Nitrogen is a limiting factor for growth and maintenance in many organisms, particularly those living on a herbivorous diet as the attine ants indirectly do. Selleck Kinase Inhibitor Library Recent findings show that leaf-cutting ants partly overcome nitrogen limitation by living in association with N2-fixing

bacteria that may supply as much as 50% of a colony’s nitrogen requirements [11]. Such bacterial nitrogen will be incorporated into proteins, so that the fungal symbionts of the ants must secrete proteinases to digest these into amino Z IETD FMK acids that can be assimilated. The fungal symbiont is also likely to compete for nitrogen with other, non mutualistic microorganisms living in the fungus garden [12, 9, 13], imposing further selection for effective protein degradation by the fungal symbiont. Finally, proteolytic enzymes are known to be strongly pH dependent, so in order to have effective protein degradation the pH optimum of the proteolytic enzymes should ideally match the pH of the fungus garden. Several studies have been devoted to the role of pH in controlling in vitro proteolytic enzyme secretion in fungi [14], but to our knowledge in vivo studies of pH-dependent proteolytic enzyme activities in fungi have not been done. The objective of our present study was thus threefold: 1. To use the unique growth CP-690550 datasheet form of ant fungus gardens to determine the feasibility of pH buffering studies in fungi,

2. To determine the pH activity optima of different classes of extracellular proteinases across a series of genera and species of fungus-growing ants, and 3. To map the observed differences on an independently obtained Sinomenine phylogenetic

tree of the fungal symbionts to obtain insight in the evolutionary pathways that may have generated differences in pH-dependent activities of proteinases. Results The pH conditions of fungus gardens and their buffering capacity All 29 attine ant colonies used in this study (see Table 1 for details) displayed the same pH (5.2 ± 0.1) for 1:1 water extracts taken from the middle layer of the fungus gardens. Adding acid/alkaline solutions to the fungus garden extracts did not noticeable change the color of pH paper compared to controls (data not shown) indicating that all tested fungus gardens exhibited approximately the same buffering strength. Table 1 Total and class-specific relative proteolytic activity and its pH optimum range measured in fungus gardens. Ant species Colony number Sample number Total activity pH optimum Metallo-proteinase activity pH optimum Serine proteinase activity pH optimum Aspartic proteinase activity Cysteine proteinase activity Apterostigma collare Apcol1 – 630.0 ± 18.3   593.0 ± 13.3   1.7 ± 0.5   16.0 ± 1.0 0.8 ± 0.5 Myrmicocrypta ednaella Myred1 1 168.6 ± 9.5 6.2 ± 0.11 151.5 ± 6.4 6.0 ± 0.04 9.4 ± 1.0 7.0 ± 0.012 — 9.3 ± 1.0   Myred2 2 165.2 ± 9.2   104.