Leveraging modern DNA assembly techniques for rapid, markerless genome modification.

The ability to alter the genomic material of a prokaryotic cell is necessary for experiments designed to define the biology of the organism. In addition, the production of biomolecules may be significantly improved by application of engineered prokaryotic host cells. Furthermore, in the age of synthetic biology, speed and efficiency are key factors when choosing a method for genome alteration. To address these needs, we have developed a method for modification of the Escherichia coli genome named FAST-GE for Fast Assembly-mediated Scarless Targeted Genome Editing. Traditional cloning steps such as plasmid transformation, propagation and isolation were eliminated. Instead, we developed a DNA assembly-based approach for generating scarless strain modifications, which may include point mutations, deletions and gene replacements, within 48 h after the receipt of polymerase chain reaction primers. The protocol uses established, but optimized, genome modification components such as I-SceI endonuclease to improve recombination efficiency and SacB as a counter-selection mechanism. All DNA-encoded components are assembled into a single allele-exchange vector named pDEL. We were able to rapidly modify the genomes of both E. coli B and K-12 strains with high efficiency. In principle, the method may be applied to other prokaryotic organisms capable of circular dsDNA uptake and homologous recombination.

A tale of two reductases: extending the bacteriochlorophyll biosynthetic pathway in E. coli.

The creation of a synthetic microbe that can harvest energy from sunlight to drive its metabolic processes is an attractive approach to the economically viable biosynthetic production of target compounds. Our aim is to design and engineer a genetically tractable non-photosynthetic microbe to produce light-harvesting molecules. Previously we created a modular, multienzyme system for the heterologous production of intermediates of the bacteriochlorophyll (BChl) pathway in E. coli. In this report we extend this pathway to include a substrate promiscuous 8-vinyl reductase that can accept multiple intermediates of BChl biosynthesis. We present an informative comparative analysis of homologues of 8-vinyl reductase from the model photosynthetic organisms Rhodobacter sphaeroides and Chlorobaculum tepidum. The first purification of the enzymes leads to their detailed biochemical and biophysical characterization. The data obtained reveal that the two 8-vinyl reductases are substrate promiscuous, capable of reducing the C8-vinyl group of Mg protoporphyrin IX, Mg protoporphyrin IX methylester, and divinyl protochlorophyllide. However, activity is dependent upon the presence of chelated Mg(2+) in the porphyrin ring, with no activity against non-Mg(2+) chelated intermediates observed. Additionally, CD analyses reveal that the two 8-vinyl reductases appear to bind the same substrate in a different fashion. Furthermore, we discover that the different rates of reaction of the two 8-vinyl reductases both in vitro, and in vivo as part of our engineered system, results in the suitability of only one of the homologues for our BChl pathway in E. coli. Our results offer the first insights into the different functionalities of homologous 8-vinyl reductases. This study also takes us one step closer to the creation of a nonphotosynthetic microbe that is capable of harvesting energy from sunlight for the biosynthesis of molecules of choice.

BioBrick™ compatible vector system for protein expression in Rhodobacter sphaeroides.

We report here the creation of a modular, plasmid-based protein expression system utilizing elements of the native Rhodobacter puf promoter in a BioBrick(TM)-based vector system with DsRed encoding a red fluorescent reporter protein. A suite of truncations of the puf promoter were made to assess the influence of different portions of this promoter on expression of heterologous proteins. The 3' end of puf was found to be particularly important for increasing expression, with transformants accumulating significant quantities of DsRed under both aerobic and anaerobic growth conditions. Expression levels of this reporter protein in Rhodobacter sphaeroides were comparable to those achieved in Escherichia coli using the strong, constitutive P lac promoter, thus demonstrating the robustness of the engineered system. Furthermore, we demonstrate the ability to tune the designed expression system by modulating cellular DsRed levels based upon the promoter segment utilized and oxygenation conditions. Last, we show that the new expression system is able to drive expression of a membrane protein, proteorhodopsin, and that membrane purifications from R. sphaeroides yielded significant quantities of proteorhodopsin. This toolset lays the groundwork for the engineering of multi-step pathways, including recalcitrant membrane proteins, in R. sphaeroides.

Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering.

The BioBrick™ paradigm for the assembly of enzymatic pathways is being adopted and becoming a standard practice in microbial engineering. We present a strategy to adapt the BioBrick™ paradigm to allow the quick assembly of multi-gene pathways into a number of vectors as well as for the quick mobilization of any cloned gene into vectors with different features for gene expression and protein purification. A primary BioBrick™ (BB-eGFP) was developed where the promoter/RBS, multiple cloning sites, optional protein purification affinity tags and reporter gene were all separated into discrete regions by additional restriction enzymes. This primary BB-eGFP then served as the template for additional BioBrick™ vectors with different origins of replication, antibiotic resistances, inducible promoters (arabinose, IPTG or anhydrotetracycline), N- or C-terminal Histidine tags with thrombin cleavage, a LacZα reporter gene and an additional origin of mobility (oriT). All developed BioBricks™ and BioBrick™ compatible vectors were shown to be functional by measuring reporter gene expression. Lastly, a C(30) carotenoid pathway was assembled as a model enzymatic pathway to demonstrate in vivo functionality and compatibility of this engineered vector system.

Evidence that human histidine triad nucleotide binding protein 3 (Hint3) is a distinct branch of the histidine triad (HIT) superfamily.

Human Hint3 (hHint3) has been classified as a member of the histidine triad nucleotide (Hint) binding protein subfamily. While Hint1 is ubiquitously expressed by both eukaryotes and prokaryotes, Hint3 is found only in eukaryotes. Previously, our laboratory has characterized and compared the aminoacyl-adenylate and nucleoside phosphoramidate hydrolase activity of hHint1 and Escherichia coli hinT. In this study, hHint3-1(Ala36) and its single nucleotide polymorphism, hHint3-2 (A36G variant), were cloned, overexpressed, and purified. Steady-state kinetic studies with a synthetic fluorogenic indolepropinoic acyl-adenylate (AIPA) and with a series of fluorogenic tryptamine nucleoside phosphoramidates revealed that hHint3-1 and hHint3-2 are adenylate and phosphoramidate hydrolases with apparent second-order rate constants (kcat/Km) ranging from 10(2) to 10(6) s(-1) M(-1). Unlike hHint1, hHint3-1 and hHint3-2 prefer AIPA over tryptamine adenosine phosphoramidate by factors of 33- and 16-fold, respectively. In general, hHint3s hydrolyze phosphoramidate 370- to 2000-fold less efficiently than hHint1. Substitution of the potential active-site nucleophile, His145, by Ala was shown to abolish the adenylate and phosphoramidate hydrolase activity for hHint3-1. However, 0.2-0.4% residual activity was observed for the H145A mutant of hHint3-2. Both hHint3-1 and hHint3-2 were found to hydrolyze lysyl-adenylate generated by human lysyl-tRNA synthetase (hLysRS) by proceeding through an adenylated protein intermediate. hLysRS-dependent labeling of hHint3-1 and hHint3-2 was found to depend on His145, which aligns with the His112 of the Hint1 active site. The extent of active-site His145-AMP labeling was shown to be similar to His112-AMP labeling of hHint1. In contrast to all previously characterized members of the histidine triad superfamily, which have been shown to exist exclusively as homodimers, wild type and the H145A of hHint3-1 were found to exist across a range of multimeric states, from dimers to octamers and even larger oligomers, while wild type and the H145A of hHint3-2 exist predominantly in a monomeric state. The differences in oligomeric state may be important in vivo, because unlike tetracysteine-tagged Hint1, which was found along linear arrays exclusively in the cytoplasm in transfected HeLa cells, tagged Hint3-1 and Hint3-2 were found as aggregates both in the cytosol and in the nucleus. Taken together, these results imply that while Hint3 and Hint1 prefer aminoacyl-adenylates as substrates and catalytically interact with aminoacyl-tRNA synthetases, the significant differences in phosphoramidase activity, oligomeric state, and cellular localization suggest that Hint3s should be placed in a distinct branch of the histidine triad superfamily.

Generation of virulence factor variants in Staphylococcus aureus biofilms.

Several serious diseases are caused by biofilm-associated Staphylococcus aureus. Colonial variants occur in biofilms of other bacterial species, and S. aureus variants are frequently isolated from biofilm-associated infections. Thus, we studied the generation of variants with altered expression of virulence factors in S. aureus biofilms. We observed that the number of variants found in biofilms, as measured by hemolytic activity, varied for different strains. Further study of hemolytic activity and signaling by the accessory gene regulator (Agr) quorum-sensing system in one S. aureus strain revealed three primary biofilm subpopulations: nonhemolytic (Agr deficient), hemolytic (Agr positive), and hyperhemolytic (also Agr positive). The nonhemolytic variant became the numerically dominant subpopulation in the biofilm. The nonhemolytic variant phenotype was stable and heritable, indicating a genetic perturbation, whereas the hyperhemolytic phenotype was unstable, suggesting a phase variation. Transcription profiling revealed that expression of the agr locus and many extracellular virulence factors was repressed in the nonhemolytic variant. Expression of the agr-activating gene, sarU, was also repressed in the nonhemolytic variant, suggesting one potential regulatory pathway responsible for the Agr-deficient phenotype. We suggest that the development of these variants in biofilms may have important clinical implications.

Engineered monomeric human histidine triad nucleotide-binding protein 1 hydrolyzes fluorogenic acyl-adenylate and lysyl-tRNA synthetase-generated lysyl-adenylate.

Hint1 is a homodimeric protein and member of the ubiquitous HIT superfamily. Hint1 catalyzes the hydrolysis of purine phosphoramidates and lysyl-adenylate generated by lysyl-tRNA synthetase (LysRS). To determine the importance of homodimerization on the biological and catalytic activity of Hint1, the dimer interface of human Hint1 (hHint1) was destabilized by replacement of Val(97) of hHint1 with Asp, Glu, or Arg. The mutants were shown to exist as monomers in solution by a combination of size exclusion chromatograph, static light scattering, and chemically induced dimerization studies. Circular dichroism studies revealed little difference between the stability of the V97D, V97E, and wild-type hHint1. Relative to wild-type and the V97E mutant, however, significant perturbation of the V97D mutant structure was observed. hHint1 was shown to prefer 3-indolepropionic acyl-adenylate (AIPA) over tryptamine adenosine phosphoramidate monoester (TpAd). Wild-type hHint1 was found to be 277- and 1000-fold more efficient (k(cat)/K(m) values) than the V97E and V97D mutants, respectively. Adenylation of wild-type, V97D, and V97E hHint1 by human LysRS was shown to correlate with the mutant k(cat)/K(m) values using 3-indolepropionic acyl-adenylate as a substrate, but not tryptamine adenosine phosphoramidate monoester. Significant perturbations of the active site residues were not detected by molecular dynamics simulations of the hHint1s. Taken together, these results demonstrate that for hHint1; 1) the efficiency (k(cat)/K(m)) of acylated AMP hydrolysis, but not maximal catalytic turnover (k(cat)), is dependent on homodimerization and 2) the hydrolysis of lysyl-AMP generated by LysRS is not dependent on homodimerization if the monomer structure is similar to the wild-type structure.