Microbial EPS are often synthesized in response to a nutritional stress, with nitrogen limitation being a common environmental cue. As EPS synthesis entails a significant investment in terms of cellular energy and other resources under a condition when resources and energy are scarce, extensive re-organization of metabolic activity is expected and sophisticated regulatory mechanisms are needed to respond to environmental triggers. The common trigger of nutrient limitation for their synthesis and their association with adverse growth conditions suggest some unifying scheme of EPS regulation. Yet, our understanding is very limited about how their biosynthesis is regulated at the molecular level. In this study, we probe the regulation mechanisms of curdlan synthesis with a focus on those potentially conserved mechanisms. The transcriptome profiling along with genetic, biochemical, and physiological analysis reveals a multifaceted network of regulation for curdlan biosynthesis, including RpoN-independent nitrogen regulation, c-di-GMP, acidocalcisomes, polyP metabolism, and the stringent response.
First, the transcriptome analysis showed that upon nitrogen depletion, the curdlan synthesis operon was up-regulated by up to 100-fold, thus transcriptional regulation plays a prominent role in its biosynthesis. As curdlan synthesis is activated in response to nitrogen depletion, the involvement of the nitrogen signaling cascade is expected. The signal transduction components, NtrB and NtrC, were previously identified as essential components for curdlan synthesis , and this was confirmed in this study. Additionally, this study uncovered some unexpected details of how nitrogen signaling affects curdlan synthesis. The operon structure for ntrBC, having nifR as the first gene, is unusual for a non-nitrogen fixer, and nifR deletion was found to reduce curdlan production. We also show that the RpoN sigma factor is not involved, implicating an alternative sigma factor in the regulation. This is a significant departure from the archetype nitrogen regulation, in which RpoN is required for transcription of the regulated genes and NtrC serves as the activator. RpoN-independent transcriptional regulation has been shown to operate extensively in only one other microorganism, Rhodobacter capsulatus, a photosynthetic bacterium and a nitrogen fixer . Interestingly, R. capsulatus also contains a nifR-like gene (nifR3) in the same operon as ntrBC , suggesting a connection between the ntrBC operon structure and its RpoN independence. Future studies to identify the elusive sigma factor responsible for the regulation holds the key to understand this new mode of nitrogen signaling.
The genome sequence of ATCC 31749 suggests the presence of aciocalcisomes. This study provides the first evidence that metabolism within the organelle influences curdlan biosynthesis through energy storage in the form of polyP and maintenance of intracellular pH. The reduced curdlan produced by ΔrrpP suggests that the membrane-bound proton-translocating pyrophosphatase (RrpP) plays an essential role in acidocalcisome-mediated intracellular pH regulation. By eliminating polyP degradation via ppx1 deletion, high curdlan synthesis could be triggered in exponential phase cells, clearly establishing the role of polyP in curdlan biosynthesis. The contribution of polyP as an energy source in curdlan biosynthesis, however, is complicated by the regulatory role of polyP in the stringent response.
Despite the fact that many EPS are produced under nutritionally stressed conditions which likely trigger a stringent response, bacterial EPS synthesis have rarely been studied in the context of the stringent response. In this study, we show that curdlan synthesis is abolished in cells lacking the gene responsible for synthesis of the stringent response signal (p)ppGpp. This indicates that curdlan synthesis requires the stringent response. Further analysis by qRT-PCR showed that the curdan synthesis operon was down-regulated by 57-fold in the relA/spoT deletion mutant. Thus without the stringent response, up-regulation of the curdlan operon could not be elicited. This is a significant finding as it convincingly establishes requirement of the stringent response for the transcriptional up-regulation of an EPS operon. Only one previous report described a relationship between the stringent response and EPS synthesis . However, in this report, the Sinorhizobium meliloti mutant incapable of synthesizing the effector molecule of stringent response by knocking out the only relA/spoT gene (the same approach as used in this study), produced more succinoglycan, an EPS, and the succinoglycan operon was significantly up-regulated in the mutant. Thus, in this case, ppGpp negatively regulates succinoglycan synthesis, which is significantly different from the complete dependence of curdlan synthesis on the presence of the effector molecule, (p)ppGpp. Hence, our data suggest a novel type of EPS regulation as a direct effect of the stringent response, which has not been reported. The molecular details of activation, however, await further elucidation. As reviewed by Srivatsan and Wang, two models of activation were proposed for (p)ppGpp . In direct activation, the effector molecule, (p)ppGpp, increases transcription from cognate promoters by acting on RNA polymerase, whereas indirect activation involves an alternative sigma factor. Much of the understanding of the stringent response, however, is based on the studies with E. coli, in which the stationary phase sigma factor, RpoS, plays a central role in stringent response . Unfortunately, the E. coli model is unlikely to be applicable here as the ATCC 31749 genome does not encode a corresponding stationary phase sigma factor. Additionally, the ATCC 31749 genome contains only one relA/spoT homolog and two predicted exopolyphosphatases, signifying a significant departure from the E. coli system.
Additional data were obtained, which demonstrated the influence of the nucleotide second messenger, c-di-GMP, on curdlan synthesis. C-di-GMP is synthesized by diguanylate cyclases which contain a conserved GGDEF motif as the catalytic active site . The genome sequence of ATCC 31749 contains 31 predicted genes coding for GGDEF domain proteins, of which three GGDEF domain proteins had more than a 2-fold up-regulation in gene expression under nitrogen limitation (Table 2). Deletion of AGRO_3967, encoding an up-regulated GGDEF domain protein, yielded a 57% decrease in curdlan production (Figure 3A), suggesting that c-di-GMP may regulate curdlan biosynthesis, thus adding another factor to the already complicated regulatory network.
The use of systems biology tools in this study identified many components important to the transcriptional regulation of curdlan synthesis, including the ntrBC operon in nitrogen signal transduction, polyP, (p)ppGpp, and c-di-GMP. Subsequent experiments with gene knockout mutants and qRT-PCR revealed some aspects that are unique to curdlan synthesis and the producer microorganism. These include RpoN-independent activation of the curdlan operon and the connection of curdlan synthesis to the metabolic activity of a bacterial organelle. We also present convincing evidence that curdlan biosynthesis is involved in the stringent response. While these three aspects appear to be unique to curdlan, the components identified, NtrBC, polyP, (p)ppGpp, and c-di-GMP, are present in most microorganisms that synthesize EPS. Thus, it is reasonable to expect some common features to emerge. Indeed, Kornberg suggested that polyP may have served as an ATP-alternative energy source , and subsequent work from his lab confirmed the role of polyP in alginate synthesis in P. aeruginosa , and in a separate work, demonstrated a connection between polyP accumulation, nitrogen limitation, and the stringent response . Additionally, the stringent response in P. aeruginosa was recently shown to regulate quorum sensing , which affects synthesis of the EPS alginate , providing another link between the stringent response and EPS biosynthesis. Thus, polyP, a ubiquitous molecule accumulated in response to nitrogen limitation and in the stationary phase, plays a central role in the regulation of EPS synthesis and may additionally serve as an energy source. Our findings from this study further support this notion. In reference to the alginate model as reviewed by Rehm and Valla, a sigma factor (AlgU) and regulatory protein (AlgR), are central to integrate the molecular components influencing alginate biosynthesis . Data presented here indicate that an unidentified sigma factor is involved in the regulation, and previous studies implicate the presence of a regulatory protein (CrdR) required for curdlan synthesis . Further studies with CrdR and identification of the yet unknown sigma factor involved in the transcriptional regulation will be important to integrate the information uncovered in this work. While we believe each regulatory network may differ in some specific details, NtrC, polyP metabolism, and stringent response are aspects that may provide a basis for unified mechanisms of EPS regulation.