The monarch butterfly (Danaus plexippus) cryptochrome 1 (DpCry1) belongs in the class of photosensitive insect cryptochromes. Here we purified DpCry1 expressed in a bacterial host and obtained the protein with a stoichiometric amount of the flavin cofactor in the two-electron oxidized, FAD ox , form. Exposure of the purified protein to light converts the FAD ox to the FAD . flavin anion radical by intraprotein electron transfer from a Trp residue in the apoenzyme. To test whether this novel photoreduction reaction is part of the DpCry1 physiological photocycle, we mutated the Trp residue that acts as the ultimate electron donor in flavin photoreduction. The mutation, W328F, blocked the photoreduction entirely but had no measurable effect on the light-induced degradation of DpCry1 in vivo. In light of this finding and the recently published action spectrum of this class of Crys, we conclude that DpCry1 and similar insect cryptochromes do not contain flavin in the FAD ox form in vivo and that, most likely, the FAD ox --3 h FAD . photoreduction reaction is not part of the insect cryptochrome photoreaction that results in proteolytic degradation of the photopigment.Cryptochromes are blue-light photoreceptors that control growth and development in plants and the circadian clock in animals (1-3). They have sequence and structural similarities to DNA photolyases that repair far UV-induced DNA damage using blue light as a co-substrate. Photolyases contain two-electron reduced deprotonated FAD (FADH Ϫ ) as the catalytic cofactor and methenyltetrahydrofolate (MTHF) 3 or, in rare cases, 8-deazaflavin as a photoantenna for gathering photons and transferring excitation energy to FADH Ϫ to initiate catalysis (2). Cryptochromes from plant and animal sources, as well, contain FAD and are known or presumed to contain MTHF (4 -6). However, the functional redox state of flavin in cryptochromes is unknown with certainty, and there is no evidence that the flavin cofactor in cryptochrome functions as a redox catalyst (1, 3).Arabidopsis thaliana cryptochrome 1 (AtCry1), which was the first cryptochrome to be discovered (7), has been the most extensively investigated. The AtCry1, as well as the related AtCry2, can be purified as recombinant proteins expressed in Escherichia coli or baculovirus/insect cell expression systems (4,5,8). The photopigments purified from these expression systems contain trace amounts of MTHF and stoichiometric flavin cofactor in the two-electron oxidized, FAD ox , form. A number of interesting observations have been made with Arabidopsis cryptochromes, in particular with AtCry1, which may be relevant to their functions. First, AtCry1 binds ATP in the cavity containing the flavin cofactor (8 -10). Second, AtCry1 exhibits an autophosphorylating kinase activity (9 -11). Third, the FAD ox of AtCry1 is photoreduced to the FADH ⅐ neutral radical by intraprotein electron transfer both in vitro and in vivo (4, 12). Fourth, this intraprotein electron transfer causes a conformational change in the C-terminal tail of At...

The public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Department of Defense, Washington Headquarters Services, Directorate for Information SPONSORING/MONITORING AGENCY REPORT NUMBER(S) AFRL-PR-WP-TR-2006-2049 DISTRIBUTION/AVAILABILITY STATEMENTApproved for public release; distribution is unlimited. SUPPLEMENTARY NOTESReport contains color. ABSTRACTMicrobially contaminated aviation fuel cause the Air Force increased maintenance and replacement costs from problems such as fuel gauge malfunctions, fuel line and filter plugging, and corrosion. As a result, there is considerable interest in identifying microbial growth and finding strategies to mitigate it. Previous research to isolate and identify aviation fuel microbial contaminants has used cultivation-based methodologies. This study aimed to investigate newer molecular methods to more comprehensively characterize the bioburden in aviation fuel supplies. Several fuel samples were analyzed for bacterial contamination using two distinct methods: a cultivation-independent method (direct PCR) and a traditional cultivation-dependant method. A total of 36 bacterial genera were identified, including 28 genera which have not been previously reported in aviation fuel. Nearly 62% of the new bacterial genera were isolated with the cultivation-independent method only, 33% with both methods, and only 5% with the cultivation-dependant method only. SUBJECT TERMS LIST OF FIGURES AND TABLES ABSTRACTMicrobial contaminated aviation fuel causes the Air Force increased maintenance and replacement costs from problems such as fuel gauge malfunctions, fuel line and filter plugging, and corrosion. As a result, there is considerable interest in identifying microbial growth and finding strategies to mitigate it. Previous research to isolate and identify aviation fuel microbial contaminants has used cultivation-based methodologies. This study aimed to investigate newer molecular methods to more comprehensively characterize the bioburden in aviation fuel supplies. Several fuel samples were analyzed for bacterial contamination using two distinct methods: a cultivation-independent method (direct PCR) and a traditional cultivation-dependent method. A total of 36 bacterial genera were identified, including 28 genera which have not been reported in aviation fuel. Nearly 62% of the new bacterial genera were isolated with the cultivation-independent method only, 33% with both methods, and only 5% with the cultivation-dependent method only. The cultivation-independent method identified an average of four times the amount of contaminants as identified using the cultivation-dependent...

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