Metabolic Engineering of Cofactor F420 Production in Mycobacterium smegmatis

Cofactor F420 is a unique electron carrier in a number of microorganisms including Archaea and Mycobacteria. It has been shown that F420 has a direct and important role in archaeal energy metabolism whereas the role of F420 in mycobacterial metabolism has only begun to be uncovered in the last few years. It has been suggested that cofactor F420 has a role in the pathogenesis of M. tuberculosis, the causative agent of tuberculosis. In the absence of a commercial source for F420, M. smegmatis has previously been used to provide this cofactor for studies of the F420-dependent proteins from mycobacterial species. Three proteins have been shown to be involved in the F420 biosynthesis in Mycobacteria and three other proteins have been demonstrated to be involved in F420 metabolism. Here we report the over-expression of all of these proteins in M. smegmatis and testing of their importance for F420 production. The results indicate that co–expression of the F420 biosynthetic proteins can give rise to a much higher F420 production level. This was achieved by designing and preparing a new T7 promoter–based co-expression shuttle vector. A combination of co–expression of the F420 biosynthetic proteins and fine-tuning of the culture media has enabled us to achieve F420 production levels of up to 10 times higher compared with the wild type M. smegmatis strain. The high levels of the F420 produced in this study provide a suitable source of this cofactor for studies of F420-dependent proteins from other microorganisms and for possible biotechnological applications

Forest Decline after a 15-Year “Perfect Storm” of Invasion by Hemlock Woolly Adelgid, Drought, and Hurricanes

Invasions by introduced pests can interact with other disturbances to alter forests and their functions, particularly when a dominant tree species declines. To identify changes after invasion by the insect hemlock woolly adelgid (Adelges tsugae; HWA), coinciding with severe droughts and hurricanes, this study compared tree species composition of eastern hemlock (Tsuga canadensis) forests on 11 plots before (2001) and 15 years after (2016) invasion in the southern Appalachian Mountains, USA. Losses of hemlock trees after HWA invasion were among the highest reported, with a 90% decline in density, 86% decline in basal area, and 100% mortality for individuals ≥ 60 cm in diameter. In contrast to predictions of theoretical models, deciduous tree density declined after HWA invasion, while basal area changed little, at least during the initial 15 years after invasion. Overall, forest density declined by 58%, basal area by 25%, and tree species richness by 8%. Factors additional to HWA likely exacerbating forest decline included: droughts before (1999–2001) and after HWA invasion (2006–2008); tree uprooting from hurricane-stimulated winds in 2004; pest-related declines of deciduous tree species otherwise likely benefitting from hemlock’s demise; death of deciduous trees when large hemlocks fell; and competition from aggressive understory plants including doghobble (Leucothoe fontanesiana), rosebay rhododendron (Rhododendron maximum), and Rubus spp. Models of forest change and ecosystem function should not assume that deciduous trees always increase during the first decades after HWA invasion