In this study, 46 TCSTs have been identified in E. amylovora, a pathogen of rosaceous plants which mainly resides in the plant xylem, but can also grow epiphytically on stigmas of flowers. Compared to other plant pathogenic enterobacteria such as D. dadantii and P. carotovora subsp. atroseptica, both of which can survive not only in plants, but also in soil, or the animal counterpart Es. coli, the number of TCSTs present in E. amylovora is relatively small, thus may reflecting the particular host niche for this pathogen. Furthermore, the genome size (3.9 Mbp) of E. amylovora is also the smallest among sequenced enterobacterial plant and mammalian pathogens . However, E. amylovora still maintains both enterobacterial-specific TCSTs (such as the Rcs system)  and plant enterobacterial-specific ones (such as HrpXY) .
On the other hand, E. amylovora has almost the same number of TCSTs as that found in E. tasmaniensis, a saprophytic bacterium isolated from apple flowers in Tasmania, Australia . Since both bacteria occupy the same ecological niche during colonization and can grow epiphytically on flowers, these bacteria might have developed and maintained similar TCSTs. This suggests that TCSTs may be evolutionarily maintained to cope with similar environmental and plant host signals in closely related bacteria. It is interesting to note that fire blight is endemic to North America, and it has subsequently spread to Europe and New Zealand in the 1950s and in 1917, respectively, but it has not yet been reported in Australia . It is possible that these two bacteria may have not yet encountered each other, as the distribution of E. tasmaniensis outside of Australia remains unknown. Surprisingly, we found that the majority of TCSTs in these two bacteria share a high level of aa similarity and identity. These results suggest that similar TCSTs might play important roles for the survival and proliferation among closely-related bacteria in similar plant niches.
Currently, several thousands of TCSTs have been identified in sequenced bacterial genomes [55–57]. Although the basic biochemistry of TCSTs is well understood, some structural insights in phosphorylation-dependent changes of TCST domains are variable. Domain architecture has proven particularly informative for analyzing multidomain proteins involved in signal transduction and in predicting the functions of these signal transduction proteins [43, 58]. In RRs, structural characterization of DNA-binding domains has revealed several variations on the common helix-turn-helix (HTH) theme, such as NarL- and OmpR-types. Recently, many novel conserved domains have been described such as PAS, GAF, GGDEF, EAL, and HD-GYP, thereby affirming the complexity of bacterial signaling systems [43, 58]. In E. amylovora, most HKs belong to four common HK groups, and are periplasmic sensors. Moreover, most RRs in this bacterium are OmpR-, NarL-, NtrC-, and LytR-like proteins, the four most common families of DNA-binding RRs found in prokaryotes . These results indicate that E. amylovora has maintained some basic signal transduction systems for the bacterium to survive. Phylogenetic and genomic analyses have revealed co-evolutionary relationships between cognate HKs and RRs. This seems obvious in E. amylovora as orphan HKs and RRs are rare.
It is well understood that TCSTs are involved in regulating virulence gene expression in plant bacterial pathogens . Previous reports on TCSTs have demonstrated the importance of TCSTs in the virulence of bacterial plant pathogens. However, data on complete or global virulence regulation networks are lacking. A recent study by Qian et al.  provides a useful beginning towards a better understanding of the regulatory networks involved. A genome-wide mutagenesis of all 54 RRs in X. campestris pv. campestris has revealed that two novel RRs are involved in virulence, thus facilitating future studies on signaling networks in this bacterium . In this study, we have utilized a reverse genetic approach and constructed 59 HK and RR mutants in E. amylovora, which will also provide valuable tools for future global gene expression assays using microarrays to deduce signaling networks in this bacterium.
Early studies have revealed that in E. amylovora, the Hrp T3SS, which delivers effector proteins into host plants, and the EPS amylovoran are two major virulence factors [52, 59, 60]. Previous reports have also indicated that the RcsCDB phosphorelay system regulates amylovoran biosynthesis, while the two-component system HrpXY regulates hrp-T3SS gene expression. Recently, we have further demonstrated that the Rcs system is essential for virulence in E. amylovora and may play a role in the survival of the pathogen . Mutations in the Rcs system have rendered the organism non-pathogenic . In this study, we have found that hrpX, hrpY and hrpXY mutants remain virulent, and could induce a spotty weak hypersensitive response (HR) on tobacco (Figure S2A, [see Additional file 2]); while, a hrpXYS triple mutant has a normal Hrp- phenotype. It is interesting to note that, in a previous report, Tn5-insertional mutants of hrpY have been reported to be non-pathogenic, and could not induce an HR on tobacco . Two classes of hrpX insertional mutants have been identified, one similar to the hrpY mutant and the other that continue to cause disease and induce a spotty HR on tobacco . Similar observations have been reported in Pantoea stewartii subsp. stewartii, causal agent of Stewart's wilt of corn ; wherein, Tn5-insertional hrpX and hrpY mutants exhibit a Hrp- phenotype and in-frame deletion hrpX mutants show reduced virulence. Since the hrpXY is transcribed as an operon, it is possible that Tn5 insertion could cause polar effects on the downstream genes such as hrpS (Figure S2B, [see Additional file 2]). Indeed, a recent study indicates that the hrp regulatory genes in P. stewartii subsp. stewartii participate in a novel regulatory loop that upregulates itself by readthrough transcription of hrpL into hrpXYS .
It has been proposed that in E. amylovora, both HrpY and HrpS regulate hrpL, encoding the master regulator of T3SS, and that the effects of HrpY and HrpS are additive . Subsequent studies of P. stewartii subsp. stewartii, D. dadantii, and Pantoea herbicola pv. gyposophilae have demonstrated that HrpY initially activates hrpS by binding to its promoter, and then the HrpL is activated by HrpS as well as by other regulatory genes [39, 63]. Furthermore, it has been reported that HrpY in D. dadantii acts as both a positive and negative regulator . In P. stewartii subsp. stewartii, besides the HrpY binding site, additional sequences further upstream of the hrpS promoter are also required for hrpS expression, suggesting that unknown regulatory proteins may act cooperatively with HrpY . Microarray studies suggest that hrpL represents only one branch of the regulatory pathways downstream of hrpRS, and a large number of genes regulated by HrpRS are hrpL-independent in P. syringae . In our study, HrpX, HrpY, and HrpS also act as negative regulators, as hrpX, hrpY, hrpXY, and hrpXYS mutants produce more amylovoran. Our virulence tests suggest that the T3SS remains functional in hrpX, hrpY, and hrpXY deletion mutants, but not in the hrpXYS mutant, whereby HrpS and HrpL may be activated by other unknown regulators except that production and/or translocation of HrpN (Harpin) in tobacco is severely attenuated as showed in an HR assay (Figure S2A, [see Additional file 2]). It is possible that host signals affecting gene expression may also be different in tobacco.
Various models have proposed that HrpX senses environmental signals in the plant apoplast or the Hrp-inducing medium to phosphorylate HrpY [18, 34, 35]. However, domain structure analysis has indicated that HrpX is a soluble cytoplasmic protein, and may sense intracellular signals. This suggests that other signaling pathways may also be involved in activating hrpXY, hrpS, or hrpL by sensing outside signals to regulate T3SS. Since the expression of HrpX, HrpS, and HrpL is regulated by low pH, also corresponding to conditions under which OmpR-EnvZ and GrrS-GrrA are activated, our results further suggest that both OmpR-EnvZ and GrrS-GrrA can regulate the hrpXY operon or hrpS either directly or indirectly as reported in other plant pathogenic bacteria [10, 28, 33, 34]. Further studies are needed to dissect the roles of OmpR-EnvZ and GrrS-GrrA in regulating T3SS.
Several regulatory genes have been previously reported to control amylovoran biosynthesis in E. amylovora, including the Rcs system, RcsA, Lon protease, and H-NS protein [17, 50, 65]. Here, we have further identified several groups of regulators, including both negative and positive regulators. These regulators may form a network that governs the production of amylovoran under different conditions to benefit pathogen survival or pathogenesis. Regulatory cascades are also likely to occur as global regulators such as OmpR-EnvZ and GrrS-GrrA may control expression of other regulatory genes or proteins, such as hrpXY and quorum sensing systems, as reflected in the amount of amylovoran produced in these mutants. However, we cannot rule out that cross-talk between different TCSTs may further complicate this scenario. Our study indicates that regulation of amylovoran biosynthesis is highly complex and further suggests that the pathogen has developed a system to control this major virulence factor.
Swarming is a flagella-driven form of motility for movement across solid surfaces as a group [66–70]. Swarmer cells are normally hyperflagellated and require extracellular components such as EPS and surfactants that enable mass migration [71, 72]. Previous studies have identified several global regulators in Es. coli and in other bacteria, including the Rcs system, OmpR and GrrSA, known to influence flagella biosynthesis, especially the master regulator flhDC [71, 73]. In this study, we have identified both negative (GrrSA) and positive (EnvZ/OmpR) regulators of swarming motility. It is easy to accept that the GacSA system may negatively regulate flagella biosynthesis, thus rendering grrSA mutants hypermotile. It is also obvious that flagella biosynthesis is not impaired in the envZ-ompR mutants as the swarming phenotype is different between the envZ-ompR mutants and the flhDC-fliA mutants. The obvious question that arises as to why envZ-ompR mutants are non-motile although they produce prolonged flagella. A recent study in Salmonella typhimurium has reported that mutations in chemotaxis pathways are impaired for swarming motility and further revealed a role of flagellum in sensing external wetness . It has been proposed that swarming requires a fluid environment generated as bacteria extract water from the underlying agar gel . Flagella are designed to work in this aqueous environment; that is swarming cells move in a thin layer of fluid over the surface of the agar. Further studies have revealed that the wetting agent that draws water out of the underlying agar is an osmotic agent . It is likely that in our envZ-ompR mutants, this unknown osmotic agent provides a signal for EnvZ-OmpR system that may regulate chemotaxis response or creates high osmolarity so that water can be removed from the agar, thus affecting swarming motility. Further studies are needed to clarify this hypothesis.