Heterologous Expression of Active Dehalobacter Respiratory Reductive Dehalogenases in Escherichia coli.

Reductive dehalogenases (RDases) are a family of redox enzymes that are required for anaerobic organohalide respiration, a microbial process that is useful in bioremediation. Structural and mechanistic studies of these enzymes have been greatly impeded due to challenges in RDase heterologous expression, potentially because of their cobamide-dependence. There have been a few successful attempts at RDase production in unconventional heterologous hosts, but a robust method has yet to be developed. Here we outline a novel respiratory RDase expression system using Escherichia coli. The overexpression of E. coli's cobamide transport system, btu, and anaerobic expression conditions were found to be essential for production of active RDases from Dehalobacter-an obligate organohalide respiring bacterium. The expression system was validated on six enzymes with amino acid sequence identities as low as 28%. Dehalogenation activity was verified for each RDase by assaying cell extracts of small-scale expression cultures on various chlorinated substrates including chloroalkanes, chloroethenes, and hexachlorocyclohexanes. Two RDases, TmrA from Dehalobacter sp. UNSWDHB and HchA from Dehalobacter sp. HCH1, were purified by nickel affinity chromatography. Incorporation of the cobamide and iron-sulfur cluster cofactors was verified; however, the precise cobalamin incorporation could not be determined due to variance between methodologies, and the specific activity of TmrA was consistent with that of the native enzyme. The heterologous expression of respiratory RDases, particularly from obligate organohalide respiring bacteria, has been extremely challenging and unreliable. Here we present a relatively straightforward E. coli expression system that has performed well for a variety of Dehalobacter spp. RDases. IMPORTANCE Understanding microbial reductive dehalogenation is important to refine the global halogen cycle and to improve bioremediation of halogenated contaminants; however, studies of the family of enzymes responsible are limited. Characterization of reductive dehalogenase enzymes has largely eluded researchers due to the lack of a reliable and high-yielding production method. We are presenting an approach to express reductive dehalogenase enzymes from Dehalobacter, a key group of organisms used in bioremediation, in Escherichia coli. This expression system will propel the study of reductive dehalogenases by facilitating their production and isolation, allowing researchers to pursue more in-depth questions about the activity and structure of these enzymes. This platform will also provide a starting point to improve the expression of reductive dehalogenases from many other organisms.

Purification and Kinetic Characterization of the Essential Condensation Enzymes Involved in Prodiginine and Tambjamine Biosynthesis.

Prodiginines and tambjamines are related families of bioactive alkaloid natural products with pharmaceutical potential. Both compound families result from a convergent biosynthetic pathway ending in the condensation of a conserved bipyrrole core with a variable partner. This reaction is performed by unique condensation enzymes, and has the potential to be manipulated to produce new pyrrolic compounds. We have purified and reconstituted the in vitro activity of the condensation enzymes PigC and TamQ from Pseudoalteromonas sp., which are involved, respectively, in the prodiginine and tambjamine biosynthetic pathways. Kinetic analysis confirmed a Uni Uni Bi Uni ping-pong reaction sequence with competitive and uncompetitive substrate inhibition for PigC and TamQ respectively. The kinetic parameters of each enzyme provide insight into their differing substrate scope, and suggest that TamQ may have evolved a wide substrate tolerance that can be used for the production of novel prodiginines and tambjamines.

Long-term survival of Dehalococcoides mccartyi strains in mixed cultures under electron acceptor and ammonium limitation.

Few strains of Dehalococcoides mccartyi harbour and express the vinyl chloride reductase (VcrA) that catalyzes the dechlorination of vinyl chloride (VC), a carcinogenic soil and groundwater contaminant. The vcrA operon is found on a Genomic Island (GI) and, therefore, believed to participate in horizontal gene transfer (HGT). To try to induce HGT of the vcrA-GI, we blended two enrichment cultures in medium without ammonium while providing VC. We hypothesized that these conditions would select for a mutant strain of D. mccartyi that could both fix nitrogen and respire VC. However, after more than 4 years of incubation, we found no evidence for HGT of the vcrA-GI. Rather, we observed VC-dechlorinating activity attributed to the trichloroethene reductase TceA. Sequencing and protein modelling revealed a mutation in the predicted active site of TceA, which may have influenced substrate specificity. We also identified two nitrogen-fixing D. mccartyi strains in the KB-1 culture. The presence of multiple strains of D. mccartyi with distinct phenotypes is a feature of natural environments and certain enrichment cultures (such as KB-1), and may enhance bioaugmentation success. The fact that multiple distinct strains persist in the culture for decades and that we could not induce HGT of the vcrA-GI suggests that it is not as mobile as predicted, or that mobility is restricted in ways yet to be discovered to specific subclades of Dehalococcoides.

Culturing marine bacteria from the genus Pseudoalteromonas on a cotton scaffold alters secondary metabolite production.

The discovery of secondary metabolites from marine microorganisms is beset by numerous challenges including difficulties cultivating and subsequently eliciting expression of biosynthetic genes from marine microbes in the laboratory. In this paper, we describe a method of culturing three species from the marine bacterial genus Pseudoalteromonas using cotton scaffold supplemented liquid media. This simple cultivation method was designed to mimic the natural behavior of some members of the genus wherein they form epibiotic/symbiotic associations with higher organisms such as sponges and corals or attach to solid structures as a biofilm. Our scaffolded cultivation is highly effective at stimulating an attachment/biofilm phenotype and causes large changes to metabolite profiles for the microbes investigated. Metabolite changes include alteration to the production levels of known molecules such as violacein, thiomarinol A, and the alterochromide and prodiginine families of molecules. Finally and critically, our technique stimulates the production of unknown compounds that will serve as leads for future natural product discovery. These results suggest our cultivation approach could potentially be used as a general strategy for the activation of silent gene clusters in marine microbes to facilitate access to their full natural product biosynthetic capacity.

Isolation and characterization of tambjamine MYP1, a macrocyclic tambjamine analogue from marine bacterium Pseudoalteromonas citrea.

Tambjamines are natural products that consist of a conserved bipyrrole core functionalized with different imines giving rise to many derivatives. The core structure of tambjamines allows ion coordination through the nitrogen atoms, which is a key aspect in many of their observed antimicrobial, anticancer, and antimalarial bioactivities. Minor variances in the compound structure have a considerable impact on the potency of these activities, so identifying new analogues is valuable for maximizing tambjamine biological potential. In this work, we describe the isolation and structure elucidation of the first naturally occurring macrocyclized tambjamine, tambjamine MYP1, from the marine microbe Pseudoalteromonas citrea. We also compare the apparent pK a of cyclic and linear tambjamine analogues and discuss how structural strain may effect the compound's ion coordination abilities.