In this study, we identified 40 candidate Hfq-dependent sRNAs in the plant pathogen E. amylovora and further demonstrated that four of them regulated various virulence traits including motility, amylovoran EPS production, biofilm formation, and the T3SS. Although sRNAs have been increasingly recognized as pivotal regulators in bacteria, genome-wide identification of sRNAs has only been performed in a limited number of bacteria. In plant pathogens in particular, sRNA identification using deep sequencing methods has been reported in only three bacterial species prior to this study. In a transcriptome analysis of Pseudomonas syringae, transcription of 19 of the 21 non-coding RNAs predicted by Rfam database was detected , and three previously unannotated non-coding RNAs (psr1, psr2, and psr3) were also discovered. In Xanthomonas campestris, 23 sRNAs were identified from a genome-wide transcriptome analysis by deep sequencing, and one sRNA, sX12, was identified as a virulence regulator . In Agrobacterium tumefaciens, 26 sRNAs were identified by combining a comparative bioinformatics approach and a deep sequencing approach [23, 54]. Compared to these studies which identified trans- and cis-encoded sRNAs, our work specifically focused on the identification of the trans- encoded sRNAs that are regulated by the RNA chaperone Hfq. The number of sRNAs identified in this study, 40, is comparable to the number of sRNAs identified in the bacterial species mentioned above and in closely related species such as E.coli (about 107 sRNAs in E. coli K-12, documented in the Rfam database).
Hfq-dependent sRNAs are a major group of bacterial sRNAs whose stability and function are dependent on the RNA chaperone Hfq. The deletion mutant of hfq in E. amylovora renders pleiotropic phenotypes including reduced motility and amylovoran production, increased attachment, disrupted T3 secretion and translocation, and reduced virulence . This suggests that Hfq, as the global sRNA chaperone, may interact with multiple sRNAs that target various mRNAs to control different aspects of cellular and virulence processes. To test this hypothesis, we aimed to specifically identify the Hfq-dependent sRNAs and focus on their expression in Hrp-inducing minimal medium, a condition that mimics the in planta environment.
Two independent searches, based on RNA-seq and Rho-independent terminator prediction, were performed for the purpose of identifying Hfq-dependent sRNAs. RNA-seq identifies small, intergenic transcripts whose stabilities are dependent on Hfq. Although some sRNAs identified in the deep sequencing contain Rho-independent terminators, it was not clear whether the RNA-seq method had identified all sRNAs that possess Rho-independent terminators. To take the presence of Rho-independent terminator into consideration and to ensure that all the sRNAs with Rho-independent terminators are identified, we performed a second search by first mapping all of the Rho-independent terminators in the E. amylovora genome, and then identifying sRNA-encoding genes by detecting short-length transcriptional activity upstream of the terminators. The combination of the two searches identified Hfq-dependent sRNAs that possess Rho-independent terminators and sRNAs that do not contain Rho-independent terminators but depend on Hfq for their cellular stability.
Rho-independent terminators, which contain potential Hfq binding sequences, are considered to be important features of Hfq-dependent sRNAs [13, 15]. In this study, 17 of the sRNAs identified did not possess typical Rho-independent terminators although the abundance of these sRNAs was reduced in Ea1189Δhfq compared to Ea1189. Prior to this work, sRNAs whose stabilities are dependent on the presence of Hfq but do not contain Rho-independent terminators have been observed in a few bacterial species. For example, 10 sRNAs were identified by RNA-seq in a study aiming to identify novel sRNAs in E. coli. The abundance of five of them (ychE-oppA, ytfL-msrA, glnA-typA, yhcF-yhcG, and yhcC-gltB) showed significant reduction in an hfq mutant compared to the wild type E. coli. However, none of these five Hfq-dependent sRNAs possessed Rho-independent terminators . In contrast, Rho-independent terminator sequences were identified in sRNAs whose stability is not dependent on Hfq, such as ygfl-yggE; and ynfM-asr. Similarly, in Yersinia pseudotuberculosis, some sRNAs whose abundance is Hfq dependent did not contain Rho-independent terminators, such as Ysr4 . Our observation, along with previous observations, suggests the presence of sRNAs whose abundance is Hfq-dependent yet do not contain typical Rho-independent terminators in multiple species of the Enterobacteriaceae family. Further protein-RNA binding assays will elucidate whether Hfq directly interacts with these sRNAs or if the stabilization of the sRNAs by Hfq is indirect.
We observed a dynamic re-patterning of Hfq-dependent sRNAs between 6 and 12 hr induction in Hrp-inducing MM. In E. amylovora, the expression of key virulence genes is induced in Hrp-inducing minimal medium, and expression levels of some of these genes are at different levels between 6 and 12 hrs after inoculation. The change of proportion of an sRNA over time in this medium may indicate its role in modulation of virulence factors. Three of the sRNAs whose expression increased (AcrZ, RprA and Hrs21), are also virulence-regulating sRNAs. By increasing their expression in the Hrp-inducing medium, they may activate virulence-related genes at different timings or host locations during pathogenesis. In contrast, the relative abundance of Hrs6 and OmrAB dropped from 2.1% and 0.3% of the total sRNA pool at 6 hr post-induction to 1.5% and 0.2% at 12 hr post-induction, respectively. We also demonstrated that Hrs6 and OmrAB promote motility and limit amylovoran production (see Results). In E. amylovora, motility and amylovoran are two critical virulence determinants that are expressed at different stages of infection. Motility is believed to be critical for the early stage of infection, which enables E. amylovora to move from the stigma of the flower or at wound sites on leaves into the plants to establish infections. Biofilm formation is turned on at the later stage of infection to help E. amylovora to migrate into the xylem and cause systemic infections, and amylovoran is a critical component of biofilms formed by E. amylovora. The fact that Hrs6 and OmrAB activate motility and repress amylovoran production, and that the abundance of Hrs6 and OmrAB dropped from 6 hr to 12 hr post-induction in Hrp-inducing minimal medium, suggest that E. amylovora may use sRNAs such as Hrs6 and OmrAB as a regulatory mechanism to transit from early to late stages of infection.
Besides the virulence-regulating sRNAs, the re-patterning of the expression of other sRNAs was also observed. The expression of GcvB increased the most from 6 hr to 12 hr (6.3-fold) among all sRNAs. A similar observation was made in E. coli, where GcvB was barely detectable at 3 hrs in M9 minimal medium, but was strongly expressed at 8 hrs induction when analyzed by Northern blot . Likewise, the expression dynamics of Hrs5 in E. amylovora were similar to the expression of the ortholog RybB in E. coli. Taken together, these observations suggest that the expression of conserved sRNAs in Enterobacteriaceae is similar across bacterial species, suggesting that some of the functions that these sRNAs possess are conserved among different species. The re-patterning of sRNA expression may also decide the regulatory activities of the sRNAs, since competitions of sRNAs for the availability of Hfq occurs, and more abundant sRNAs may have better access of Hfq and exert stronger regulation .
From this study and a previous study, we have identified four sRNAs (ArcZ, Hrs6, OmrAB, RprA) as virulence regulators in E. amylovora, and in some cases have identified the specific virulence determinants regulated. OmrAB, ArcZ and Hrs6 were identified as positive regulators of motility in this study. In contrast, OmrAB and ArcZ were shown to be negative regulators of motility and FlhDC, the master regulator of motility, in E. coli. The over-expression of OmrAB and ArcZ led to reduced motility on soft agar plates, as well as reduced translation of flhDC. This suggests that although OmrAB and ArcZ are motility regulators in both E. amylovora and E. coli, the regulatory mechanism may be different.
Hrs6 is a novel Hfq-dependent sRNA that was identified for the first time, and we demonstrated that Hrs6 inversely controls amylovoran production and motility in E. amylovora. Although not documented in the Rfam database, Hrs6 has high sequence conservation in many Enterobacteriaceae species (Figure 3). Since Hrs6 has not been previously characterized in other Enterobacteriaceae and in light of the functions identified in this study, here we name it RmaA (Regulator of motility and amylovoran A). The sequence and function of RmaA was documented in NCBI, with the accession number KJ372221. It would be interesting to further characterize the detailed regulatory mechanism of RmaA on motility and amylovoran production in E. amylovora, as well as the regulatory function of RmaA in other Enterobacteriaceae species.
ArcZ was identified as a virulence-regulating sRNA in our previous study , and we found in this study that ArcZ confers pleiotropic regulation on multiple virulence determinants including motility, amylovoran production, attachment, biofilm formation, and the type III secretion system. Our observations that the virulence regulation repertoire of ArcZ is very similar to that of the global sRNA chaperone Hfq suggests that ArcZ could be the most critical virulence regulating sRNA in E. amylovora. ArcZ was previously described as a positive regulator of the stationary sigma factor RpoS and a negative regulator of motility in E. coli[58, 59]. It is also known as a negative regulator of serine uptake, oxidative stress, and motility in Salmonella. Additionally, ArcZ is characterized as one of the 34 sRNAs that are not required for murine virulence in Salmonella enterica. To our knowledge, this is the first report describing the regulatory mechanism of ArcZ affecting virulence. This also suggests that a small RNA may play different regulatory roles in various pathogens.