The ability to make poly-P does not appear to affect the expression of RpoS in V. cholerae
grown in rich or high-phosphate minimal medium. We reached this conclusion based on the findings that our ppk
mutant made similar amounts of RpoS-dependent HA/protease and did not exhibit rpoS
phenotypes unless it was grown in phosphate-limited medium. These results are in close agreement with a previous report showing that a V. cholerae ppk
mutant did not exhibit phenotypes predicted from E. coli
). In a previous study the motility of and biofilm formation by a V. cholerae ppk
mutant were found to be slightly affected (25
). We did not observe these effects in our ppk
mutant, which was isolated from a V. cholerae
El Tor biotype strain belonging to a different lineage. The finding that our ppk
mutant expressed RpoS and motility is fully consistent with its ability to effectively colonize the suckling mouse intestine.
A previous report showed that V. cholerae
makes about 100 times more poly-P than E. coli
makes, yet a mutant had fewer phenotypic changes (25
). Our results show that a V. cholerae ppk
mutant is indeed more sensitive to environmental stresses, but only when the extracellular concentration of phosphate is low (Fig. ). We investigated possible mechanisms for the sensitivity of our ppk
mutant to multiple environmental stresses. The results indicated that inactivation of ppk
does not affect growth in low-phosphate medium, the ability to derepress the pho
regulon, and the ability to express the RpoS protein in low-phosphate medium (see Fig, S1, S2, and S3 in the supplemental material). However, these experiments did not rule out the possibility that RpoS activity is affected in the ppk
mutant grown in low-phosphate medium. To examine this possibility, we measured the induction of catalase activity in the wild type and mutant after a downshift to low-phosphate conditions. The inability of the ppk
mutant to induce catalase in response to phosphate limitation (Fig. ) explains its increased sensitivity to H2
in this medium and could have been due to lower RpoS activity.
Studies with E. coli
have shown that different starvation diets induce the RpoS-mediated stress responses by different mechanisms
transcription, translation protein stability, and activity (27
mutant AJB37 survived prolonged carbon, nitrogen, and phosphorus starvation in ASW salts like the wild type survived but expressed stress-related phenotypes in low-phosphate MOPS medium containing a carbon and nitrogen source. Therefore, we suggest that poly-P specifically protects V. cholerae
from environmental stresses under phosphorus imbalance conditions. Furthermore, we observed that the ATP levels of the ppk
mutant did not increase when the mutant was transferred to low-phosphate medium (Fig. ). Consequently, we propose that the increased stress sensitivity of strain AJB37 (ppk
) in low-phosphate medium is a consequence of a more drastic metabolic defect, an inability to generate ATP by mobilizing intracellular poly-P reservoirs. The paucity of ppk
-related phenotypes in V. cholerae
suggests that large amounts of poly-P are made in this bacterium only to be used under very specific conditions. Why has V. cholerae
evolved to make such large amounts of poly-P? Clearly, poly-P is not required for the expression of virulence and intestinal colonization. However, it has been proposed that V. cholerae
can survive outside the human host in estuaries and brackish waters (9
). It is well established that phosphorus and nitrogen play crucial roles in the ecology of aquatic ecosystems (6
). Phosphorus has been proposed to be the most common cause of eutrophication in freshwater lakes, reservoirs, streams, and the headwaters of estuaries, while nitrogen is believed to be the key mineral nutrient controlling primary production in the ocean (6
). Depending on the specific aquatic environment, both nitrogen and phosphorus could become limiting nutrients simultaneously or in a cyclic manner (6
). It is likely that in an environment in which phosphorus is limiting, bacteria capable of synthesizing large poly-P stores could have a competitive advantage. Salinity gradients have been recognized to be an important environmental stressor in aquatic ecosystems. Poly-P-defective mutant AJB37 was found to be remarkably sensitive to high NaCl concentrations (Fig. ). It will be of interest to examine if more widespread inhabitants of aquatic ecosystems (e.g., V. cholerae
non-O1 and non-O139 and other members of the genus Vibrio
) have similarly large poly-P stores.
The major differences in poly-P metabolism between E. coli and V. cholerae could reflect the evolutionary adaptation of these organisms to different habitats. While E. coli is a normal inhabitant of the lower gastrointestinal tract of humans and animals, long-term human carriage of V. cholerae is very unusual. Consequently, V. cholerae could have evolved to make more poly-P to resist longer exposure to phosphate-limited conditions outside the gastrointestinal tract.
In summary, many mechanisms that potentially enhance the survival of V. cholerae outside the human host have been proposed. These mechanisms include the general stress response, formation of biofilm communities, association with phytoplankton and zooplankton, and a viable but not culturable stage. In this paper we describe a novel mechanism: synthesis of large poly-P stores for ATP biosynthesis. Our results show that availability of a large poly-P high-energy phosphate depository enhances the capacity of V. cholerae to survive environmental stresses in a low-phosphate environment.