Control of biofilm formation by an Agrobacterium tumefaciens pterin-binding periplasmic protein conserved among diverse Proteobacteria.

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages.

Biochemical and genetic studies define the functions of methylthiotransferases in methanogenic and methanotrophic archaea.

Methylthiotransferases (MTTases) are radical S-adenosylmethionine (SAM) enzymes that catalyze the addition of a methylthio (-SCH3) group to an unreactive carbon center. These enzymes are responsible for the production of 2-methylthioadenosine (ms2A) derivatives found at position A37 of select tRNAs in all domains of life. Additionally, some bacteria contain the RimO MTTase that catalyzes the methylthiolation of the S12 ribosomal protein. Although the functions of MTTases in bacteria and eukaryotes have been established via detailed genetic and biochemical studies, MTTases from the archaeal domain of life are understudied and the substrate specificity determinants of MTTases remain unclear. Here, we report the in vitro enzymatic activities of an MTTase (C4B56_06395) from a thermophilic Ca. Methanophagales anaerobic methanotroph (ANME) as well as the MTTase from a hyperthermophilic methanogen - MJ0867 from Methanocaldococcus jannaschii. Both enzymes catalyze the methylthiolation of N6-threonylcarbamoyladenosine (t6A) and N6-hydroxynorvalylcarbamoyladenosine (hn6A) residues to produce 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) and 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine (ms2hn6A), respectively. To further assess the function of archaeal MTTases, we analyzed select tRNA modifications in a model methanogen - Methanosarcina acetivorans - and generated a deletion of the MTTase-encoding gene (MA1153). We found that M. acetivorans produces ms2hn6A in exponential phase of growth, but does not produce ms2t6A in detectable amounts. Upon deletion of MA1153, the ms2A modification was absent, thus confirming the function of MtaB-family MTTases in generating ms2hn6A modified nucleosides in select tRNAs.

Control of Biofilm Formation by an Agrobacterium tumefaciens Pterin-Binding Periplasmic Protein Conserved Among Pathogenic Bacteria.

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (cdGMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase (DGC-PDE), is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are formed via a non-essential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering cdGMP breakdown and dampening its synthesis. Pterins are excreted and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon that is conserved amongst multiple Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a new pterin-responsive regulatory mechanism that controls biofilm formation and related cdGMP-dependent phenotypes in A. tumefaciens and is found in multiple additional bacterial pathogens.

Highlighting the Unique Roles of Radical S-Adenosylmethionine Enzymes in Methanogenic Archaea.

Radical S-adenosylmethionine (SAM) enzymes catalyze an impressive variety of difficult biochemical reactions in various pathways across all domains of life. These metalloenzymes employ a reduced [4Fe-4S] cluster and SAM to generate a highly reactive 5'-deoxyadenosyl radical that is capable of initiating catalysis on otherwise unreactive substrates. Interestingly, the genomes of methanogenic archaea encode many unique radical SAM enzymes with underexplored or completely unknown functions. These organisms are responsible for the yearly production of nearly 1 billion tons of methane, a potent greenhouse gas as well as a valuable energy source. Thus, understanding the details of methanogenic metabolism and elucidating the functions of essential enzymes in these organisms can provide insights into strategies to decrease greenhouse gas emissions as well as inform advances in bioenergy production processes. This minireview provides an overview of the current state of the field regarding the functions of radical SAM enzymes in methanogens and discusses gaps in knowledge that should be addressed.