanism underlying temperature control, we tested the expression of the rovC gene in response to temperature using a translational reporter fusion (pProvCrovC’-‘mCherry). As shown in Fig 5A, no major shifts of the rovC-mCherryhigh and rovC-mCherrylow populations were detectable after 6 h of incubation at 37°C. Although similar numbers of rovC-mCherryhigh expressing bacteria were detectable 6 h after the temperature upshift, we could only detect clpV4-gfp (T6SS4-ON) expressing bacteria in cultures grown at 25°C (Figs 5A, 5B and S5), indicating that the activity of the rovC promoter is not subjected to temperature control.
(A) Wt clpV4-gfp expressing a plasmid-encoded ProvC rovC’-‘mCherry fusion was incubated for 6 h at 25°C or 37°C, and gfp- and mCherry-expressing bacteria were quantified by flow cytometry. 1 x 105 bacteria were analyzed for each time point using flow cytometry. Experiments were performed in four independent replicates; the mean and standard deviation are shown. Significant differences of GFP+ bacteria at 25°C and 37°C were determined using an unpaired t-test and of mCherry-expressing bacteria using a Two-Way ANOVA with Tukey’s correction. (B) Fluorescence microscopy of wt clpV4-gfp provC’-‘mCherry after 6 h of incubation at 25°C or 37°C. Representative images of the GFP and mCherry channels and an overlay of the brightfield and the GFP and mCherry channels were shown. Bacteria were imaged on agarose pads containing 1% agarose, and the scale bar represents 10 µm. (C) qRT-PCR was performed with total RNA extracted from wt, ∆pnp, and ∆rnc incubated for 2 h at 25°C or 37°C. Specific primer pairs were used to determine expression levels of the rovC gene, and log2 fold changes to sopB as a non-temperature-regulated reference gene [6] were calculated. Experiments were performed in three independent replicates, and significant differences were determined using a Two-Way ANOVA with Tukey’s correction. (D-E) RNA coverage of the rovC gene of wt, ∆pnp, and ∆rnc, incubated for 2 h at 25°C with the same scale (D) and an adjusted scale (E). Data were taken from Meyer et al. [46]. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001, ns = not significant p > 0.05.
Based on the fact that the amount of rovC transcript was significantly reduced at 37°C compared to 25°C, as demonstrated by qRT-PCR (Figs 3E and 4E), and by an RNA-sequencing analysis [6], we hypothesized that the rovC mRNA is a potential target of temperature-controlled RNase-mediated degradation. To test this, we analyzed rovC transcript levels in mutant strains lacking different RNase genes identified in Y. pseudotuberculosis YPIII [46]. We found that rovC transcript levels in bacteria grown at 25°C were significantly increased in mutants in which the polynucleotide phosphorylase (∆pnp) or the RNase III gene (∆rnc) was deleted (Fig 5C). The increase in rovC transcript levels was considerably more pronounced in the ∆pnp mutant compared to the ∆rnc mutant. We also performed a comparative rovC transcription profile analysis of the wildtype and both RNase mutants using an RNA-sequencing data set from our previous study [46]. As shown in Fig 5D and 5E, the overall read pattern covering the rovC gene is comparable, and no typical changes of the sequencing read patterns due to the processing by RNases were detectable [62–64]. This suggests that the influence of the RNases on rovC transcript levels is not direct. Furthermore, we revealed that the rovC mRNA amount was still significantly reduced at 37°C in both mutants compared to 25°C (Fig 5C). Hence, both RNases control the synthesis of RovC but do not seem to be mainly involved in their temperature control.
To further investigate rovC transcript stability, we artificially overexpressed rovC encoded on a medium copy plasmid from a temperature-insensitive, arabinose-inducible promoter to exclude regulatory mechanisms influencing rovC transcription (Fig 6A). We found that RovC-overexpression under the control of the PBAD promoter resulted in the synthesis of RovC at 37°C (Fig 6B). However, the overall amount of detectable RovC was 1.38 x higher when rovC was overexpressed at 25°C (Fig 6C). Accordingly, rovC transcript levels were higher at 25°C after induction (Fig 6D), emphasizing a post-transcriptional regulation of rovC mRNA levels in response to temperature. We further tested how overexpression of rovC under these conditions influences the induction of the T6SS4 gene cluster. For this purpose, we used a PT6SS4tssA4’-‘gfp reporter fusion in which the predicted T6SS4 promoter [18] and the first codons of the first gene of the operon (tssA4, Fig 1A) are fused to gfp. As shown in Fig 6E and 6F, three hours after induction of rovC overexpression both, the number of GFP-expressing bacteria and the overall expression of the gfp reporter fusion were significantly increased at 25°C and 37°C compared to empty vector controls. In line with rovC transcript and RovC protein levels in the bacteria (Fig 5B and 5D), the number of T6SS-ON bacteria and the overall expression of the PT6SS4tssA’-‘gfp reporter fusion was still higher at 25°C compared to 37°C. This further indicates that RovC is fully functional at 37°C when overexpressed from an alternative promoter, and thermally induced changes in the protein folding and/or quaternary structure of RovC triggered by the temperature upshift do not seem to be important for T6SS4 expression control.
(A) To investigate RovC functionality at 37°C, rovC was overexpressed from a non-temperature-sensitive PBAD promoter (PBAD rovC, provC+). Created in BioRender. Dersch, P. (2025) https://BioRender.com/vt98mb7. (B) Western blotting of wt clpV4-gfp harboring PBAD rovC+ (provC+) or empty vector (pV) 0 h and 3 h after inducing overexpression of rovC at 25°C or 37°C. Protein levels of RovC were detected with a specific antibody against RovC, and GAPDH was used as a loading control. Experiments were performed in three independent replicates; one representative Western blot is shown. (C) Ratio of RovC between 25°C and 37°C. The amount of detected RovC of all three Western blot replicates was normalized to the loading control, and the ratio between 25°C and 37°C was calculated. (D) Total RNA of samples in (B) was extracted to perform qRT-PCR to determine expression levels of the rovC gene. Expression levels were normalized to sopB as a non-temperature-regulated reference gene [6] and log2 fold changes 3 h after inducing overexpression of rovC between provC+ and empty vector control (pV) were calculated. Experiments were performed in three independent replicates. Significant differences were determined using an unpaired t-test. (E-F) Promoter region of T6SS4 and first codons of tssA4 were translationally fused to gfp on a plasmid to identify bacteria with an active T6SS4 promoter by flow cytometry. Strains additionally harbored provC+ or empty vector (pV), and overexpression of rovC was induced for 3 h at either 25°C or 37°C. The amount of GFP+ bacteria (E) and the expression intensity (F) of 1 × 105 bacteria were analyzed, and the data depict the mean and standard deviation of three independent experiments. Significant differences were determined using Two-Way ANOVA with Tukey’s correction. * = p ≤ 0.05, *** = p ≤ 0.001, **** = p ≤ 0.0001, ns = not significant p > 0.05.
Processing of the T6SS4 polycistronic mRNA leads to differential expression of T6SS4 genes in response to temperature
In our attempt to analyze how RovC influences T6SS4 transcript levels at different temperatures, we also tested the expression of the translational clpV4-gfp fusion after rovC overexpression (Fig 7A). Even though the PT6SS4 promoter was highly induced at 37°C in approximately 70% of the bacteria (Fig 6E), we could not detect an equivalent induction with the clpV4-gfp fusion. Compared to 25°C with 90% GFP-positive bacteria (T6SS4-ON), less than 5% of the population could be identified as T6SS4-ON at 37°C (Fig 7B). This suggests that not only the level of rovC mRNA but also that of T6SS4 transcripts is influenced by temperature. A qRT-PCR analysis comparing the transcript levels of five T6SS4 genes (tssA4, vipA4, hcp4, clpV4, and tssK4) after RovC induction supported this assumption (Fig 7C). The abundance of most transcripts was significantly lower at 37°C compared to 25°C (Fig 7C). One exception is hcp4, for which similarly high levels of the hcp4 transcript and the Hcp4 protein were detected at 25°C and 37°C temperatures (Fig 7C and 7D).
(A) Scheme of potential downregulation of T6SS4 expression at 37°C caused by an unstable T6SS4 transcript or T6SS4 proteins. Created in BioRender. Dersch, P. (2025) https://BioRender.com/947o0tl. (B) Quantification of clpV4-gfp expressing bacteria after inducing overexpression of rovC for 3 h at either 25°C or 37°C. Data depict the mean and standard deviation of three independent experiments. (C) Total RNA of samples analyzed in B) was extracted for qRT-PCR. Specific primer pairs for five T6SS4 genes were used to determine gene expression within the T6SS4 operon. All expression levels were normalized to sopB as a reference gene, and log2 fold changes 3 h after overexpressing rovC between provC+ and empty vector control (pV) were calculated. Experiments were performed in three independent replicates, and significant differences were determined using a Two-Way ANOVA with Šidák correction. (D) Western blotting of the same samples analyzed in (B). Protein levels of ClpV4-GFP and Hcp4 were detected using specific antibodies against GFP and Hcp4. GAPDH was used as a loading control. One representative Western blot out of three independent experiments is shown. * = p ≤ 0.05, *** = p ≤ 0.001, **** = p ≤ 0.0001, ns = not significant p > 0.05.
It is unlikely that this expression pattern is caused by an additional promoter located upstream within the T6SS4 operon, as no promoter or an additional transcriptional start site upstream of hcp4 is predicted [6,18]. Moreover, a translational hcp4’-‘gfp fusion construct harboring the 5’UTR upstream of hcp4 without the T6SS4 promoter was not expressed (S6 Fig). Taken together, this suggests that in addition to the rovC mRNA, the transcripts of the individual T6SS4 genes are post-transcriptionally controlled, but to a different extent.
To prove this, we compared the transcript levels of eight genes covering different regions of the T6SS4 operon under native (non-RovC-inducing) conditions using qRT-PCR (Fig 8A and 8B). We found that the overall abundance of transcripts covering the different genes of the operon varied significantly. The transcript levels of the first four encoded genes in the operon (tssA4, vipA4, vipB4, hcp4) were significantly higher compared to the genes further downstream (tssE4, tssF4, clpV4, tssK4) (Fig 8B). A comparison of the qRT-PCR data with the transcription profile of the individual T6SS4 genes using our RNA-sequencing data supported this observation and confirmed a particularly high abundance of the hcp4 transcript (S7 Fig).
(A) Schematic overview of the T6SS4 operon. (B) Total RNA of Y. pseudotuberculosis wt incubated for 6 h at 25°C was extracted to perform qRT-PCR. Specific primer pairs for eight T6SS4 genes were used to determine expression levels within the operon, log2 fold changes to sopB as a non-temperature-regulated reference gene [6] were calculated. Experiments were performed in three independent replicates, and significant differences were determined using an unpaired t-test. (C) Northern blot of total RNA of YP53 (∆csrA) and YP154 (∆rovC). A specific probe for hcp4 was used; 16S and 23S rRNA were used as loading controls. (D) Secondary structure of the wildtype intergenomic region between hcp4 and tssE4 was predicted using the mFold web server [65]. The region which was mutated is marked in yellow. (E) Total RNA of Y. pseudotuberculosis wt and a strain with the mutated loop structure grown for 6 h at 25°C was extracted to perform qRT-PCR. Specific primer pairs for six T6SS4 genes were used to determine expression levels within the operon, and log2 fold changes to sopB as a non-temperature-regulated reference gene [6] were calculated. Experiments were performed in three independent replicates, and significant differences were determined using a Two-Way ANOVA with Šidák correction. (F) Scheme of potential post-transcriptional T6SS4 regulation. The T6SS4 transcript is processed due to endoribonucleolytic cleavage (red scissors). The 5’ and 3’ protection loop up- and downstream of hcp4 protects the hcp4 transcript from further endonuclease activity and from being degraded by exoribonucleases (green pie). The loop structures of the 5’-UTR and 3’-UTR of hcp4 were predicted using the mFold web server [65]. Created in BioRender. Dersch, P. (2025) https://BioRender.com/efeis70. ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001, ns = not significant p > 0.05.
Subsequent Northern blotting with an hcp4 probe revealed multiple bands of different sizes, further indicating a processed T6SS4 transcript (Fig 8C). As T6SS4 expression in the Y. pseudotuberculosis wt is generally very low, the csrA mutant was used for better visualization of the hcp4 transcript on the Northern blot, as deletion of this global regulator leads to an overall upregulation of T6SS4 gene cluster expression (Figs 4 and S4). A rovC mutant strain was used as a negative control, as this abolishes T6SS4 expression [30].
The results strongly suggest that the differences in T6SS4 transcript levels result from post-transcriptional control mechanisms such as differential mRNA degradation, stabilization, and/or transcriptional termination. Secondary structure prediction of the intergenomic region between hcp4 and tssE4 revealed a GC-rich stem loop directly downstream of the hcp4 gene (Fig 8D). The hairpin stem was chromosomally mutated to test whether this loop functions as a potential transcription terminator, which would explain the observed transcriptional stop after hcp4 (Fig 8D, marked in orange). qRT-PCR revealed no differences in the transcript levels of the tested T6SS4 genes (Fig 8E). Only the transcript level for hcp4 was significantly reduced in the strain with the mutated stem loop compared to wt. This indicates that the loop may not function as a transcriptional terminator to pause transcription downstream of hcp4, but rather acts as a 3’-protective loop to resist exoribonucleolytic degradation of hcp4 mRNA after endolytic cleavage at this site (Fig 8F). The inspection of the T6SS4 cluster further revealed another potential stem-loop structure that could be formed in the intergenic region between vipB4 and hcp4 (Fig 8F). The intergenomic region additionally consists of several AUUA-motifs, which could be recognized and then cleaved by endoribonucleases. As the hcp4 probe interacted with a small RNA fragment of the expected size of the hcp4 gene of around 500 bp (Fig 8C), it is likely that this stem-loop structure also contributes to processing and/or stabilization of the hcp4 transcript.
Discussion
Y. pseudotuberculosis is a widespread environmental bacterium that can also infect mammals [1,2]. This dual lifestyle requires a precise and rapid change in gene expression in response to changing conditions, such as entry into the human body. In this context, temperature plays an important role in the regulation of gene expression in Y. pseudotuberculosis, since many virulence-associated and metabolic genes are strictly controlled by temperature [5,6]. In this study, we could show that the T6SS4 in Y. pseudotuberculosis is strongly regulated by temperature but in the opposite manner to the T3SS. At moderate temperatures (25°C), the T6SS4 gene cluster is heterogeneously expressed, with only 10–15% of the population producing the transcriptional regulator RovC at levels which are sufficient to induce T6SS4 expression. At 37°C, rovC is rapidly downregulated on post-transcriptional level, leading to a rapid and complete shut-off of T6SS4 gene expression. Moreover, T6SS4 is part of a complex regulatory network that is embedded in global regulation. We found that global regulators such as the carbon starvation system regulator CsrA, the global protease Lon, the Yersinia modulator protein YmoA, and the two RNases PNPase and RNase III repress RovC and T6SS4 synthesis at 25°C. This indicated a very sophisticated and tight control of T6SS4 gene expression in response to temperature and other environmental signals that differs fundamentally from the control of T6SS expression in other bacteria.
In this study, a deeper analysis of T6SS4 expression showed that differential expression levels of the Yersinia-specific transcriptional activator RovC drive T6SS4 heterogeneity (T6SS4-ON versus T6SS4-OFF) (Figs 1 and 2). Phenotypic heterogeneity has been intensively studied in recent years and can be triggered, for example, by microenvironments with different demands, leading to different gene expression within a subpopulation [66,67]. Phenotypic heterogeneity can also be a strategy to survive sudden environmental fluctuations. In this so-called bet-hedging strategy, only some bacteria express certain genes that are not necessary under normal conditions. This additional expression of non-required genes can cost energy and resources. Nevertheless, since no adaptation phase is necessary in the event of sudden changes, the survival of at least a subpopulation is ensured [68–72]. Such bistability of gene expression has been demonstrated for rovA in Y. pseudotuberculosis controlling invasin expression in response to temperature [73–75]. RovA undergoes a conformational change upon an upshift from 25°C to 37°C that increases its accessibility for proteases, and hence, its rapid degradation abolishes its positive autoregulation (positive feedback loop) [73–75]. The exact mechanism of how heterogeneity of rovC and thus, T6SS4 expression is achieved is still unclear. However, no autoregulation or -activation of RovC was detectable [30], indicating a substantially different mechanism from RovA. Moreover, the functional role of T6SS4 remains unknown. However, the presence of four different T6SS clusters in Y. pseudotuberculosis likely requires tight regulation to ensure that T6SS4 expression is restricted to the environments where it is most advantageous.
In contrast to Y. pseudotuberculosis, T6SS is not heterogeneously but equally expressed in most studied bacteria, including Vibrio and Pseudomonas, where the secretion system genes have been extensively studied [19,22,23,26,48–50]. Only one recent study in enteroaggregative E. coli (EAEC) has reported that its T6SS is ON in 60% and OFF in 40% of the bacteria [76]. Yet, the implicated regulatory factors in EAEC are very distinct from Y. pseudotuberculosis. They found that heterogeneous T6SS expression in EAEC is regulated by the interaction of Fur (ferric uptake regulator) with the T6SS promoter region and genetically controlled by GATC methylation sites [76]. In contrast, heterogeneous T6SS4 expression in Y. pseudotuberculosis is already determined by the heterogeneous expression of the transcriptional regulator RovC (Fig 2). In addition, neither Fur binding sites nor GATC methylation sites have been found for RovC, suggesting a different regulation of heterogeneous T6SS4 expression in Y. pseudotuberculosis.
Our study further showed that RovC synthesis is the major control hub for T6SS4 expression. All global regulators of T6SS4 and temperature changes seem to influence rovC transcript levels strongly but do not affect its promoter activity or protein stability. Several regulatory scenarios are possible. For instance, differential folding of the mRNA in response to temperature could expose or hide RNase cleavage sites [77,78] and/or influence translation efficiency [77,79–85]. Alternatively, the synthesis or activity of RNases targeting the rovC mRNA could be increased at higher temperatures, leading to degradation of the rovC transcript.
Like other functionally linked genes, all core components of T6SS4 are encoded in close proximity under the control of only one promoter and form a polycistronic mRNA [18]. As a result, only one mRNA is transcribed, which, in theory, should lead to stoichiometrically equal amounts of transcripts. However, not all components are needed to the same extent, and regulatory measures are implemented to adjust T6SS4 component synthesis to their functional need. In the case of the T6SS, for example, proteins that assemble the membrane or the base plate complex are required in lower quantities than those building the tubular structure, which consists of hundreds of copies of stacked Hcp proteins [21,86]. While different translation efficiencies have been described to resolve this problem in some cases [87,88], we show that T6SS4 gene expression differences have already occurred at the transcript level. Very low transcript levels were detected for the genes downstream of hcp4. In contrast, the mRNA abundance of the first four genes of the operon was significantly higher, particularly hcp4, encoding the tube protein. No additional promoter activity was detected upstream of hcp4 (S6 Fig), and no other additional transcriptional start site within the operon has been identified [6,18]. Thus, differential T6SS4 mRNA levels seem to result from endoribonucleolytic cleavage of the polycistronic T6SS4 cluster mRNA at two sites with stem-loop structures flanking the hcp4 gene. A very rapid exoribonucleolytic degradation can explain the low abundance of the T6SS gene transcripts downstream of hcp4. In contrast, both stem loops flanking the resulting short hcp4 transcript will likely protect the transcript from further cleavage, leading to very high hcp4 transcript and thus Hcp4 protein levels (Figs 8C and 5D). Differential processing and degradation of polycistronic mRNA are described for several bacterial operons [77,89–93]. Processing of polycistronic mRNAs, resulting in stabilization of upstream genes (e.g., by the presence of stem loop structures, as identified downstream of hcp4) was described for the maltose and iscRSUA operon in E. coli or the puf operon in Rhodobacter capsulatus [77,89,90,94]. Endonucleolytic cleavage of mRNAs can also lead to a lower abundance of upstream transcripts (as observed for tssA4, vipA4, vipB4, compared to hcp4) due to degradation by 3’-5’-exoribonucleases. Such a mechanism has been demonstrated for the pap and glycogen operon in E. coli [77,91,92,95]. Alternatively, as reported in other systems [89,96,97], strong translation of the hcp4 gene could prevent this gene from being degraded by 3’-5’ exoribonucleases.
The observed post-transcriptional regulation of the T6SS4 polycistron, leading to a different abundance of T6SS4 transcripts, fits with studies reporting differential T6SS protein amounts [86]. Some proteins, such as those coding for the baseplate, require only a small number of copies, whereas the tube and sheath structure components require many copies [21,29,52,98–100]. After a firing event, the sheath structure components VipA and VipB are disassembled by the ATPase ClpV and remain in the cell [29,49,101,102]. In contrast, Hcp is secreted with VgrG and the effector proteins into the target organism and can be found in the supernatant after a firing event [21,26,103,104]. This requires immediate de novo synthesis of the secreted proteins in case of a new firing event. To solve this, other organisms such as Vibrio spp. encode, in addition to the main T6SS island, auxiliary clusters with hcp or effector proteins under the control of a separate promoter [20,50,105,106]. Since a single promoter controls all T6SS4 genes in Y. pseudotuberculosis, the observed differential stabilization of the respective mRNAs is an effective way for Y. pseudotuberculosis to meet the different protein requirements.
To date, the precise role of T6SS4 is still unclear. The highest expression of rovC and T6SS4 at moderate temperatures and their strong repression at 37°C observed in this and other studies (Figs 1C and 5A, [18,31]) indicate a function outside mammalian hosts. This is supported by multiple RNA sequencing datasets, including an in vivo transcription profile of Y. pseudotuberculosis within the Peyer’s patches during a mouse infection [6,44,46,47], revealing a complete repression of T6SS4 gene expression at body temperature. Most interestingly, we found T6SS4-type clusters in the environmental-associated strains Winslowiella toletana, Serratia fonticlola, Enterobacillus tribolii, Trabulsiella guamensis, T. odontotermitis, which belong to the family of Enterobacteriaceae (Fig 9). The identified T6SS clusters are homologous in synteny and protein identity to the T6SS4 of Y. pseudotuberculosis and T6SS-A of Y. pestis. Among them, W. toletana shows the highest protein identity to the T6SS4 of Y. pseudotuberculosis, including the gene of the so far unique transcriptional activator RovC (Fig 9, > 70% amino acid identity). Initially described as Erwinia toletana, W. toletana exhibits an optimal growth temperature between 28°C and 30°C [107,108]. It was first isolated from olive knots in association with the plant pathogen P. savastanoi, and its presence has been shown to enhance olive tree infection [107,109]. T. guamensis was found in soil, vacuum cleaner dust, or human stool samples, T. odontotermitis and E. tribolii were isolated from the gut of termites and red fluor beetle, respectively [110–113]. S. fonticola is a widely distributed environmental bacterium, commonly found in soil or aquatic habitats [114,115]. Despite a few clinical cases of S. fonticola, none of the organisms are considered primary human pathogens. The high amino acid identity, particularly for W. toletana strongly indicates a similar function of the T6SSs, possibly linked to a plant- or insect-associated ecological niche [116].
The amino acid identity of T6SS4 components between Y. pseudotuberculosis and different strains is given as a percentage based on protein sequence similarity. Visualization of the cluster similarities was done using clinker webserver [117]. The scale bar represents 2.5 kb.
In contrast to these assumptions, one group reported that some potential T6SS4 effectors are essential for virulence in mice [40,42,43]. One of these potential effectors (YPK_3548) is encoded at the 3’-end of the T6SS4 cluster. This gene is however not present in the T6SS4-type cluster in Y. pestis and W. toletana (Fig 9), and its expression was not affected by the loss of the csrA gene, in contrast to all other T6SS4 cluster genes [30]. As all in vitro experiments to characterize the effectors were also carried out at 26°C [40,43], a more detailed analysis of the in vivo function of the T6SS4 system and its putative effectors is required to elucidate whether the T6SS4 system is important for a plant- and/or animal-associated lifestyle.
Materials and methods
Media and growth conditions
E. coli was grown overnight in 5 ml Luria-Bertani (LB) medium (5 g/l yeast extract, 10 g/l tryptone, 5 g/l NaCl) at 37°C. Super Optimal Broth with Catabolic Repression (SOC) medium (5 g/l yeast extract, 20 g/l tryptone, 10 mM NaCl, 2.5 ml KCl, 10 mM MgSO4, 10 mM MgCl2, 20 mM glucose) was used to allow E. coli cells to recover after transformation. The medium is based on Super Optimal Broth (SOB) medium [118], supplemented with glucose. Y. pseudotuberculosis was grown in LB (BD Bioscience, USA), supplemented with 1 mM CaCl2 or in brain-heart infusion (BHI) medium (37 g/l BHI, BD Bioscience, USA). Unless stated otherwise, Y. pseudotuberculosis cultures were incubated overnight at 25°C in LB, main cultures at either 25°C or 37°C. If necessary, selective antibiotics were added in the following final concentration: 100 µg/ml ampicillin, 50 µg/ml kanamycin, and/or 50 µg/ml chloramphenicol. To induce overexpression of rovC from pBAD30 plasmid, 0.1% arabinose was added to the culture in early exponential phase (OD600 0.7).
Plasmid and strain construction
In this study, molecular cloning of DNA into vectors was performed following the Gibson Assembly Protocol (E5510) (New England Biolabs), based on the method developed by Gibson et al. [119]. Plasmid DNA was isolated using the Nucleospin Plasmid kit (Macherey Nagel), and genomic DNA of Y. pseudotuberculosis was isolated using the ISOLATE II Genomic DNA Kit (Bioline). The oligonucleotides used for cloning, sequencing and qRT-PCR were purchased from Eurofins Genomic. PCRs for fragment amplification were performed using Q5 High-Fidelity 2X Master Mix (New England Biolabs) following the manufacturer’s instructions. PCR products were purified using Nucleospin Gel and PCR clean-up kit (Macherey Nagel). To verify correct clones, colony PCRs were performed using 2x DreamTaq Green PCR Master Mix (Thermo Scientific) according to the manufacturer’s instructions. Sanger sequencing (Microsynth Seqlab GmbH) confirmed successful cloning of plasmids. Plasmids and primers used in this study are listed in S1 and S2 Tables.
For cloning of pANK4 and pANK15, vector pFU98 was linearized by digestion with NheI and NotI (New England Biolabs, Ipswich, USA). For pANK4, the 5’-UTR of rovC (-579 to +13) was amplified with primers IX691/692 using Q5 High-Fidelity 2X Master Mix (New England Biolabs). Primers IX687/IX688 were used to amplify mCherry. A splicing by overlap extension PCR (SOE PCR) [120] was performed to generate a combined DNA fragment of 5’-UTR of rovC and mCherry. The combined DNA fragment was cloned into linearized pFU98 following the Gibson Assembly Protocol (E5510) (New England Biolabs). For generating pANK15, primers X108/X109 were used to amplify the 5’-UTR of YPK_3566, including the predicted promoter region of T6SS4 (-581 to +15) [18]. Primers X110/111 were used to amplify gfpmut3.1. SOE PCR was performed to generate the combined DNA fragment with primers X108/X111, which was subsequently cloned into linearized pFU98. For cloning of pANK25, vector pFU31 was linearized with SalI and NheI (New England Biolabs, Ipswich, USA). The 5’UTR of hcp4 (-212 to +30) was amplified with primers X382/X383. The amplified DNA fragment was subsequently cloned into linearized pFU31. For cloning of pANK45, the suicide plasmid pAKH3 was linearized by digestion with XmaI and SphI (New England Biolabs, Ipswich, USA). The upstream and downstream region of the intergenomic region between hcp4 (YPK_3563) and tssE4 (YPK_3564) were amplified with primers X558/X559 and X560/X561, respectively. A combined DNA fragment of the upstream and downstream region was generated with primers X558/X561 performing a SOE PCR.
All generated vectors were transformed into electrocompetent E. coli SM10 λpir. 500 ml of LB were inoculated 1:100 with an E. coli overnight culture and grown to an OD600 of 0.6 at 37°C. The culture was pelleted for 10 min at 8,000x g and 4°C, followed by two washing steps with 50 ml and 25 ml ice-cold water. After an additional washing step in 1 ml ice-cold water, the cells were centrifuged again for 10 min at 8,000 x g and 4°C. The pellet was resuspended in a final volume of 1.8 ml ice-cold 10% glycerol. 50 µl aliquots were immediately frozen in liquid nitrogen and stored at -80°C. For transformation, 2 µl of plasmid DNA was added to 50 µl competent cells and exposed to 2.2 kV (200 Ω, 25 μF) for 5 ms in a pre-cooled electroporation cuvette. Transformed E. coli cells were mixed with 1 ml SOC and incubated for 1 h at 37°C with permanent aeration to recover. Afterwards, the cells were pelleted for 2 min at 8,000 x g, resuspended in 100 µl medium, plated out, and incubated overnight at 37°C on agar plates with the desired antibiotic. Correct cloning of generated plasmids was confirmed by Sanger sequencing.
Electrocompetent Y. pseudotuberculosis strains were used to transform pVK25, pBAD30, pANK4, pANK15, pANK25, pFU31, pKD4, and pCP20. The 15 ml BHI medium was inoculated 1:50 with Y. pseudotuberculosis overnight cultures. After 3 h of incubation at 25°C, the culture was pelleted for 5 min at 8,000 x g and 4°C and washed twice with 5 ml ice-cold sterile water. After the second wash, the pellet was resuspended in 200 µl ice-cold water and immediately used for transformation. For each transformation, 2 µl plasmid DNA and a 50 µl aliquot of competent cells were added to a pre-cooled electroporation cuvette. Transformation was performed by exposing the cells for 5 ms to 2.2 kV (200 Ω, 25 μF). Subsequently, the transformed cells were allowed to recover in 1 ml BHI medium for 2 h at 25°C under constant aeration. 100 µl of each transformation was plated on agar plates with the corresponding antibiotics and incubated at 25°C for two days.
Construction of Y. pseudotuberculosis mutants
All strains used in this study are listed in S1 Table. Chromosomal Y. pseudotuberculosis mutants were generated by homologous recombination using suicide plasmids derived from pAKH3 [121], except for YP78. For chromosomal fusion of clpV4 to gfp, pASS90 was conjugated into the respective Y. pseudotuberculosis strains. For chromosomal deletion of the intergenomic region between hcp4 and tssE4, pANK45 was conjugated into Y. pseudotuberculosis wildtype [122]. Conjugation of Y. pseudotuberculosis was followed by sucrose selection on agar plates containing 6% sucrose. After conjugation, correct clones were confirmed by PCR and Sanger sequencing (Microsynth Seqlab GmbH).
To chromosomally delete lon (YPK_3232), a Red-mediated recombination method was used as described [123]. In brief, primers I407/I408 were used to amplify a kanamycin resistance cassette with homologous regions of YPK_3232 using pKD4 as template. The amplified DNA fragment was transformed into YPIII harboring pKD4. Loss of pKD4 and successful gene disruption were confirmed with colony PCR. The kanamycin resistance cassette was eliminated by transformation of the mutated strain with pCP20. Afterwards, the plasmid was cured by incubating for 3h at 42°C due to the temperature-sensitive replicon [123,124].
Fluorescence-based flow cytometry
Flow cytometry was performed as a high-throughput method to analyze gene expression and promoter activity using the CytoFLEX S (Beckmann Coulter, USA). To avoid the detection of spillover signal, a compensation matrix was applied for all used fluorochromes according to the manufacturer’s manual. To separate bacteria from the debris, the samples were stained with 5 µg/ml 4′,6-diamidino-2-phenylindole (DAPI) and detected by the PB450 channel. The ECD channel was used to differentiate live and dead cells, which were stained with 1 µg/ml propidium iodide (PI). FITC was used to detect GFP-expressing bacteria. In experiments, where rovC promoter activity was analyzed by gating for mCherry+ bacteria, the samples were not stained with PI, and mCherry+ bacteria were analyzed in the ECD channel. The threshold was set to 2500, defined by sideward scatter height (SSC-H). In this study, the gain for the SSC was set to 400, for the forward scatter (FCS) to 100, for PB450–300, for ECD to 1250, and for fluorescein isothiocyanate (FITC) to 700. For the analysis of GFP- or mCherry-expressing bacteria, the samples were incubated according to the respective experiment. Depending on the OD600, 1–10 µl were taken directly from the culture at indicated timepoints and added to 500 µl PBS with 5 µg/ml DAPI and 1 µg/ml PI. 1 x 105 bacteria were analyzed for every sample using the gating strategy displayed in S2 Fig.
Fluorescence microscopy
Fluorescence microscopy was performed using the Keyence epi-fluorescence microscope BZ-X (Osaka, Japan) at 60x magnification (Plan Apochromat 60x Oil). The samples were pelleted for 5 min at 8,000x g at room temperature and resuspended to an OD600 of 10. From this dense suspension, 1 µl was transferred to an agarose pad containing 1% agarose and 0.5 x PBS to avoid the swimming of motile Y. pseudotuberculosis. For an even surface, the agarose was poured on a microscopy slide, fitted with a gene frame (Thermo Fisher Scientific, Waltham, USA).
RNA isolation
Bacterial cultures were grown according to the respective experiment. At indicated timepoints, 2 ml of the culture were pelleted for 2 min at 10,000x g at room temperature. The pellet was directly frozen in liquid nitrogen and stored at -80 °C until isolation of total RNA. Total RNA was extracted using the Monarch Total RNA Miniprep Kit (New England Biolabs) according to the manufacturer’s protocol: “Total RNA Purification from Tough-to-Lyse Samples (bacteria, yeast, plant, etc.)”. The first part of the sample digestion and homogenization protocol was modified as follows: After thawing, the bacterial pellet was resuspended in 200 µl TE-buffer (100 mM Tris HCl pH 7.5, 1 mM EDTA pH 8) containing 10 mg/ml lysozyme (L6876, Sigma-Aldrich) and incubated for 10 min at room temperature. Afterwards, 2x volumes RNA Lysis Buffer were added and the sample was vortexed vigorously for 10 sec. After centrifugation for 2 min at 16,000x g, the supernatant was transferred to a gDNA removal column. The extraction was proceeded with the second part of the RNA Binding and Elution protocol. Total RNA was diluted with 50 µl of nuclease-free water, and the purity and concentration were determined using the Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, USA). Because of the high abundance of genomic DNA, an additional DNA digestion step was included after isolation of total RNA. For this, 7.5 µg RNA was filled up to 44 µl of nuclease-free water, 5 µl 10X TURBO DNase buffer, and 1 µl TURBO DNase enzyme (Invitrogen, Thermo Fisher Scientific, Waltham, USA). Samples were incubated for 30 min at 37°C, followed by adding 1 µl of TURBO DNase enzyme and incubating for 30 min at 37°C. The reaction was stopped by adding 5 µl DNase Inactivation Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, USA). After inactivation, the sample was centrifuged for 3 min at 16,000 x g at room temperature, and 35 µl were transferred to a fresh reaction tube. Purity and concentration of the RNA were again analyzed using the Nanodrop spectrophotometer, and the RNA was diluted to 15 ng/µl and stored at -80°C for further experiments.
Quantitative real-time PCR (qRT-PCR)
To determine the amount of transcript of different T6SS4 genes, qRT-PCR was performed using the Luna Universal One-Step RT-qPCR Kit (New England Biolabs) with the LightCycler 96 System (Roche). Each reaction was performed in technical triplicates in a 10 µl reaction, containing 5 µl Luna Universal One-Step Reaction Mix (2x), 0.5 µl Luna WarmStart RT Enzyme Mix (20x), 0.5 µl of each primer (10 µM) and 1 µl of the desired RNA template (15 ng/µl). The reaction was filled up with nuclease-free water to 10 µl. Relative changes in gene expression were calculated using sopB as a reference gene [6,125]. Primers used for qRT-PCR are listed in S2 Table.
Northern blot
In order to detect the hcp4 transcript, a DIG-labelled hcp4-specific probe was generated (Primers are listed S2 Table). Total RNA was isolated, and 15 µg/20 µl of RNA was used for the Northern blot. The samples were mixed with 4 µl of 5x loading dye (31% formamide, 2.7% formaldehyde, 0.1 mg/ml ethidium bromide, 4 mM EDTA pH 8, 20% glycerol, 0.03% bromphenole blue, 10% MOPS buffer (20x)). The samples were boiled 2x for 10 min at 70°C with 5 min in between at 10°C. The samples were immediately incubated for 2 min on ice before they were separated on a MOPS agarose gel containing 1.2 g agarose, 5.5 ml 20% MOPS buffer (400 mM MOPS, 100 mM sodium acetate, 20 mM EDTA) and filled up to 100 ml distilled water. After the separation, the 16 and 23S rRNA loading control were detected using UV-light. To transfer the RNA on a positively charged nylon membrane, vacuum blotting was carried out for 1.5 h and 5 bars. After UV-crosslinking of the membrane, the membrane was prehybridized for 1 h at 42°C in prehybridization buffer containing 20 ml formamide, 10 ml 20x SSC buffer (3 M NaCl and 0.3 M NaCitrate, pH 7), 8 ml 10x blocking agent (Roche Blocking reagent), 2 ml N-Laurylsarcosinate (20 mg/ml), 40 µl 20x SDS, and 2 ml distilled water. 3 µl of the DIG-labelled DNA probe was added to 200 µl water, heated (10 min, 95°C), and cooled down (5 min on ice) twice a row. After a final heating to 95°C for 10 min, the probe was directly added to 20 ml prehybridization buffer. The nylon membrane was incubated and hybridized with the DNA probe overnight at 42°C. Next, the membrane was washed twice for 5 min at room temperature with washing buffer 1 (0.2x SSC buffer, 0.1% SDS) followed by an additional washing step for 15 min at 68°C with washing buffer 2 (0.1x SSC buffer, 0.1% SDS). The membrane was then blocked for 1 h at room temperature in 1x blocking reagent (Roche Blocking reagent) in maleic acid buffer. To visualize the hcp4 transcript, the membrane was incubated for 1.5 h with a α-Digoxygenin antibody (Anti-Digoxigenin-AP (fab fragments), Roche) 1:6000 in 1x blocking agent in maleic acid buffer. After the incubation with the antibody, the membrane was washed twice for 15 min each at room temperature with CPD* washing buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% TWEEN-20, pH 7.5). Washing of the membrane was subsequently followed by equilibration for 5 min in the detection buffer (0.1 M Tris-HCl pH 9.5, 0.1 M NaCl). To finally detect the hybridized probe, the membrane was incubated for 5 min with 1 ml of the substrate solution (1:100 CDP* (CDP-Star system, Roche) in detection buffer). The signal was recorded via exposure on X-ray films (CL-Xposure, Thermo Scientific, USA).
Western blot
For the detection of RovC, Hcp4 and ClpV4-GFP proteins, cultures were incubated depending on the desired conditions. At indicated timepoints, whole cell extracts were prepared by pelleting 1 ml of each sample for 5 min at 8,000x g at RT. The pellet was resuspended in an adjusted 1x Laemmli buffer [126] (40% v/v glycerol, 240 mM Tris-HCl, 8% w/v SDS, 5% v/v β-mercaptoethanol and 0.04% w/v bromophenol blue) to an OD600 of 10 and heated for 10 min at 95°C. Proteins were separated on a 15% polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (Sigma) by Western blotting. Primary antibodies against RovC (1:1000 dilution) and Hcp4 (1:5000) were generated by Davids Biotechnology, ClpV4-GFP was visualized using a monoclonal antibody against GFP (#MAB 3580, Merck Millipore, 1:2000). GAPDH was used as loading control and visualized with a monoclonal antibody against bacterial GAPDH (#MA5–15738, Invitrogen, 1:2000).
β-galactosidase assays
The β-galactosidase assay, first described by Miller 1972 [127], was used to determine rovC promoter activity. Y. pseudotuberculosis strains harbored pAKH189 or pTS03, respectively were incubated at 25°C and 37°C. At indicated timepoints, 200 µl samples were permeabilized by adding 50 µl 0.1% SDS and chloroform, respectively. After incubation for 10 min, 1.8 ml Z-buffer was added (60 mM Na2HPO4, 20 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4). The reaction was started by adding 400 µl ONPG as substrate (4 mg/ml). The reaction was stopped by adding 1 ml of 1 M Na2CO3. The activities were calculated as follows: ß-galactosidase activity [Miller units] = 1000 x OD450 x ∆t (min)-1 x V (ml)-1 x OD600.
Supporting information
S2 Table. Oligonucleotides for DNA amplification.
https://doi.org/10.1371/journal.ppat.1013356.s002
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S1 Fig. clpV4-gfp expression is repressed at 37°C.
Fluorescence microscopy of Y. pseudotuberculosis wt clpV4-gfp incubated for two and 6 h at 25°C (A) or 37°C (B). Representative images of the brightfield and GFP channels and an overlay of both channels were shown. Bacteria were imaged on agarose pads containing 1% agarose, and the scale bar represents 10 µm.
https://doi.org/10.1371/journal.ppat.1013356.s003
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S2 Fig. Gating strategy to analyze heterogeneous T6SS4 and rovC expression.
(A) Samples were stained with DAPI to gate for bacteria. (B) PI staining to exclude dead bacteria. (C) Negative control (YPIII wildtype) for GFP-expressing bacteria. (D) Gate for GFP-expressing bacteria. (E) Negative control (YPIII wildtype) for mCherry-expressing bacteria. (F) Gates for mCherry-expressing bacteria, divided into low and high expression intensity. H = height, A = area, SSC = side scatter. Exemplary plots are shown.
https://doi.org/10.1371/journal.ppat.1013356.s004
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S3 Fig. Deletion of lon results in increased rovC promoter activity.
Wt clpV4-gfp pProvC rovC’-‘mCherry and ∆lon clpV4-gfp p pProvC rovC’-‘mCherry were incubated overnight at 25°C. 1 x 105 bacteria were analyzed with flow cytometry, and experiments were performed in four independent experiments. Statistical significance was tested with an unpaired t-test. ns = not significant p > 0.05.
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S4 Fig. T6SS4 gene expression is downregulated at 37°C.
(A-C) Total RNA of an overnight culture of wt, ∆csrA, and ∆ymoA was isolated to perform qRT-PCR. Specific primer pairs for five T6SS4 genes were used to determine the expression within the T6SS4 operon. Log2-fold changes were calculated between the T6SS4 transcript and sopB as a non-temperature-regulated reference gene [6]. Experiments were performed in three biological replicates, and significant differences were determined using a Two-Way ANOVA with Šidák correction **** = p ≤ 0.0001.
https://doi.org/10.1371/journal.ppat.1013356.s006
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S5 Fig. The rovC promoter activity is not subject to temperature-dependent control.
Fluorescence microscopy of Y. pseudotuberculosis wt clpV4-gfp harboring pProvCrovC’-‘mCherry. Strains were incubated for 2 h and 6 h at either 25°C (A) or 37°C (B). Representative images of the GFP and mCherry channels, overlays of the GFP and mCherry channels, and an overlay of all channels with the brightfield, were shown. Bacteria were imaged on agarose pads containing 1% agarose, and the scale bar represents 10 µm.
https://doi.org/10.1371/journal.ppat.1013356.s007
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S6 Fig. Accumulation of hcp4 transcript is not due to an additional hcp4 promoter.
A low copy plasmid harboring the upstream region of hcp4, phcp4’-‘gfp (-212 to +30 base pairs with respect to the translational start site) was transformed into YPIII wt. Samples were incubated in 20 ml LB overnight at 25°C. wt clpV4-gfp was used as a control to compare the amount of clpV4-gfp expressing bacteria to the potential hcp4 promoter activity. Samples were taken for flow cytometry, and 1 x 105 cells were analyzed. Experiments were performed in three biological replicates. The data depict the mean and standard deviation. pV = empty vector control.
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S7 Fig. hcp4 transcript is the most abundant within the operon.
RNA coverage of all T6SS4 genes from samples of YPIII wt, incubated for 2 h at 25°C. Data was taken from Meyer et al., 2024 [46].
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Acknowledgments
We would like to thank Dr. Ann-Kathrin Heroven for constructing the lon deletion mutant. We also thank the members of the Institute of Infectiology for helpful discussions throughout the course of this project.
Financial disclosure statement
This work was supported by the University of Münster (AK, BK, IM, YT, CSK, IPS, PD, ASH) and the German Research Foundation (DFG), with grant No. DFG-STO1208/2–1 given to ASH, and grant of the priority programme SPP2002 grant No. DFG DE616/7–2 given to P. Dersch.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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