Date Published: May 1, 2014
Publisher: Public Library of Science
Author(s): Surbhi Wadhawan, Satyendra Gautam, Arun Sharma, Richard A. Wilson.
Xanthomonas campestris strains have been reported to undergo programmed cell death (PCD) in a protein rich medium. Protein hydrolysates used in media such as nutrient broth comprise of casein digest with abundance of proline and glutamate. In the current study, X. campestris pv. campestris (Xcc) cells displayed PCD when grown in PCD inducing medium (PIM) containing casein tryptic digest. This PCD was also observed in PCD non-inducing carbohydrate rich medium (PNIM) fortified with either proline or proline along with glutamate. Surprisingly, no PCD was noticed in PNIM fortified with glutamate alone. Differential role of proline or glutamate in inducing PCD in Xcc cells growing in PNIM was studied. It was found that an intermediate product of this oxidation was involved in initiation of PCD. Proline oxidase also called as proline utilization A (PutA), catalyzes the two step oxidation of proline to glutamate. Interestingly, higher PutA activity was noticed in cells growing in PIM, and PCD was found to be inhibited by tetrahydro-2-furoic acid, a competitive inhibitor of this enzyme. Further, PCD was abolished in Xcc ΔputA strain generated using a pKNOCK suicide plasmid, and restored in Xcc ΔputA strain carrying functional PutA in a plasmid vector. Xanthomonas cells growing in PIM also displayed increased generation of ROS, as well as cell filamentation (a probable indication of SOS response). These filamented cells also displayed enhanced caspase-3-like activity during in situ labeling using a fluorescent tagged caspase-3 inhibitor (FITC-DEVD-FMK). The extent of PCD associated markers such as DNA damage, phosphatidylserine externalization and membrane depolarization were found to be significantly enhanced in wild type cells, but drastically reduced in Xcc ΔputA cells. These findings thus establish the role of PutA mediated proline oxidation in regulating death in stressed Xanthomonas cells.
Xanthomonas campestris pv. glycines (Xcg), a pathogen of soybean causing bacterial pustule disease, and other Xanthomonas campestris pathogenic strains [namely X. campestris NCIM 2961 and X. campestris pv. malvacearum] were earlier reported in this laboratory to undergo growth medium dependent programmed cell death (PCD) during post-exponential phase , , , , . The process was later found to be associated with an increase in NADH generation leading to formation of reactive oxygen species (ROS) , . Since the PCD process in this bacterium was observed to be induced under conditions where the protein content of the medium was higher than the carbohydrate content, a fine balance of these two major nutrients was thought to be an essential factor governing metabolism associated survival of this microorganism in laboratory culture conditions. The designed conditions of growth of this organism in laboratory are quite distinct from its natural habitat on soybean leaf, where it is more accustomed to carbohydrate rich environment. As Xanthomonas represents one of the major groups of bacterial plant pathogens, understanding the balance between its survival and death could not only have broad practical significance in agriculture but also provide significant clues to microbial growth control and regulation. Casein digest is one of the PCD inducing constituents of PIM for Xanthomonas and predominantly provides very high levels of glutamate and proline. Hence, the effect on PCD process of Xanthomonas campestris pv. campestris (Xcc) upon addition of these two amino acids, either individually or in combination in PCD non-inducing medium (PNIM), was examined. The study further focused on the activity of one of the major enzymes, PutA (proline utilization A, also called proline oxidase or proline dehydrogenase) which is involved in the metabolism of proline in bacteria, including Xcc.
Proline metabolism is distinct from that of primary amino acids and plays a regulatory role in certain physiological conditions . Proline catabolism has been observed to be an important source of energy in some Gram negative bacteria such as Helicobacter pylori, Bradyrhizobium japonicum, Sinorhizobium meliloti, and Mycobacterium smegmatis during stress , , , , . PutA is significantly upregulated in Mycobacterium smegmatis and is preferentially used as an electron donor to the respiratory chain during energy limiting conditions . The role of PutA during metabolic stress induced cell death in Xanthomonas was investigated in this study. A significant increase in the intracellular proline levels and PutA activity was observed in PCD inducing medium or PIM (LB broth) implying the involvement of this enzyme in the programmed cell death (PCD) of Xanthomonas (Fig. 2). LB broth used in this study is predominantly composed of casein digest and yeast extract both of which are rich in several amino acids and peptides . As a result, the microorganism has to use them both as carbon and nitrogen source. During such growth conditions certain metabolic pathways can be preferentially upregulated over others. High intracellular levels of proline in Xanthomonas cells can be attributed to abundant free proline and peptides present in PIM. High proline levels have been reported to induce PutA activity as well . A good correlation was found between intracellular proline levels and PutA activity in Xcc wt cells cultured in PIM (Fig. 2). Addition of proline in PNIM also resulted in induction of PCD in Xanthomonas campestris cells (Fig. 1). However, the presence of glutamate in PNIM did not induce PCD, instead it supported the growth of Xanthomonas (Fig. 1). This observation is consistent with our earlier study where α-ketoglutarate when added in PNIM was not found to induce PCD in Xanthomonas. Glutamate and glutamine are important amino acids for bacterial metabolism . Glutamate participates in both catabolism and anabolism. It can be deaminated to form α-ketoglutarate, a TCA intermediate. Glutamate accounts for ∼88% of cellular nitrogen and is required for the biosynthesis of purines, pyrimidines and amino sugars . Hence, when starch minimal medium (PCD non–inducing medium or PNIM) was supplemented with excess glutamate, it provided a ready source of nitrogen favoring anabolic events as it was observed to accelerate Xanthomonas growth. In a recent study, α-ketoglutarate has been shown to contribute to a variety of metabolic processes including TCA cycle, biogenesis of several amino acids, carnitine biosynthesis and as a cofactor in several dioxygenases . It is also reported to be involved in the detoxification of ROS during oxidative stress . Due to this possible reason, addition of glutamate or α-ketoglutarate in PNIM was not found to induce PCD in Xanthomonas. The exact reason for this observation is still unresolved. Surprisingly, other TCA intermediates like pyruvate, citrate and malate were found to induce PCD in an earlier study from this laboratory . The differential role of these TCA intermediates with respect to induction of PCD process indicates the complexity of the process and is subject to further study.