RNase-mediated reprogramming of Yersinia virulence

RNA degradation is an essential process that allows bacteria to regulate gene expression and has emerged as an important mechanism for controlling virulence. However, the individual contributions of RNases in this process are mostly unknown. Here, we tested the influence of 11 potential RNases in the intestinal pathogen Yersinia pseudotuberculosis on the expression of its type III secretion system (T3SS) and associated effectors (Yops) that are encoded on the Yersinia virulence plasmid. We found that exoribonuclease PNPase and endoribonuclease RNase III inhibit T3SS and yop gene transcription by repressing the synthesis of LcrF, the master activator of Yop-T3SS. Loss of both RNases led to an increase in lcrF mRNA levels. Our work indicates that PNPase exerts its influence via YopD, which accelerates lcrF mRNA degradation. Loss of RNase III, on the other hand, results in the downregulation of the CsrB and CsrC RNAs, thereby increasing the availability of active CsrA, which has been shown previously to enhance lcrF mRNA translation and stability. This CsrA-promoted increase of lcrF mRNA translation could be supported by other factors promoting the protein translation efficiency (e.g. IF-3, RimM, RsmG) that were also found to be repressed by RNase III. Transcriptomic profiling further revealed that Ysc-T3SS-mediated Yop secretion leads to global reprogramming of the Yersinia transcriptome with a massive shift of the expression from chromosomal to virulence plasmid-encoded genes. A similar reprogramming was also observed in the RNase III-deficient mutant under non-secretion conditions. Overall, our work revealed a complex control system where RNases orchestrate the expression of the T3SS/Yop machinery on multiple levels to antagonize phagocytic uptake and elimination by innate immune cells.


Introduction
Bacterial pathogens employ a large variety of regulators and integrate control mechanisms to tightly control the synthesis of crucial virulence factors on the transcriptional and post-transcriptional levels.In particular, RNA-mediated control processes provide pathogens with a highly efficient strategy to quickly respond to changes in their environment outside and inside their hosts [1][2][3].Among these mechanisms are sensory and regulatory RNAs, which were shown to modulate virulence factor expression in response to external stimuli [1][2][3].Other important players, which have been less addressed in this scenario, are ribonucleases (RNases).They play a critical role in RNA metabolism as they degrade RNA and are involved in the processing and quality control of transcripts [4].RNases are enzymes that catalyze the cleavage of a phosphodiester bond within a double-or single-stranded RNA molecule (endonucleases) or from the 3'-or 5'-end (exonucleases) [5,6].While some RNases are highly conserved across different species (e.g.RNase E, PNPase), some are only present in certain bacteria, e.g.RNase III in Gram-negative bacteria.The susceptibility of a particular RNA to an RNase thereby depends on multiple factors: (i) intrinsic RNA properties, e.g.RNA secondary structure, (ii) rate of translation, (iii) RNA-binding proteins, and (iv) sRNAs [4,[7][8][9][10].
More recently, certain RNases have also been found to be crucial regulators of virulence genes.Among them are specific exonucleases, i.e.PNPase, and endoribonucleases such as RNase Y in Gram-positive bacteria (Staphylococcus aureus, Streptococcus pyogenes, and Clostridium perfringens) or RNase III in Gram-negative bacteria (Escherichia coli, Salmonella enterica serovar Typhimurium) [8][9][10][11].RNases are encoded in a single bacterium, and the functions of some of the enzymes are coregulated and can be complemented and/or compensated to a certain degree by others [12][13][14].Considering the essential role of RNases in RNA metabolism, it is expected that the expression and the activity of RNases are tightly regulated [6,15,16].Bulk RNA degradation was found to be dependent on a membrane-bound, multiprotein complex, the degradosome, including RNases, such as RNase E and the polynucleotide phosphorylase (PNPase), and auxiliary proteins, which assist and/or regulate the degradation process.The composition of the degradosome varies between
Yersiniae are Gram-negative bacteria of which Y. pestis, the causative agent of plague, and its enteric relatives Y. enterocolitica and Y. pseudotuberculosis are human pathogens.Y. pseudotuberculosis, the most closely related, direct ancestor of Y. pestis is an intestinal pathogen [22].It causes a variety of gut-associated diseases, such as watery diarrhea, enteritis, and mesenteric lymphadenitis, called Yersiniosis.Upon oral uptake, the bacteria enter and transfer through the intestinal epithelial layer into the underlying lymphatic tissues (Peyer's patches) where they rapidly proliferate extracellularly [23][24][25].During this infection phase, the yersiniae induce the expression and formation of the Ysc type III secretion system (Ysc-T3SS) encoded on the 70 kb virulence plasmid pYV/pIB1 [26,27].The Ysc-T3SS, a syringe-like machinery called the injectisome, injects 7-8 Yop effector proteins into professional phagocytes to protect the bacteria from phagocytosis, manipulate inflammatory processes, and induce cell death [28,29].Expression of the Ysc-T3SS/Yop machinery is tightly regulated in response to temperature and host cell contact by a complex network of regulatory factors [19,22,27,30].The majority of the ysc/yop genes is induced by the AraC-type transcriptional activator LcrF [31].Synthesis of LcrF is thermally regulated on the transcriptional and post-transcriptional levels.This is mediated by the thermo-sensitive transcriptional repressor YmoA and a cis-acting RNA thermometer element including the ribosomal binding site of lcrF [32,33].Synthesis of the Ysc-T3SS/Yop apparatus is also tightly linked to the activity of the secretion machinery.Pettersson et al. demonstrated that expression of the injectisome components and the Yops are strongly induced upon host cell contact [30].Cell contact-mediated induction can be mimicked by the depletion of Ca 2+ , a phenomenon known as the low calcium response.This converts the T3SS apparatus into the secretion state and enables a strong, synchronized activation of the ysc-T3SS and yop genes [34,35].In a previous study, we demonstrated that one of the secreted Yop proteins, YopD, plays a pivotal role as a host cell contact sensing device and negative regulator of Ysc-T3SS/Yop synthesis in the absence of inducing cues [19].YopD was found to coopt important global riboregulators to control the Ysc-T3SS/Yop master regulator LcrF.Secretion of the YopD translocator upon cell contact increases the ratio of the post-transcriptional RNA-binding regulator CsrA to its antagonistic small RNAs CsrB and CsrC.YopD secretion also reduces the levels of the degradosome RNases PNPase and RNase E [19].This indicated that riboregulators and RNA-mediated control processes play a pivotal role in the coordinated control of Yersinia virulence traits.
In this study, we extended our analysis and found that in particular RNase III and PNPase reduce the stability of the lcrF mRNA.PNPase exerts its main influence on Ysc-T3SS/Yop synthesis through the RNA-regulatory function of YopD [19], whereas RNase III influences translation and stability of lcrF mRNAs via (i) CsrA-antagonizing Csr RNAs, and (ii) likely by the control of ribosomal and translation-related factors.

PLOS PATHOGENS
RNase-mediated reprogramming of Yersinia their target specificity, cleavage mechanism, and role in bacterial physiology [7,10], and previous global expression analysis by RNA-seq showed that these RNases are all controlled in response to virulence-relevant parameters such as temperature and/or nutrient availability [36,37].As an uncontrolled/induced expression of the Ysc-T3SS/Yop machinery was previously shown to result in a growth reduction [38][39][40], we first characterized the growth behavior of the RNase mutants.Most RNase mutants did not influence bacterial growth (S1 Fig) .Only the PNPase and RNase III-deficient strains (YP139 (Δpnp), YP356 (Δrnc)) exhibited a growth delay at 25˚C and 37˚C, and complementation by the respective RNase gene could restore the phenotype of the wildtype (Fig 1A).In contrast to the Δpnp mutant, the growth defect of the rnc mutant was much stronger at 37˚C (Fig 1A).A similar growth arrest has been observed upon a constitutive expression of the Ysc-T3SS of Yersinia [38][39][40].We therefore also tested growth at 37˚C under Ca 2+ -limiting conditions, as a substitute for host cell contact, to induce Ysc-T3SS/Yop expression and Yop secretion.In agreement with previous results [39,40,38], induction of Yop secretion is accompanied by a strong growth reduction of the wildtype which was more severe in both RNase-deficient mutants (Fig 1A).
This prompted us to select the Δpnp and Δrnc mutant for further analysis by electron microscopy (Fig 1B).In contrast to the wildtype and the Δpnp mutant, additional surfaceexposed structures resembling needles of T3S injectisomes [41] were detected on the Δrnc mutant at 37˚C with scanning and negative staining electron microscopy, which are also seen with the wildtype under secretion conditions (37˚C/-Ca 2+ ) (Fig 1B).This indicated that the production of the Ysc-T3SS/Yop machinery is induced at 37˚C without Ca 2+ depletion in the Δrnc mutant, conditions under which the production and assembly of the apparatus are normally repressed [34,42].

Analysis of the influence of RNase III and PNPase on Yop secretion and expression
To test and characterize the influence of the PNPase and RNase III on the Ysc-T3SS/Yop machinery, Y. pseudotuberculosis wildtype strain YPIII and the isogenic Δpnp and Δrnc mutants were cultivated at 25˚C (i.e.non-expression conditions), 37˚C (i.e.T3SS/Yop-nonsecretion conditions) and at 37˚C/Ca 2+ -limitation which mimics host cell contact and triggers expression and activity of the Ysc-T3SS/Yop machinery (i.e.T3SS/Yop-secretion conditions) [34,42].A mutant strain deficient in the T3SS component YscS was used as a negative control as the encoded protein is essential for Yop secretion [43].Yop secretion by the wildtype was only detectable at 37˚C/-Ca 2+ (T3SS-secretion conditions), but not at 25˚C and 37˚C (Fig 2A).In contrast, slightly higher amounts of Yops were already secreted by the Δpnp mutant under non-secretion conditions (37˚C), and a significantly higher amount of secreted Yop proteins was detectable under identical conditions in the absence of RNase III (Fig 2A [38][39][40]. To confirm the influence of RNase III on the secretion of a specific Yop protein, we quantified the secretion of an introduced, plasmid-encoded YopE-β-lactamase fusion protein (YopE-BlaM) by nitrocefin assays after a shift from 25˚C to 37˚C +/-Ca 2+ (Fig 2B).In agreement with our previous results, secretion of the YopE-BlaM fusion protein was significantly increased in the Δrnc mutant at 37˚C, but not in the wildtype, whereas similar high amounts of

PLOS PATHOGENS
RNase-mediated reprogramming of Yersinia (Δpnp), and YP356 (Δrnc) were grown at 25˚C, 37˚C, and 37˚C/-Ca 2+ ; the secreted proteins in the supernatant of the cultures were precipitated with TCA and separated on SDS gels (left panels).The Yop secretion-deficient mutant YP101 (ΔyscS) was used as negative control.Secreted Yop proteins were quantified using ImageJ.Data represent the mean ± SD from three independent biological replicates relative to the amounts of Yops secreted by the wildtype at 25˚C (right panel).Significant differences were determined using the one-way Anova test and are indicated by asterisks (*P <0.05, **P = 0.01).c: indicates an unidentified secreted protein used to control the efficiency of protein precipitation (B) Secretion of a plasmid-encoded YopE-beta-lactamase (BlaM) fusion protein (illustrated in the upper panel) expressed in Y. pseudotuberculosis strains YPIII (wt), and YP356 (Δrnc) grown at 37˚C and 37˚C/-Ca 2+ for 4 h was determined.Data represent the mean ± SD from three independent biological replicates (lower panel).Significant differences were determined using the Student's t-test and are indicated by asterisks (****P<0.0001). https://doi.org/10.1371/journal.ppat.1011965.g002
To test, whether the synthesis of the Ysc-T3SS components and/or the Yops, or solely the secretion of Yop proteins is affected, Yop protein levels in whole cell extracts of Y. pseudotuberculosis YPIII (wt), and the Δpnp and Δrnc RNase mutant strains grown under Yop secretion (37˚C/-Ca 2+ ) or non-secretion conditions (37˚C) were analyzed by Western blotting using an anti-all Yop antibody (Fig 3).We found that higher amounts of the Yops are synthesized in the Δpnp mutant at 37˚C/non-secretion conditions.This is in agreement with a previous study, reporting that certain T3SS and yop genes and equivalent proteins (e.g.YpkA, YopD,

PLOS PATHOGENS
YscC) were about 1.5-2.3fold higher expressed in the absence of PNPase [18].The overall increase of the amounts of secreted Yops under 37˚C/non-secretion conditions is even higher and also significant in the Δrnc mutant when compared to wildtype (Fig 3B).This supported the secretion data (Fig 2), demonstrating that RNAse III has a stronger influence on the T3SS/ yop machinery than PNPase.The overall Yop protein levels of both mutants are almost identical under secretion and non-secretion conditions and comparable to the Yop levels of the wildtype under secretion conditions (37˚C/-Ca 2+ ); except for YopE.This effector seems to be considerably reduced in the absence of RNase III compared to the wildtype (Fig 3B and 3C).Notably, also the synthesis of the YopE-BlaM reporter is strongly increased at 37˚C in the Δrnc strain based on the β-lactamase activity (Fig 2B), similar to the synthesis of other Yops (Fig 3C).In contrast, the YopE protein was barely detectable in the same strain (Fig 3C).This indicated that not yopE expression but rather post-transcriptional steps of YopE synthesis/stability are affected in the absence of RNase III.
To elucidate how the loss of PNPase and RNase III triggers the synthesis of the Ysc-T3SS/ Yop components under non-inducing conditions, we next addressed the impact of the pnp and rnc knock-out mutations on known Ysc-T3SS/Yop control mechanisms and regulators.

PNPase and RNase III influence on Ysc-T3SS/Yops does not occur through an increase of the pYV copy number
Synthesis of the Ysc-T3SS/Yop injectisome components is controlled on different levels, including the regulation of the copy number of the virulence plasmid pYV (also named pIB1) in response to temperature and host cell contact/secretion [44].As the copy number of plasmids is subjected to complex regulation involving RNases [45], we employed qPCR to determine whether a knockout of the rnc or the pnp gene influences the pYV copy number.However, similar to the wildtype, we found only a subtle increase of the copy number from 1 to 2 copies in the mutants upon a shift from 25˚C to 37˚C, while an increase to 3-4 copies of pYV was observed under T3SS/Yop-secretion conditions (37˚C/-Ca 2+ ) (S3 Fig) .Accordingly, increased Yop expression and synthesis at 37˚C (Fig 3) cannot be explained by an increase in the pYV copy number and must be based on the deregulation of other control factors.

RNase III and PNPase mainly influence Yop protein expression through the control of LcrF
Expression of the yop and the Ysc-T3S component genes is mainly activated by the AraC-type transcriptional activator LcrF in response to temperature and host cell contact [19,31,33].Therefore, we tested whether the absence of RNase III and PNPase influenced LcrF synthesis.We found that the amount of the LcrF protein was significantly increased at 37˚C/non-inducing conditions in both RNase mutant strains (Fig 4A -4C).The overall influence of the loss of RNase III was more pronounced compared to the loss of PNPase (Fig 4A and 4B), and could be fully complemented by the rnc-encoding plasmid (Fig 4C).Interestingly, high LcrF levels in the Δrnc strain at 37˚C (no secretion) and 37˚C/-Ca 2+ (secretion) were comparable, demonstrating that LcrF synthesis is independent of the secretion control process in the absence of RNase III (Fig 4B and 4C).

RNase III and PNPase influence lcrF transcript levels
To unravel the molecular mechanism by which the RNases control LcrF levels, we first investigated the lcrF transcript level in the Δrnc and Δpnp mutant compared to wildtype at 37˚C.In agreement with previous studies [19,33], we observed that the lcrF transcript is generally very

PLOS PATHOGENS
RNase-mediated reprogramming of Yersinia unstable in the wildtype as visualized by a diffuse pattern of different-length transcripts hybridizing with the lcrF probe on Northern blots (Fig 5A).Although this general pattern did not change in the absence of both RNases a significantly higher amount of lcrF transcripts was observed in the absence of RNase III at 37˚C (Fig 5A), and this effect could be fully complemented by a plasmid-encoded copy of the rnc gene (Fig 5B).Loss of PNPase caused a smaller upregulation of lcrF mRNAs under these conditions, whereas no influence of the RNases was observed under secretion conditions at 37˚C/-Ca 2+ (Fig 5A).
In a parallel experiment, we also performed a comparative transcription profile analysis of the wildtype and the Δrnc mutant grown at 37˚C by RNA-sequencing (for more detail see also below).In agreement with the results of the Northern blots, we could detect a massive increase (Δpnp) (A), and YP356 (Δrnc) (B) were grown at 37˚C (+) and 37˚C/-Ca 2+ (-).The secretion-deficient mutant YP101 (ΔyscS) and the LcrF-deficient mutant strain YP179 (ΔlcrF) were used as a negative control.Whole-cell extracts were prepared and separated by SDS-PAGE.The transcriptional activator LcrF was detected by Western blotting using an LcrF-specific polyclonal antiserum.An antiserum against H-NS was used for loading control (left panel).Data were quantified by ImageJ and represent the mean ± SD from three independent biological replicates (right panel).Significant differences were determined using Student's t-test and indicated by asterisks (*P <0.05; ***P<0.001).(C) Y. pseudotuberculosis strains YPIII (wt), YP139 (Δrnc), and YP356 (Δrnc) pIVO20 (prnc + ) were grown at 37˚C (+) and 37˚C/-Ca 2+ (-); the LcrF-deficient mutant strain YP179 was used as a negative control.Whole-cell extracts were prepared and separated on SDS gels.Synthesized LcrF was detected by Western blotting using a polyclonal LcrF antiserum.An antiserum against RNA polymerase (RNAP) was used for loading control.https://doi.org/10.1371/journal.ppat.1011965.g004Upregulation of lcrF transcript levels in the Δrnc and Δpnp mutant could have occurred by the direct influence of the RNases on the stability of the lcrF mRNA or by an indirect effect on the activation of lcrF transcription.We first tested whether the overall increase of lcrF transcript levels in the absence of RNase III and PNPase is indirect and caused by an increase of lcrF transcription.For this purpose, we first used two reporter constructs, which both harbor the promoter region of the yscW-lcrF operon from -311 to +8 fused to phoA (transcriptional fusion, pMV53) and from -304 to +281 (including the entire 5' untranslated region) relative to the transcriptional start site fused to lacZ (translational fusion, pKB35) to compare the activity of the promoter at 37˚C.However, no increase in promoter P yscW-lcrF activity was detectable with both reporters in the Δrnc and the Δpnp mutant, respectively (S4 Fig).This demonstrated that an influence of the RNases on the initiation of lcrF transcription can be excluded.
To investigate whether the absence of RNase III and PNPase affects the stability of the lcrF transcript, we determined the effects of RNase III on lcrF mRNA decay and measured the halflife of the transcript in the wildtype, the Δrnc, Δpnp, and Δpnp/Δrnc mutant by Northern blot analysis (Fig 6A and 6B).The half-life of the lcrF mRNA in the Δrnc mutant was two-fold higher than in the wildtype.Loss of PNPase also increased lcrF stability although to a lesser extent and not significantly, and the effect of both RNases on lcrF transcript stabilization seems to be additive (Fig 6B).
Next, we investigated whether the RNases act directly on the lcrF mRNA, but according to our analysis, it is very unlikely that the lcrF transcript is a direct target of RNase III.Firstly, we did not identify potential RNase III cleavage sites (e.g.~22 nt double-stranded RNA segments including base-pairing preferences such as 5' GAAAnnAAAG/CUUUnnUUUG 3') within the yscW-lcrF transcript, which were identified in direct targets in previous surveys [46][47][48].Only one longer double-stranded RNA segment without a potential RNase III cleavage sequence was identified in the 5' untranslated region (5'-UTR) of the yscW-lcrF transcript.However, its presence in the 5'-UTR of the P yscW-lcrF -lacZ reporter fusion (pKB35) did not result in an expression change in the Δrnc mutant compared to wildtype (S4B Fig) .Secondly, no smallersize degradation products of the lcrF transcript were detectable in the mRNA pool of the wildtype compared to the Δrnc strain in gels with a high resolution (S5 Fig) .Thirdly, although we detected a massive increase of reads mapping to the lcrF mRNA in our RNA-seq analysis when visualized with the same nucleotide score height, no obvious differences in the RNA read patterns covering the lcrF gene region of the wildtype and the Δrnc mutant were detectable (Fig 5C and 5D).Moreover, no typical endoribonuclease-mediated processing events generating additional sharp/trimmed read ends in the wildtype were identified (Fig 5D) as described for direct mRNA targets [49,50].These results indicated that RNase III is unlikely to target the lcrF mRNA directly at 37˚C, but rather affects post-transcriptional regulators that have an overall influence on lcrF transcript levels, such as the effector protein YopD and the carbon storage regulatory system CsrABC [19].

Mutual influence of RNase III, PNPase, and the translocon protein YopD
Besides its role as a structural component of the translocation pore of the T3SS for Yop delivery [51,52], YopD also acts as a negative post-transcriptional regulator that represses the synthesis of the master regulator LcrF and components of the Ysc-T3SS/Yop system under nonsecretion conditions [19,53,54].To test whether PNPase and RNase III affect YopD synthesis, we compared intracellular YopD levels between wildtype and RNase mutants.

PLOS PATHOGENS
The amount of YopD was strongly increased in the absence of RNase III at 37˚C with a similar level observed in the wildtype at 37˚C/-Ca 2+ (Fig 7A).However, YopD upregulation does not explain the observed phenotypes of the Δrnc mutant, as the presence of YopD was shown to reduce and not increase lcrF mRNA levels, and Ysc-T3SS/Yop synthesis [19,53,54].On the contrary, YopD seems to be upregulated in the absence of RNase III as part of the entire Ysc-T3SS/Yop secretion complex due to higher LcrF levels (Fig 3B).
Moreover, the amount of the transcriptional activator LcrF is similarly increased in the Δrnc and ΔyopD mutant compared to wildtype, but the overall LcrF level is much higher in the Δrnc/ΔyopD double compared to the single mutants (Fig 7B), indicating an additive effect.This shows that RNase III regulates ysc-T3SS/yop gene expression not via but rather together with YopD.
Loss of PNPase resulted also in the upregulation of YopD, but its influence was much less pronounced (Fig 7A).A comparison of the ΔyopD and the Δpnp/ΔyopD mutant revealed no difference in LcrF levels, whereby the overall repressive effect of YopD was generally stronger Surprisingly, the production of LcrF and YopD did not further increase in the Δpnp/Δrnc double mutant as expected from the single mutation.In contrast, the Δrnc/Δpnp double mutant contained less YopD and LcrF than the Δrnc single mutant (Fig 7A and 7B).This indicated an interplay of the RNases in which the loss of PNPase seems to counteract the upregulation of LcrF promoted by the loss of RNase III.It is known that RNase III cleaves within the 5' end of the pnp transcript and initiates its rapid degradation in E. coli; as a result, PNPase mRNA levels are significantly increased in the E. coli Δrnc mutant strain [55,56].Significantly higher amounts of the pnp transcripts were also identified in the Δrnc mutant of Y. pseudotuberculosis grown at 37˚C by the RNA-seq analysis (log 2 = 2.2 (T1) 1.9 (T2);

Impact of RNase III on the Csr system
As the mechanism of how RNase III controls lcrF mRNA levels independently of YopD remained unclear, we tested whether RNase III influences the amount of CsrA.CsrA is another post-transcriptional regulator known to increase the stability of the lcrF transcript in response to host cell contact/secretion [19].CsrA belongs to the carbon storage regulator (Csr) system together with the two regulatory RNAs CsrB and CsrC which sequester multiple CsrA dimers to eliminate their function (Fig 8A, right panel).Loss of RNase III did not influence the overall amount of CsrA (Fig 8A).However, expression of the counteracting CsrB and CsrC sRNAs was significantly reduced in the absence of RNase III (Fig 8B).This, in turn, increases the amount of unbound, active CsrA, which is known to strongly reduce the degradation of the lcrF mRNA [19].
The molecular mechanism of how CsrA stabilizes the lcrF mRNA is currently unknown [19].However, as CsrA was found to enhance translation of the lcrF transcript [19], and a higher ribosome occupancy upon upregulation of translation was shown to protect mRNAs from RNase-mediated degradation [57,58], it seemed possible that increased availability of active CsrA in the Δrnc mutant enhances translation and thus lcrF mRNA stability.Based on this assumption, we tested the influence of RNase III on the expression of a translational reporter fusion in which the lacZ gene is fused to codon 25 of the lcrF gene, harboring the intergenic RBS and the entire promoter region of the yscW-lcrF operon up to position -304 according to the transcriptional start site (Fig

RNase III influence on the translational machinery
The increase of active CsrA could explain the Δrnc phenotype as the CsrB and CsrC levels are reduced to approximately 50% in the RNase III-deficient strain (Fig 8B).However, additional identified expression changes of components of the translation machinery may also have an influence.Our RNA-Seq analysis comparing the transcription profiles of the wildtype and the Δrnc mutant revealed that the transcript levels of 11 ribosomal proteins (r-proteins), as well as several translation initiation factors and rRNA/tRNA-modifying enzymes, were significantly altered with a log 2 fold change of +/-�1 (S1 Table).Among them are (i) the r-protein transcripts of rpsJ (S10, log 2 = 3.059) and rplC (L3, 2.784) encoded by the S10 operon involved in rRNA and r-protein regulation [59]; (ii) the translational initiation factor IF-3 (InfC) (log 2 of 1.02), an essential bona fide factor that promotes 30S initial complex formation, accurate tRNA selection, and fidelity of bacterial translation initiation [60,61], (iii) the 16S rRNA methyltransferase RsmG/GidB with the ability to enhance the loading of r-proteins and 30S ribosome assembly in the presence of the 5'-leader [62], and (iv) the 16S rRNA-processing protein RimM assisting in the assembly of the 30S subunit, and promoting translation efficiency [63,64].The higher abundance of these translation factors in the Δrnc mutant could also increase (or assist CsrA-mediated activation of) lcrF translation and thus lcrF mRNA stability.

RNase III-mediated global reprogramming of the Y. pseudotuberculosis transcriptome in response to virulence-relevant conditions
Based on our results, we assumed that loss of RNase III has a global influence on the gene expression pattern of the Yersinia chromosome and the virulence plasmid.To obtain more information about the overall influence of RNase III, we performed transcriptomic profiling by strand-specific RNA sequencing followed by a differential expression analysis (DESeq) by comparing sequence reads from strand-specific libraries of Y. pseudotuberculosis wildtype and the Δrnc mutant strain.For this purpose, the bacteria were grown at 25˚C (T0) (environmental, non-T3SS expression conditions), and then shifted to 37˚C (host, T3SS non-secretion), and 37˚C/-Ca 2+ (host, T3SS secretion conditions) for 1 h (T1) and 4 h (T2). 1 h was chosen as this is the earliest time point at which an LcrF/Yop upregulation can be detected, and 4 h was chosen for maximal T3SS induction indicated by the strong expression of the activator LcrF, and synthesis of the Yops (S6 Fig) .From each library, between 2-3 million cDNA reads were generated and mapped to the YPIII genome sequence (NC_010465) and the pYV virulence plasmid (NC_006153) (S1 Dataset).The global gene expression profiles of the different samples were distinct, and profiles of the three biological triplicates clustered together (S7 Fig).
The detailed analysis of the transcriptome revealed that approximately >99% of the reads of the Y. pseudotuberculosis YPIII (wt) and the Δrnc mutant grown at 25˚C mapped to the chromosome (4,7 Mb, 4,250 genes).The read distribution was only slightly changed in the wildtype when the bacteria were shifted to 37˚C.Under these conditions, more reads were found to map to the virulence plasmid pYV (99 genes, 66 kb) at T1 and T2.However, this still accounts for less than 3% of the total reads (Fig 9A and S2 Dataset).This changed significantly under secretion conditions (induced by the depletion of Ca 2+ ).Approximately 56% (T2) of the reads were assigned to the pYV, and only 44% (T2) were mapped to the chromosome.This indicated a major shift of the transcriptome from chromosomally-to virulence plasmidencoded genes.Most strikingly, a similar shift was observed in the Δrnc mutant grown at 37˚C under non-secretion conditions (+Ca 2+ ) (Fig 9A and S3 Dataset).About 47% (T2) of the entire transcripts originated from the virulence plasmid, which resembled the situation in the wildtype under secretion conditions (-Ca 2+ ).This is particularly striking because the virulence plasmid represents only 1.5% of the entire Y. pseudotuberculosis genome.
To identify which global pathways are particularly affected by the absence of RNase III at 37˚C under non-secretion conditions, we summarized the genes up-or downregulated (log2-fold change (log2FC) � +/-2, p-value � 0.05) in groups based on their association with the pKB34 encoding the yscWlcrF-lacZ translational fusion was transformed into Y. pseudotuberculosis strains YPIII (wt) pAKH85 (pV, empty vector), YP356 (Δrnc) pAKH85 (pV, empty vector) and the complementing strains YPIII (wt) pIVO20 (prnc + ) and YP356 pIVO20 (prnc + ).The transformants were grown at 37˚C and beta-galactosidase activity was determined, respectively.Data represent the mean ± SD from three independent biological replicates.Significant differences were determined using Student's t-test and indicated by asterisks (*P <0.05).https://doi.org/10.1371/journal.ppat.1011965.g008Our results further showed that loss of RNase III at 25˚C (T0: 33 genes) was rather moderate, whereas a shift to 37˚C had a significant impact on the transcripts of numerous genes compared to wildtype (37˚C, T1: 194 genes, T2: Notably, the abundance of almost all pYV-encoded Ysc-T3SS/Yop-encoding transcripts, including the transcripts of the yscW-lcrF operon was strongly increased in the Δrnc mutant at 37˚C compared to wildtype at 37˚C (S2 Based on this analysis, we hypothesized that secretion-relevant genes are already induced under non-secretion conditions at 37˚C in the absence of RNase III.To prove this assumption, we compared transcriptome changes of the wildtype upon the shift from nonsecretion to secretion conditions (YPIII, 37˚C/+Ca 2+ vs. 37˚C/-Ca 2+ ) (S2 Dataset) with changes of the wildtype and the Δrnc mutant at 37˚C under non-secretion conditions (YPIII, 37˚C/+Ca 2+ vs. Δrnc, 37˚C/+Ca 2+ ) (S4 Dataset).Notably, of the 28 pYV-encoded transcripts which were found in a higher abundance in the Δrnc mutant at 37˚C at T2, 27 were also increased under secretion conditions in the wildtype to almost the same magnitude.This indicated that lcrF and T3SS/yop expression upon host cell contact could be triggered by a reduction of functional RNase III during the infection.
We further found that besides pYV-encoded factors, only a few other chromosomallyencoded transcripts were induced in the Δrnc mutant at 37˚C at T1; among them are several translation-relevant factors, e.g. the ribosome-association factor Rai/YfiA (log2FC = 1.5), the ribosomal protein S2/RpsB (log2FC = 1.3), and the translation initiation factor IF3 (log2FC = 1).These components of the translational machinery could also promote/assist in the global reprogramming of the Yersinia transcriptome in the absence of RNase III.

RNase III-mediated influence on virulence-relevant Yop-injection and phagocytosis
Next, we analyzed whether global reprogramming in the absence of RNase III under nonsecretion conditions (37˚C), which includes the upregulation of all pYV-encoded secretionrelevant genes, improves the defense against the attack of phagocytic cells.To do so, we first to the chromosome or pYV under the different tested conditions are shown.(B) Influence of RNase III on global pathways at T1 and T2 at 37˚C.Differentially expressed genes between wildtype and the Δrnc mutant with a log2FC cut-off of -2/+2 and Pvalue � 0.05 were used for the analysis of global biological pathways based on the categories established according to the KEGG database.(C) Quantitative analysis of differentially expressed genes between wildtype and the Δrnc mutant.Bar plots show the absolute number of differentially expressed genes (left panel) after Ca 2+ depletion after 1 h (T1) or 4 h (T2) of the wildtype (wt) or the Δrnc mutant, or (right panel) between the wildtype (wt) and the Δrnc mutant at 25˚C (T0), before the shift to 37˚C for 1 h (T1) or 4 h (T2) in the presence or absence of Ca 2+ .https://doi.org/10.1371/journal.ppat.1011965.g009

PLOS PATHOGENS
RNase-mediated reprogramming of Yersinia infected activated human THP-1 macrophages with the wildtype and the Δrnc mutant harboring the plasmid-encoded YopE-β-lactamase fusion protein (YopE-BlaM).Translocation assays demonstrated that the loss of RNase III resulted in a strong increase in YopE-BlaM injection into THP-1 macrophages.As shown in Fig 10A and 10B, the majority of THP-1 cells treated with the Δrnc mutant turned blue as a measure of translocation.Upon infection with the wildtype, considerably fewer THP-1 macrophages showed a blue fluorescence, and most of them exhibited a lower blue fluorescence intensity (Fig 10A and 10B).As expected, macrophages treated with the strains harboring the control plasmid remained green fluorescent.A similar YopE-BlaM translocation pattern was observed when the translocation was analyzed in human epithelial cells (S8 Fig) .This indicated that the Yop translocation efficiency is considerably increased in the absence of RNase III.
As Yop injection has repeatedly been shown to prevent the phagocytosis of Yersinia [28,29], we tested whether the loss of RNase III affects the phagocytic activity of macrophages.For this, wildtype and Δrnc mutant bacteria were grown at 37˚C and added to activated THP-1 pseudotuberculosis strains YPIII (wt) and YP356 (Δrnc) were added to human THP-1 macrophages in an MOI of 30, and after 1 h intracellular amounts of the bacteria were determined as described in Material and Methods.Data represent the average ± SD from three independent biological replicates relative to the phagocytosis of the wildtype defined as 1.0.Significant differences were determined using the Student's t-test and are indicated by asterisks (**** P<0.0001).https://doi.org/10.1371/journal.ppat.1011965.g010

PLOS PATHOGENS
macrophages, and the number of intracellular bacteria was quantified.We found that the number of phagocytosed bacteria was significantly reduced with the Δrnc mutant compared to wildtype (Fig 10C).This strongly suggested that global reprogramming initiated by a loss/ downregulation of RNase III is accompanied by reduced growth, but a higher capacity to successfully defend against the attack of macrophages.

Discussion
Gram-negative bacteria possess roughly 20 RNases which control RNA turnover and maturation, and this plays an important role in many virulence-relevant processes [4,7,10].RNases can possess specificity towards certain RNA types (e.g.sRNA or dsRNA) and their activity can also depend on the intrinsic folding of the RNA, length of the transcript, bound ribosomes, and regulatory proteins [4,7,10].Among them are the highly conserved 3'->5' exonuclease PNPase and the endoribonuclease RNase III.In this study, we demonstrate that both RNases are global regulators in Y. pseudotuberculosis with multiple cellular targets, including virulence factors, such as the type III secretion system affecting Yop secretion.
Loss of PNPase had a moderate influence on the expression of T3SS/yop genes, and a comparable effect has been reported for the expression of certain T3SS and yop genes previously [18].In contrast, the absence of RNase III led to a strong increase of T3SS injectisome formation and Yop protein synthesis and secretion under normally non-inducing conditions (37˚C/ +Ca 2+ ).The ysc-T3SS/yop expression pattern of the Δrnc strain was very similar to that of the

PLOS PATHOGENS
wildtype under secretion conditions (37˚C/-Ca 2+ ), suggesting that loss of RNase III triggered a process equivalent to host-cell contact/Ca 2+ depletion.
A detailed analysis assigned to identify the underlying molecular mechanism revealed that Ysc-T3SS/Yop induction in the Δpnp and Δrnc mutant occurred through the upregulation of the AraC-type master activator LcrF of the ysc-T3SS/yop genes.As expression of the lcrF gene is part of a complex regulatory network, implicating multiple transcriptional regulators (YmoA, H-NS, IscR, RcsB), post-transcriptional regulatory elements (e.g. a fourU RNA thermometer, RNA binding proteins YopD and CsrA) [19,31,33] as well as plasmid-copy number control systems (RepA, CopA/B) [44], we tested the influence of both RNases on different control levels of LcrF synthesis.
We found that the loss of both RNases did not affect the transcription of the lcrF gene and the copy number of the virulence plasmid, but led to increased lcrF mRNA levels/stability.However, against our first assumption, the lcrF mRNA is most likely not a direct target of the RNases.RNase III is known to target intramolecular and intermolecular double-stranded RNA segments located mostly 5' or 3' to a coding sequence [47,48].However, no sequences with similarity to the described base-pairing preferences of RNase III cleavage sites could be identified within the yscW-lcrF transcript.Moreover, no characteristic RNase-mediated degradation products and no typical sequencing read pattern changes of the lcrF mRNA due to processing by RNase III or PNPase were detectable, as described for targeted mRNAs of other bacteria [49,50].
This suggested that the observed RNase-mediated influence on lcrF transcript levels may be an indirect consequence of the control of regulators known to affect the stability of the lcrF mRNA.One such regulator is YopD, whose intracellular presence in the absence of host cell contact/secretion was shown to destabilize the lcrF transcript [19].The comparative analysis of Δpnp, Δrnc, and ΔyopD single and double mutants and their influence on LcrF synthesis revealed a complex control circuit in which RNase III, PNPase, and YopD are tightly interconnected and mutually affect their synthesis (Figs 7D and 11).In this network, PNPase reduces the stability of the lcrF mRNA, most likely through the downregulation of YopD.The overall influence of PNPase on lcrF transcript levels via YopD is rather moderate.This can be explained by feedback control, as the loss of YopD also resulted in a significantly lower abundance of pnp mRNAs [19].Similar to PNPase, RNase III reduces the stability of the lcrF transcript, but its overall influence is much more pronounced.Loss of the rnc gene led to much higher lcrF mRNA levels, and this effect could be further enhanced by the deletion of the yopD gene.We also observed that pnp transcript levels were significantly increased in the Δrnc mutant, indicating that RNase III cleaves within the pnp transcript and initiates its degradation as described for E. coli [55,56].These observations suggested two separate pathways in which (1) RNase III has a very strong and dominant negative influence on the lcrF mRNA independent of YopD, and (2) RNase III exerts a YopD-dependent compensatory effect on lcrF mRNA stability via the repression of the PNPase (Fig 11).The second pathway may have evolved to balance the negative influence of RNase III on lcrF mRNA stability in the absence of host cell contact, whereby an additional upregulation of LcrF production is still possible upon YopD secretion.
The exact molecular mechanism of how RNase III promotes the destabilization of the lcrF mRNA by pathway (1) is still unknown.However, the results of this study indicate that this may (at least in part) occur through the control of the RNA-binding protein CsrA (Fig 11), a post-transcriptional regulator of the Csr system.CsrA is known to interact directly with the 5'untranslated region of the lcrF transcript and enhances lcrF mRNA translation [19] which could also increase lcrF mRNA stability.In fact, studies with E. coli showed that translation initiation regions (5'-UTR + first 11 codons of the downstream gene) that dictate high translation initiation rates (e.g.high RBS indexes and/or absence of strong local secondary structures) recruit more ribosomes to the mRNA, and the high ribosome occupancy can protect the transcript from degradation by RNases [65,66].Here, we show that loss of RNase III has no effect on the overall levels of CsrA but leads to a significant decrease in the regulatory RNAs CsrB and CsrC.As both Csr RNAs were shown to bind and sequester multiple CsrA dimers to block their activity [67], a higher amount of unbound, active CsrA is expected in the Δrnc mutant.An increase in active CsrA could explain (i) higher stability of the lcrF mRNA, and (ii) higher expression of the translational lcrF-lacZ fusion in the absence of RNase III.
It is also possible that other translation-relevant factors that were shown to be differentially expressed in the Δrnc mutant support this CsrA function or enhance translation initiation separately, which could increase ribosome coverage and thus lcrF transcript stability.Among these factors are 11 ribosomal proteins, and the essential bona fida translation initiation factor IF-3 (InfC) promoting 30S initial complex formation [60,61].Moreover, transcripts of the 16S rRNA methyltransferase RsmG/GidB and the 16S rRNA-processing protein RimM were significantly increased in the absence of RNase III which both also have a positive influence on translation initiation, e.g. by subverting problems that occur during the in vivo assembly of the 30 S ribosome [62][63][64]68] or by guiding the folding of the rRNAs to facilitate the formation of productive r-protein-RNA interactions [69].In contrast, the transcript of the ribosome-associated inhibitor A (RaiA/YfiA), which accumulates in the stationary phase, and stabilizes 70S ribosomes in an inactive state [70], was less abundant in the absence of RNase III.
The observed changes regarding the translation-relevant factors could be a consequence of defects in rRNA processing associated with the loss of RNase III.It is well known that RNase III initiates maturation of the ribosomal RNAs encoded on the pre-rRNA transcript by an initial series of processing events, which are subsequently cleaved and trimmed by other ribonucleases [71][72][73][74][75].In RNase III-deficient bacteria 30 S rRNA precursors (including 16S, 23S, 5S rRNA, and tRNA sequences) are produced [72][73][74][75][76].The 30 S rRNA can be processed by alternative maturation pathways.However, this involves the formation and incorporation of not fully matured rRNAs (prRNAs) into the ribosomes, which alter their overall activity [71,[77][78][79].The RNase III-deficient bacteria may indirectly compensate for these alterations by the upregulation of translation factors.However, the influence of RNase III on translation factors could also be direct.Multiple RNase III in vivo cleavage sites were recently found in mRNAs encoding ribosomal proteins, translation elongation factors, and enzymes involved in RNA modification in E. coli [80,81].This indicated that the role of RNase III in the control of translation is much larger than expected, and could support global reprogramming of bacterial gene expression, e.g.upon host cell contact, by altering the translational machinery.
Apart from the ysc-T3SS/yop-related alterations, the RNase III-deficient strain is characterized by a wide range of transcriptional changes, including mRNAs and sRNAs across all categories (e.g.metabolism, additional virulence traits, responses to environmental stresses, genetic/environmental information processing).Among them are several other RNA-controlling factors, i.e. small regulatory RNAs encoded on the virulence plasmid (e.g.Ysr291-293 overlapping with the lcrV-lcrH-yopD transcript), which could also affect lcrF mRNA stability.
Approximately 10% of all mRNAs are affected by the introduction of the Δrnc mutant in Yersinia.This is similar to other bacteria, whereby the impact on individual genes varies substantially among the species [82][83][84].Our comparison further revealed that only 1-2% of the total uniquely mapped reads were generated from the virulence plasmid pYV at conditions reflecting the environment or initial stages of the infection.However, this drastically changed under Ysc-T3SS/Yop-inducing conditions mimicking host cell contact.Although only approximately 1% of the genetic information of Y. pseudotuberculosis is encoded on pYV, it accounts for more than 50% of the transcripts detected under secretion conditions, whereas the ratio of the chromosomally-encoded transcripts was drastically reduced (from >99% to less than 50%).This may also explain the growth arrest associated with the induction of Ysc-T3S-mediated secretion as the abundance of many transcripts important for metabolism and physiological traits is affected.It also became apparent that in particular mRNAs of pYV were already upregulated in the Δrnc mutant under non-secretion conditions.This suggested that reprogramming observed under secretion conditions in the wildtype might be triggered by a reduction of functional RNase III.However, no change in rnc transcript levels was detectable upon a shift from non-secretion to secretion conditions.In this respect, RNase III is either not the superior trigger of LcrF/Ysc-T3SS/Yops or it is modulated on the post-translational level.
In summary, several aspects of this study indicate that RNase III-mediated control of LcrF and Ysc-T3SS/Yop production is (i) linked to components or regulators, such as CsrA and YopD, cooperating with the translational machinery, and (ii) accompanied by a global reprogramming of gene expression.Interestingly, translation has also previously been reported to play a role in LcrF, and Ysc-T3SS/Yop synthesis in another context.For instance, it was demonstrated that the RNA-binding translocator protein YopD associates with the 30S ribosomal particles in an LcrH-dependent manner and that this prevents translation initiation of its target mRNAs, including lcrF and the yop transcripts [85].Hence, RNase III-mediated alterations of the composition and activity of the ribosome could in turn also modulate the interaction of YopD with the ribosome and affect its function as a regulator of lcrF mRNA translation and stability.
Although the Y. pseudotuberculosis Δrnc strain exhibits a severe growth reduction it also showed a strongly increased T3SS-Yop secretion activity at 37˚C.We found that this upregulation extended to an increased Yop translocation and phagocytosis by human macrophages.This suggests that global reprogramming in the absence of RNase III renders the pathogen more prone to defend against the attack of phagocytic innate immune cells.How this property influences bacterial colonization and survival in vivo is difficult to assess and interpret due to its general growth defect and differential regulation of many other virulence-and fitness-relevant genes-i.e.RNases play a variety of roles in controlling bacterial survival.Yet, oral mouse infection experiments comparing Y. pseudotuberculosis wildtype and an isogenic Δpnp mutant exist [21].A significantly reduced mortality (nearly 50-fold increase in the LD 50 ) and morbidity was observed for the Δpnp mutant, even though T3SS/yop expression levels were increased as seen in this work and for certain T3SS and yop transcripts and proteins in previous studies [18].Despite increased T3SS/yop expression, Yop-mediated cytotoxicity was lower when HeLa cells were infected with a pnp-deficient strain [21].Some evidence exists that the S1 RNAbinding domain of PNPase greatly affects T3SS activity without altering T3SS expression levels, but additional PNPase-controlled factors implicated in the energization of the T3SS apparatus could also contribute to this effect.
In summary, this illustrates that our current understanding of the role of RNases in the complex post-transcriptional control of Yersinia virulence is still in its infancy.However, it is clear, that their involvement not only enables Yersinia to rapidly adjust its gene expression profile to efficiently block immune cell attacks, but it also allows the bacteria to overcome accompanying energetic and stress burdens and to rapidly resume its genetic pre-attack program after a successful defense.

Cell culture, media, and growth conditions
Overnight cultures of E. coli were routinely grown in LB at 37˚C, Yersinia strains were grown at 25˚C or 37˚C in LB (Luria Bertani) broth.For the analysis of Yop and LcrF synthesis and

Strain and plasmid constructions
All DNA manipulations, restriction digestions, ligations, and transformations were performed using standard genetic and molecular techniques [86,87].Plasmid DNA was isolated using QIAprep Spin Miniprep Kit (Qiagen) or Nucleospin Plasmid Kit (Macherey-Nagel).DNAmodifying enzymes and restriction enzymes were purchased from New England Biolabs.The oligonucleotides used for amplification by PCR, and sequencing were purchased from Metabion and Eurofins.PCRs were done in a 50 μl mix for 30 cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs).Purification of PCR products was routinely performed using the QIAquick PCR Purification Kit (Qiagen) or the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel).All constructed plasmids were sequenced by Microsynth AG (Balgach, Switzerland).
Strains and plasmids used in this study are listed in S3 Table and primers for plasmid generation are listed in S4 Table .Plasmid pIVO13 was constructed by the insertion of a PCR fragment harboring the regulatory region of the encoding region of yopE.The fragment was amplified from total DNA of Y. pseudotuberculosis strain YPIII using primer set VIII193/ VIII194 cloned into the BamHI/XhoI sites of plasmid pPCH1, replacing the full-length coding sequence of yopE.The resulting plasmid pIVO13 was sequenced using primers I984 and V824.Plasmids encoding the rnc and pnp genes for complementation analyses were constructed by amplification of DNA segments including the respective native promoter identified by Nuss et al. [37], the coding sequence and the 3'-UTR of the RNase gene using primer pairs VIII365/ VIII387 for rnc and VIII636/VIII/637 for pnp (S4 Table ).The generated PCR fragments were cloned into plasmid pAKH85 generating pIVO20 and pIVO21, and their sequence was confirmed using primer VIII385 and VIII388.Plasmid pTT15 was constructed by ligation of the tet promoter and the MCS with primers VI527 and II143 into the BamHI and NotI sites of pFU86.The yscW-lcrF promoter region was amplified with primers IX729 and IX730 and ligated into the BamHI and KpnI sites of pTT15, generating pMV53.

Construction of the Y. pseudotuberculosis deletion mutants
The Y. pseudotuberculosis strains used in this study are derived from wild-type strain YPIII.All deletion mutants were generated via homologous recombination.The mutant strains YP356, YP372, and YP375 in which the RNase III gene rnc was deleted were constructed in a three-step PCR.First, the Yersinia genomic DNA was used as a template to amplify 500-bp regions flanking the rnc gene.The upstream fragment was amplified with primer pairs (VII882/VII881) of which the reverse primer contained an additional 20 nt at the 5´-end which were homologous to the upstream region of the rnc gene.The downstream fragment was amplified with primer pairs (VII879/VII880) of which the forward primer contained additional 20 nt at the 5´-end which were homologous to the downstream region of rnc.Subsequently, a PCR reaction was performed with the forward primer of the upstream fragment and

PLOS PATHOGENS
RNase-mediated reprogramming of Yersinia the reverse primer of the downstream fragment using the upstream and downstream PCR products as templates.The PCR fragment was digested with SacI and was ligated into the SacI site of the suicide plasmid pAKH3 generating plasmid pIVO11.The resulting plasmid was sequenced using primers III981/III982.Next, the plasmid was transformed into S17-1λpir and was transferred into Y. pseudotuberculosis YPIII, YP91 (ΔyopD), and YP139 (Δpnp) via conjugation.Chromosomal integration of the plasmid was selected by plating on LB supplemented with kanamycin.Single colonies were tested for the correct integration of the plasmid via colony PCR using primer pair VII366/VII367.Mutants were subsequently grown on LB agar plates containing 10% sucrose.Single colonies were tested for growth on carbenicillin and sequenced using primer pair VII366/VII367.

RNA isolation and Northern blotting
Bacterial cultures were grown under the desired conditions.The bacteria were pelleted by centrifugation for 1 min at 16000 x g.The pellets were resuspended in 0.2 volume parts of stop solution (5% water-saturated phenol, 95% ethanol) and immediately snap-frozen in liquid nitrogen.After thawing on ice, bacteria were pelleted for 1 min at 16,000 x g at 4˚C.The pellet was resuspended in lysozyme solution (50 mg lysozyme/ml TE-buffer) and incubated for at least 5 min at RT.The total RNA of lysed bacteria was isolated with the 'SV Total RNA Isolation System' (Promega, USA) according to the manufacturer's instructions.RNA concentration and quality were determined by measurement of A 260 and A 280 .
For the analysis of lcrF transcripts total cellular RNA (5-30 μg) was separated on MOPS agarose gels (1.2%) or 8% urea acrylamide (RNA <250 nt), and transferred in 10 x SSC buffer (1.5 M NaCl, 0.15 M sodium citrate pH 7.0) for 1.5 h at a pressure of 5 cm Hg to a positively charged nylon membrane by vacuum blotting or by semi-dry blotting (250 mA, 2.5 h) and UV cross-linked.Prehybridization, hybridization to DIG-labeled lcrF PCR probes, and membrane washing were conducted using the DIG luminescent Detection kit (Roche) according to the manufacturer's instructions.The lcrF transcripts of interest were detected with a DIG-labeled PCR fragment (DIG-PCR nucleotide mix, Roche) amplified with primer pairs for the individually detected according to the manufacturer's instructions.(S4 Table ).For the detection of the CsrB and CsrC RNAs and the 5S and 16S control rRNA, fluorophore-coupled DNA oligonucleotide primers purchased from Integrated DNA technologies were used (S4 Table ).The primers were incubated with the membrane in a final concentration of 15 nM for CsrB and CsrC and 5 nM for 5 S rRNA in church moderate buffer (10 mg/ml BSA, 500 nM Na 2 HPO 4 , 0.34% (w/v) H 3 PO 4 , 15% (v/v) formamide, 1 mM EDTA, 7% SDS, adjusted to pH 7.2) for 6 h.The membrane was washed with 2 x SSC and 0,2% SDS for 5 min and the RNAs were detected at 700 nm (CsrB, CsrC) or 800 nm (5 S rRNA) with the Odyssey Fc Imaging system (Li-COR).

RNA isolation for RNA-seq
To yield intact high-quality RNA for RNA-seq analyses, hot phenol RNA extraction was performed.The bacteria were harvested by centrifugation at 4,000 rpm and the bacterial pellets were snap-frozen in liquid nitrogen.Bacterial pellets were resuspended in 1/8 volume of RNA resuspension buffer (0.3 M saccharose, 0.01 M NaOAc) on ice.An equal volume of RNA lysis buffer (2% SDS, 0.01 M NaOAc pH 4.5) was added, mixed, and incubated at 65˚C for 90 sec.An equal volume of pre-heated (65˚C) saturated aqua-phenol was added, mixed with the lysate, incubated for 3 min at 65˚C, and then immediately frozen in liquid nitrogen.Subsequently, the samples were centrifuged at 16,000 x g for 10 min at room temperature and the aqueous layer was extracted and the procedure was repeated three times.For the final extraction of phenol, an equal volume of chloroform/isoamyl alcohol (24:1) was added, mixed, and

PLOS PATHOGENS
RNase-mediated reprogramming of Yersinia centrifuged for 10 min at 16,000 x g at room temperature.The RNA in the aqueous layer was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 4.5) and 2.5 volumes of icecold ethanol at -20˚C.The RNA was recovered by centrifugation at 16,000 x g for 30 min at 4˚C, the pellets were washed at 4˚C with ice-cold 70% (v/v) ethanol two times and the RNA pellets were air-dried and resuspended in nuclease-free water.

RNA stability assay
Y. pseudotuberculosis strains YPIII, YP139 (Δpnp), YP356 (Δrnc), and YP375 (Δpnp, Δrnc) were grown at 37˚C.Subsequently, rifampicin was added in a final concentration of 1 mg/ml to block transcription, and samples were taken in intervals before (0), and 1, 3, and 7.5 min after the addition of rifampicin.To finally analyze the decay rate of the RNA transcripts, Northern blots were performed with an lcrF-specific probe as described [19], and the detected relative amount of RNA in each sample was determined using the Odyssey Fc Imaging system and software (LiCor).

Quantitative PCR (qPCR) analysis of the copy number of pYV
The copy number of the Y. pseudotuberculosis virulence plasmid pYV was determined using quantitative PCR (qPCR).Total DNA of YPIII or its derivatives, grown at the indicated conditions, was isolated by the addition of chloroform:isoamylalcohol (24:1).The sample was mixed, centrifuged at 16,000 x g and the supernatant was mixed with four volumes of 100% ethanol.The precipitated DNA was pelleted by centrifugation (16,000 x g) and the pellet was resuspended in nuclease-free water.For the qPCR, three primer pairs for chromosomal genes (glnA (VII922/VII923), rpoB (VII924/VII925), YPK_3178 (VII926/VII927) and two primer pairs for plasmid genes (yscM (VII918/VII919), repA (VII920/VII921)) were chosen adapted from Wang et al. [44]. 1 ng/μl of total DNA was amplified using the primer sets in SensiFAST SYBR No-ROX (Bioline).qPCR was realized in technical duplicates in the Rotor-Gene Q realtime PCR cycler (Qiagen).Two reactions with nuclease-free water without DNA were used as controls.A melting curve was employed to ensure specificity.The plasmid copy number of the strains was calculated as described by Wang et al. [44].

RNA sequencing (RNA-seq)
Y. pseudotuberculosis YPIII and the isogenic rnc mutant YP356 were grown in LB medium to exponential phase (OD 600 0.5).The cultures were split and grown at 25˚C (T0), 37˚C, or 37˚C in the presence of 20 mM Na 2 C 2 O 4 and 20 mM MgCl 2 in triplicates.After 1 h (T1) and 4 h (T2), total bacterial RNA was isolated by a hot phenol extraction protocol (see above), DNA was digested using the TURBO DNase (Ambion), purified with phenol:chlorophorm:isopropanol, and the quality was assessed using the Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies) or Qubit (Thermo Scientific).Samples with an RIN of >8 were considered for RNA-Seq sample preparation.From the total RNA the rRNA was depleted using Ribo-Zero (Illumina).ERCC spike-ins (external RNA controls consortia Mix 1 and Mix 2) were added before rRNA depletion as a means to control for sequencing and platform performance.
Further processing included fragmentation by sonication to a median size of 200 nt, and cDNA library preparation (NEBNext Ultra II Directional RNA Library Prep Kit-Bacteria (New England Biolabs) according to the manufacturer's instructions.Multiple oligos for Illumina sequencing (#E6440L) were obtained from New England Biolabs and used as indices that allow uniqueness in both directions.Illumina sequencing was performed with the Nova-Seq 6000 sequencing system (S1 flow cell, Illumina) with 100 cycles and paired-end reads PLOS PATHOGENS (PE50, 2 x 50 bp reads).The fluorescent images were processed into sequences and transformed to FastQ format using the Genome Analyzer Pipeline Analysis software 1.8.2 (Illumina).The sequence output was controlled for general quality features, sequencing adapter clipping, and demultiplexing using the fastq-mcf and fastq-multx tools of ea-utils [88].

Read mapping, bioinformatics and statistics
Quality of the sequencing output was analyzed using FastQC (Babraham Bioinformatics).All sequenced libraries were mapped to the YPIII genome (NC_010465) and the pYV plasmid (accessions NC_006153) using Bowtie2 (version 2.1.0)with default parameters [89].After read mapping, SAMtools was employed to filter the resulting bam files for uniquely mapped reads (both strands), which were the basis for downstream analyses.To detect ORFs and transencoded sRNAs that are differentially expressed in YPIII and the rnc mutant YP356, we used DESeq2 [90] for all differential expression (DE) analyses following the default analysis steps described in the package's vignette.For each comparison, HTSeq in union count mode was used to generate raw read counts required by DESeq as the basis for DE analysis.

Gel electrophoresis, preparation of cell extracts, and Western blotting
For the detection of proteins of interest, Y. pseudotuberculosis cultures were grown under indicated growth conditions.Cell extracts of equal amounts of bacteria were prepared and separated on 12-15% SDS polyacrylamide gels [86].The gels were either stained with Coomassie Blue or the samples were transferred onto a PVDF membrane via electro-blotting and probed with the respective antibody as described [91].For visualization of the proteins mentioned below, generated polyclonal (Davids Biotechnology) or purchased monoclonal antibodies were used (anti-LcrF 1:1,000, gift of G. Plano; anti-CsrA 1:8000 anti-rabbit, anti-β-lactamase 1:10,000 anti-mouse, Abcam; anti-YopD 1:6,000 and anti all Yops 1:12,000 gift of A. Forsberg).

Yop effector secretion assay
The Yop secretion assay was performed as described [92].Bacteria were grown overnight at 25˚C in LB medium, diluted 1/50 in fresh LB medium, and grown at 25˚C for 2 h and then shifted to 37˚C in the presence or absence of 20 mM NaOx and 20 mM MgCl 2 .Proteins in the filtered medium supernatant were precipitated with TCA and harvested by centrifugation.Precipitated proteins were washed with 95% acetone with 0.5% SDS and resuspended in equal amounts of sample buffer.Subsequently, the proteins were separated on 15% SDS polyacrylamide gels and visualized by Coomassie brilliant blue staining or Western blotting with polyclonal antibodies directed against secreted Yop proteins of Y. pseudotuberculosis.

Nitrocefin-based and fluorescent-based Yop effector secretion assay
A Nitrocefin-based secretion assay was used to quantify Yop effector secretion of Y. pseudotuberculosis strains.For this purpose, Y. pseudotuberculosis strains harboring plasmid pIVO13 encoding a yopE-blaM fusion were grown under indicated conditions and adjusted to an OD 600 of 0.8.Bacterial cells were pelleted and 95 μl of the supernatant were mixed with 5 μl of a 2 mmol Nitrocefin diluted in PBS and incubated for 1 h at room temperature.The absorption at 486 nm (A 486 ) and 390 nm (A 390 ) was measured at specific time points using the Varioskan Flash (Thermo Scientific).The specific β-lactamase activity was calculated as ratio between A 486 /A 390 (sample) / A 486 /A 390 (medium).
A fluorescent-based translocation assay was employed to quantify Yop effector translocation by the Y. pseudotuberculosis strains.For this purpose, a Live-BLAzer-FRET B/G Loading Kit with CCF4-AM (Invitrogen) was used.Human macrophages (THP-1) were differentiated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) for 3 days in μ-slide 8 well (ibidi) at a seeding density of 5x10 4 cells/well.Subsequently, culture medium was refreshed and cells were further incubated for two additional days.Human epithelial cells (HEp-2) were seeded in μslide 8 well (ibidi) at a concentration of approximately 3x10 4 cells/well and allowed to attach overnight.The next day, cells were washed and infected with a multiplicity of infection (MOI) of 30 of Y. pseudotuberculosis WT and RNase mutant strains harboring plasmid pIVO13 encoding a yopE-blaM fusion or the empty plasmid as negative control.After incubation for 1 h, cells were washed with phosphate-buffered saline (PBS) and stained with loading dye containing 50 μg/ml gentamicin according to the manufacturer's protocol.After staining for 1 h, YopE translocation was visualized by fluorescence microscopy using a compact fluorescence microscope (BZ-9000, Keyence).

Analysis of the Yersinia phagocytosis by human macrophages
In preparation for the phagocytosis assay, THP-1 cells were seeded at a cell density of 6x10 4 cells/well in a 48-well plate and stimulated with 50 ng/ml PMA.Cells were replaced with fresh medium before the addition of bacteria at MOI of 30.One hour post-infection, the cells were washed extensively with PBS and added medium containing gentamicin at a concentration of 50 μg/ml to eliminate the remaining extracellular bacteria.After incubation for an additional hour, macrophages were washed and the total number of intracellular bacteria was determined by lysing the cells with 0.1% Triton X-100.Subsequently, bacteria were plated on LB agar and colonies were counted.The phagocytosis rate was calculated based on the initial bacterial inoculum.

Cell viability assay
Cell viability of bacterial cultures was measured using the BacTiter-Glo Microbial Cell Viability Assay (Promega) by detection of present ATP.Bacterial cultures were cultivated under indicated conditions and the OD 600 adjusted to 0.8 in the growth medium.The culture was mixed 1:1 with the BacTiter-Glo substrate and luminescence was measured using the Varioskan Flash (Thermo Scientific).Cell viability was determined in duplicates by calculating the ratio of luminescence of the sample and medium.

Scanning and transmission electron microscopy
In order to identify T3SS needles on the bacterial surface, negative staining for transmission electron microscopy and field emission scanning electron microscopy was used.Bacterial overnight cultures were diluted 1:50 in LB and grown at 25˚C, 37˚C, and 37˚C/-Ca 2+ for 4 h.

PLOS PATHOGENS
Three independent cultures were pooled and cultures were mixed with cooled glutaraldehyde (2%) and formaldehyde (5%).For transmission electron microscopy thin carbon support films were prepared by sublimation of carbon on freshly cleaved mica.Using 300 mesh copper grids, the samples were negatively stained with 2% (w/v) aqueous uranyl acetate, according to the method of Valentine et al. [93], and examined in a transmission electron microscope (TEM910, Zeiss, Germany) at an acceleration voltage of 80 kV at calibrated magnifications.Images were recorded digitally with a Slow-Scan CCD-Camera (ProScan, 1024 x 1024, Scheuring, Germany) applying the ITEM-Software (Olympus Soft Imaging Solutions, Mu ¨nster, Germany).For field emission scanning electron microscopy glass coverslips were coated with a poly-Llysine solution (Sigma, Munich, Germany), 30 μl of the samples were added, left for 10 min, washed gently with TE buffer (TRIS EDTA 0,01 M, pH 6.9) and then fixed in 2% glutaraldehyde in cacodylate buffer.After washing with TE buffer dehydration was carried out in a graded series of acetone (10, 30, 50, 70, 90, 100%) on ice for 15 min for each step.Samples were then critical-point dried with liquid CO 2 (CPD 300, Leica, Wetzlar) and covered with a gold film by sputter coating (SCD 040, Balzers Union, Liechtenstein).For examination in a field emission scanning electron microscope (Merlin, Zeiss, Germany), an Everhart Thornley SE-detector was used with the inlens SE-detector in a 25:75 ratio at an acceleration voltage of 5 kV.Images were recorded applying the SmartSEM software version 6.06.empty vector), YP356 (Δrnc) pAKH85 (pV, empty vector) and the complementing strains YPIII (wt) pIVO20 (prnc + ) and YP356 pIVO20 (prnc + ).The transformants were grown at 37˚C and alkaline phosphatase or beta-galactosidase activity was determined, respectively.Data represent the mean ± SD from three independent biological replicates.Significant differences were determined using Student's t-test and indicated by asterisks (*P <0.05).
).This did not result from bacterial cell lysis (S2A Fig) and the derepressed effect of the Δrnc mutant could be complemented by an rnc-positive plasmid (S2B Fig).The observed Yop secretion pattern is in full agreement with the detection of numerous T3S injectisome-like structures on the surfaces of the bacteria (Fig 1B).This might also explain the growth reduction observed at 37˚C for the Δrnc mutant (Fig 1A), the phenotype which is characteristic for Yop secretion conditions (37˚C/-Ca 2+ )

Fig 5 .
Fig 5. Influence of RNase III and PNPase on lcrF transcript levels.(A) Y. pseudotuberculosis strains YPIII (wt), YP139 (Δpnp), YP356 (Δrnc), and YP375 (Δrnc, Δpnp) were grown at 37˚C and 37˚C/-Ca 2+ .Total RNA of the samples was prepared and the lcrF transcript was detected by Northern blotting; 16S rRNA was used as loading control.(B) Y. pseudotuberculosis strains YPIII (wt), YP139 (Δpnp), YP356 (Δrnc), and YP356 (Δrnc) pIVO20 (prnc + ) and the ΔlcrF strain (YP179) were grown at 37˚C.Total RNA of the samples was prepared and the lcrF transcript was detected by Northern blotting (left panel) and quantified by ImageJ (right panel).Data represent the mean ± SD from three independent biological replicates.Significant differences were determined using Student's t-test and indicated by asterisks (*P<0.05).(C) RNA-Seq coverage tracks of the yscW-lcrF locus in Y. pseudotuberculosis YPIII (wt) and YP356 (Δrnc) grown at 37˚C.RNA-Seq reads were mapped to the pYV virulence plasmid (NC_006153.2) and analyzed using a DESeq2 pipeline.The mapping visualizes the nucleotide score of the combined biological replicates for the two strains with an equal score for both.(D) RNA-Seq coverage tracks of the yscW-lcrF locus of the YPIII (wt) and YP356 (Δrnc) were adjusted to nucleotide scores of 900 and 150, respectively to compare the read pattern of both strains.https://doi.org/10.1371/journal.ppat.1011965.g005

Fig 6 .Fig 7 .
Fig 6.Influence of RNase III and PNPase on the stability of the lcrF transcript.Y. pseudotuberculosis strains YPIII (wt), YP356(Δrnc), YP139 (Δpnp), and YP375 (Δrnc Δpnp) were grown at 37˚C.Rifampicin was added to the cultures to stop transcription.Samples were taken before (0 min) and 1, 3 and 7.5 min after rifampicin addition.Total RNA of the samples was extracted and subjected to Northern blotting, using an lcrF-specific probe.16S and 23S rRNA served as loading controls.A representative Northern blot for each strain is shown from three independent biological replicates.(B) The three independent biological replicates were used for the quantification of lcrF transcript levels with ImageJ software.16S rRNA was used as a control for quantification.Relative transcript levels were plotted in Graphpad Prism 8 to determine the lcrF mRNA decay rate.Data represent the mean ± SD from three independent biological replicates.Significant differences were determined at 7.5 min using Student's t-test and indicated by asterisks (*P <0.05).https://doi.org/10.1371/journal.ppat.1011965.g006 S4 Dataset) and by Northern blotting (Fig 7C), indicating a similar process in this pathogen.This suggests an interactive, regulatory pattern illustrated in Fig 7D, in which RNase III strongly represses LcrF production via a YopD-independent pathway, which is partially compensated by a second pathway controlling PNPase and YopD.
8C).In contrast to the transcriptional fusions (S4 Fig), the translational reporter revealed a significant increase in the synthesis of the LcrF-LacZ fusion protein in the absence of RNase III (Fig 8C).

Fig 8 .
Fig 8. Influence RNase III on the carbon storage system components CsrA, CsrB, and CsrC.(A) (Left) Y. pseudotuberculosis strains YPIII (wt) and YP356 (Δrnc) were grown at 37˚C in the presence (+) and absence of Ca 2+ (-).Whole-cell extracts were prepared and separated by SDS-PAGE.Synthesized CsrA protein was detected by Western blotting using a polyclonal antiserum against CsrA.An antiserum against H-NS was used for loading control.(Right) Scheme of the interaction of the carbon storage regulatory systems components: the RNA binding protein CsrA and the CsrA-sequestering regulatory RNAs CsrB and CsrC.(B) (Left) Y. pseudotuberculosis strains YPIII (wt), YP356 (Δrnc), and the complemented strains were grown at 37˚C in the presence of Ca 2+ (non-secretion conditions).Total RNA of the samples was prepared and CsrB and CsrC were detected by Northern blotting.(Right) Amounts of the CsrB and CsrC RNA were quantified by ImageJ and represent the mean ± SD from three independent biological replicates.Significant differences were determined using Student's t-test and indicated by asterisks (**P<0.01).(C) Plasmid

Fig 9 .
Fig 9. Global influence of RNase III on the Y. pseudotuberculosis transcriptome.Y. pseudotuberculosis strains YPIII (wt), and YP356 (Δrnc) were grown at 25˚C, and at 37˚C in the presence and absence of Ca 2+ for 1 h (T1) or 4 h (T2).Total RNA of the different strains was prepared and transcriptional profiling by strand-specific RNA sequencing followed by a differential expression analysis (DESeq) by comparing sequencing reads of wildtype and the Δrnc mutant strain was performed.(A) RNA-Seq read the distribution on the chromosome and the virulence plasmid (pYV).RNA-Seq reads which uniquely mapped 252 genes) (S4 Dataset and Fig 9C, right panel).This indicated that the influence of RNase III is strongly temperature-dependent.At 37˚C, many more metabolic gene transcripts were detected in the wildtype (S4 Dataset and Fig 9C, right panel).This may explain the generally lower fitness of the RNase III-deficient strain at this growth condition (Fig 1A).

Fig 10 .
Fig 10.Influence of RNase III on Yop translocation into macrophages and phagocytosis.(A) Translocation of a plasmid pIVO13-encoded YopE-beta-lactamase (BlaM) fusion protein expressed in Y. pseudotuberculosis strains YPIII (wt), and YP356 (Δrnc) into human THP-1 macrophages was determined by a green to blue fluorescence shift.Bacteria transformed with the empty plasmid (pV) were used as negative control.White bar: 50 μm.(B) Relative translocation of the YopE-BlaM construct determined by the ratio of blue to green fluorescent cells is illustrated.Data represent the mean ± SD from three independent biological replicates relative to the YopE-BlaM translocation determined for the wildtype defined as 1.0.Significant differences were determined using the Student's t-test and are indicated by asterisks (*** P<0.001).(C) Y.pseudotuberculosis strains YPIII (wt) and YP356 (Δrnc) were added to human THP-1 macrophages in an MOI of 30, and after 1 h intracellular amounts of the bacteria were determined as described in Material and Methods.Data represent the average ± SD from three independent biological replicates relative to the phagocytosis of the wildtype defined as 1.0.Significant differences were determined using the Student's t-test and are indicated by asterisks (**** P<0.0001).

Fig 11 .
Fig 11.Scheme of PNPase and RNase III-dependent synthesis of LcrF and the T3SS/Yops under non-secretion and secretion conditions.Regulatory network of the LcrF-T3SS/Yop system is illustrated under non-secretion (left panel) and secretion condition (right panel).Under T3SS/Yop non-inducing conditions (37˚C) RNase III has a negative influence on the translation of the lcrF gene through the control of (i) the regulatory RNAs CsrB and CsrC repressing the function of the positive trans-acting RNA-binding regulator CsrA and (ii) mostly likely of translation initiation/elongation factors.Moreover, RNase III was found to have a positive influence on YopD through the inhibition of PNPase.YopD itself interacts with the 30S subunit of the ribosome and has a negative influence on the stability of the lcrF mRNA, possibly through its negative influence on lcrF mRNA translation.Upon host cell contact, YopD is secreted and translocated into the host cell.This is accompanied by the derepression of YopD-mediated reduction of lcrF mRNA translation and host contactdependent relief of CsrB/C mediated repression of CsrA function.This in turn leads to a rapid increase of lcrF translation, and LcrF synthesis consequently leading to a strong increase of T3SS/Yop components.The strongest effects on the control of the indicated factor are given by thick solid lines, whereas a low/ reduced level of control is indicated by the dashed lines.The arrows indicate activation and T inhibition of the indicated factor/pathway.The figure was created with Biorender.com.https://doi.org/10.1371/journal.ppat.1011965.g011 lcrF transcript detection.Y. pseudotuberculosis (YPIII), YP356 (Δrnc), and YP139 (Δpnp) were grown at 37˚C, and the total RNA of the strains was prepared.10 μg of total RNA of the different strains were loaded onto a denaturing acrylamide gel to allow the detection of smaller degradation products.The lcrF transcripts were identified by Northern blot using a probe covering the lcrF coding sequence.The blot represents one of two biological replicates.The 5 S rRNA was used as loading control.(PDF) S6 Fig. Analysis of the expression of the Yops and LcrF at different time points after induction of the T3SS.Y. pseudotuberculosis (YPIII) was grown over day at 25˚C to exponential phase and was then shifted to 37˚C in the presence (37˚C) or absence of Ca 2+ (-Ca 2+ ).Wholecell extracts were prepared and the amounts of Yops (A) and LcrF (B) were analyzed by Western blotting using all-Yop and LcrF polyclonal antisera.H-NS was used as loading control.The ΔlcrF mutant was used as a negative control.(PDF) S7 Fig. RNA-Seq quality control.(A) Two-dimensional biplot and (B) three-dimensional principal component analysis of mean-centered and scaled rlog-transformed read count values of the Y. pseudotuberculosis wildtype strain YPIII and the isogenic Δrnc mutant grown over day at 25˚C to exponential phase (T0) and then shifted to 37˚C in the presence or absence of Ca 2+ for 1 h (T1) or 4 h (T2).(PDF) S8 Fig. Influence of RNase III on Yop translocation into human epithelial cells.(A) Translocation of a plasmid pIVO13-encoded YopE-beta-lactamase (BlaM) fusion protein expressed in Y. pseudotuberculosis strains YPIII (wt), and YP356 (Δrnc) into human epithelial cells (HEp-2) was determined by a green to blue fluorescence shift.Bacteria transformed with the empty plasmid (pV) were used as negative control.White bar: 50 μm.(B) Relative translocation of the YopE-BlaM construct determined by the ratio of blue to green fluorescent cells is illustrated.Data represent the mean ± SD from three independent biological replicates relative to the YopE-BlaM translocation determined for the wildtype defined as 1.0.Significant differences were determined using the Student's t-test and are indicated by asterisks (*** P<0.001).(TIF) S1 Table.RNase III-dependent mRNA changes of translation-relevant factors.The transcription profile obtained by an RNA-seq analysis of Y. pseudotuberculosis wildtype strain YPIII and its isogenic Δrnc mutant grown at 37˚C were compared.Transcripts of ribosomal proteins and other translation-relevant factors that were found in a significantly higher or lower abundance in the Δrnc mutant (log2-fold change (log 2 FC) � +/-2, p-value � 0.05) are listed.(PDF) S2 Table.RNase III-dependent mRNA changes of components of the Ysc-T3SS/Yop machinery.Upper panel: Illustration of the localization of the Ysc-T3SS/Yop components forming the injectisome of Yersinia upon host cell contact.Lower panel: The transcription PLOS PATHOGENS RNase-mediated reprogramming of Yersinia

Table and S2
Dataset and Fig 9B, right panel), but it was very similar to the wildtype at 37˚C/-Ca 2+ (S2 Table and S2 Dataset and Fig 9C, right panel).