Abstract
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.
Highlights
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 [1,2,3,4,5]
When Xanthomonas cells were grown in PCD non-inducing medium (PNIM) supplemented with proline (100 mM), the cell count peaked at 24 h of incubation followed by a sharp decline in viable cell number during further post-exponential incubation under similar growth conditions (150 rpm, 26uC) (Fig. 1)
A significant increase in the intracellular proline levels and proline utilization A (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)
Summary
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 [1,2,3,4,5]. Malvacearum] were earlier reported in this laboratory to undergo growth medium dependent programmed cell death (PCD) during post-exponential phase [1,2,3,4,5]. 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. As Xanthomonas represents one of the major groups of bacterial plant pathogens, understanding the balance between its survival and death could have broad practical significance in agriculture and provide significant clues to microbial growth control and regulation. 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, called proline oxidase or proline dehydrogenase) which is involved in the metabolism of proline in bacteria, including Xcc
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