The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.

We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis [Nicely, N.I., Parsonage, D., Paige, C., Newton, G.L., Fahey, R.C., Leonardi, R., Jackowski, S., Mallett, T.C., and Claiborne, A. (2007) Biochemistry 46, 3234-3245], and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 Å resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides. The structures of the NADH and NADPH complexes at ca. 2.3 Å resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2′-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367′-OH and Tyr425′-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367, 425F BACoADR mutants indicate that Tyr425′ is the primary proton donor in catalysis, with Tyr367′ functioning as a cryptic alternate donor in the absence of Tyr425′.

Coenzyme A (CoASH) replaces glutathione as the major low-molecular weight thiol in Staphylococcus aureus; it is maintained in the reduced state by coenzyme A-disulfide reductase (CoADR), a homodimeric enzyme similar to NADH peroxidase, but containing a novel Cys43-SSCoA redox center. The crystal structure of S. aureus CoADR has been solved using multiwavelength anomalous dispersion data and refined at a resolution of 1.54 Å. The resulting electron density maps define the Cys43-SSCoA disulfide conformation, with Cys43-S γ located at the flavin si-face, 3.2 Å from FAD-C4aF, and the CoAS-moiety lying in an extended conformation within a cleft at the dimer interface. A well-ordered chloride ion is positioned adjacent to the Cys43-SSCoA disulfide and receives a hydrogen bond from Tyr361′-OH of the complementary subunit, suggesting a role for Tyr361′ as an acid-base catalyst during the reduction of CoAS-disulfide. Tyr419′-OH is located 3.2 Å from Tyr361′-OH as well, and based on its conservation in known functional CoADRs, also appears important for activity. Identification of residues involved in recognition of the CoAS-disulfide substrate and in formation and stabilization of the Cys43-SSCoA redox center has allowed development of a CoAS-binding motif. Bioinformatics analyses indicate that CoADR enzymes are broadly distributed in both bacterial and archaeal kingdoms, suggesting an even broader significance for the CoASH/CoAS-disulfide redox system in prokaryotic thiol/ disulfide homeostasis.In contrast to the central role played by the tripeptide thiol glutathione [GSH; (1)] in maintaining thiol/disulfide homeostasis and providing an important line of antioxidant defense in, for example, Escherichia coli, earlier studies clearly indicated the absence of GSH in a number of Bacteria (2,3) and in all Archaea (4,5). Coenzyme A (CoASH) was shown to be the † This work was supported by National Institutes of Health Grant GM-35394 (A.C.), by National Science Foundation Grant , by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (Monbusho), Japan, and the Japan Society for the Promotion of Science (T.T.), and by National Science Foundation Grant MCB-9982727 (P.A.K.). T.C.M. was the recipient of International Fellowship P-99934 from the Japan Society for the Promotion of Science. A.C. was the recipient of Short-Term Invitation Fellowship RC20137002 from the Japan Society for the Promotion of Science. Data for this study were measured at beamline X26C of the National Synchrotron Light Source. (12) subsequently noted that the PNDORs could be subdivided on the basis of mechanistic and sequence-structural information into three groups, with "Group 3" including CoADR, NADH peroxidase (Npx), and NADH oxidase (Nox). Until the identification of CoADR (6), all flavoprotein disulfide reductases were members of PNDOR Groups 1 and 2, and all Group 3 enzymes were specific for either H 2 O 2 or O 2 (→2H 2 O) reduction. CoADR is clearly by sequence a Group 3 PNDOR enzyme (7); it (like Npx and No...

Rhodanese homology domains (RHDs) play important roles in sulfur trafficking mechanisms essential to the biosynthesis of sulfur-containing cofactors and nucleosides. We have now determined the crystal structure at 2.10 Å resolution for the Bacillus anthracis coenzyme A-disulfide reductase isoform (BaCoADR-RHD) containing a C-terminal RHD domain; this is the first structural representative of the multidomain proteins class of the rhodanese superfamily. The catalytic Cys44 of the CoADR module is separated by a distance of 25 Å from the active-site Cys514′ of the RHD domain from the complementary subunit. In stark contrast to the B. anthracis CoADR [Wallen, J.R., Paige, C., Mallett, T.C., Karplus, P.A., and Claiborne, A. (2008) Biochemistry 47, 5182-5193], the BaCoADR-RHD isoform does not catalyze the reduction of coenzyme A-disulfide, although both enzymes conserve the Cys-SSCoA redox center. NADH titrations have been combined with a synchrotron reduction protocol to examine the structural and redox behavior of the Cys44-SSCoA center. The synchrotron-reduced (Cys44 + CoASH) structure reveals ordered binding for the adenosine 3′-phosphate 5′-pyrophosphate moiety of CoASH, but the absence of density for the pantetheine arm indicates that it is flexible within the reduced active site. Steady-state kinetic analyses with the alternate disulfide substrates methyl methanethiolsulfonate (MMTS) and 5, 5′-dithiobis(2-nitrobenzoate) (DTNB), including the appropriate Cys→Ser mutants, demonstrate that MMTS reduction occurs within the CoADR active site. NADH-dependent DTNB reduction, on the other hand, requires communication between Cys44 and Cys514′, and we propose that reduction of the Cys44-SSCoA disulfide promotes the transfer of reducing equivalents to the RHD, with the swinging pantetheine arm serving as a ca. 20 Å bridge.