Warfarin analogs target disulfide bond-forming enzymes and suggest a residue important for quinone and coumarin binding.

Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes. In bacteria, disulfide bonds are catalyzed by DsbA and DsbB/VKOR enzymes. First, DsbA, a periplasmic disulfide oxidoreductase, introduces disulfide bonds into substrate proteins. Then, the membrane enzyme, either DsbB or VKOR, regenerate DsbA's activity by the formation of de novo disulfide bonds which reduce quinone. We have previously performed a high-throughput chemical screen and identified a family of warfarin analogs that target either bacterial DsbB or VKOR. In this work, we expressed functional human VKORc1 in Escherichia coli and performed a structure-activity-relationship analysis to study drug selectivity between bacterial and mammalian enzymes. We found that human VKORc1 can function in E. coli by removing two positive residues, allowing the search for novel anticoagulants using bacteria. We also found one warfarin analog capable of inhibiting both bacterial DsbB and VKOR and a second one antagonized only the mammalian enzymes when expressed in E. coli. The difference in the warfarin structure suggests that substituents at positions three and six in the coumarin ring can provide selectivity between the bacterial and mammalian enzymes. Finally, we identified the two amino acid residues responsible for drug binding. One of these is also essential for de novo disulfide bond formation in both DsbB and VKOR enzymes. Our studies highlight a conserved role of this residue in de novo disulfide-generating enzymes and enable the design of novel anticoagulants or antibacterials using coumarin as a scaffold.

Conformation and dynamic interactions of the multipartite genome in Agrobacterium tumefaciens.

Bacterial species from diverse phyla contain multiple replicons, yet how these multipartite genomes are organized and segregated during the cell cycle remains poorly understood. Agrobacterium tumefaciens has a 2.8-Mb circular chromosome (Ch1), a 2.1-Mb linear chromosome (Ch2), and two large plasmids (pAt and pTi). We used this alpha proteobacterium as a model to investigate the global organization and temporal segregation of a multipartite genome. Using chromosome conformation capture assays, we demonstrate that both the circular and the linear chromosomes, but neither of the plasmids, have their left and right arms juxtaposed from their origins to their termini, generating interarm interactions that require the broadly conserved structural maintenance of chromosomes complex. Moreover, our study revealed two types of interreplicon interactions: "ori-ori clustering" in which the replication origins of all four replicons interact, and "Ch1-Ch2 alignment" in which the arms of Ch1 and Ch2 interact linearly along their lengths. We show that the centromeric proteins (ParB1 for Ch1 and RepBCh2 for Ch2) are required for both types of interreplicon contacts. Finally, using fluorescence microscopy, we validated the clustering of the origins and observed their frequent colocalization during segregation. Altogether, our findings provide a high-resolution view of the conformation of a multipartite genome. We hypothesize that intercentromeric contacts promote the organization and maintenance of diverse replicons.

Union Is Strength: Target-Based and Whole-Cell High-Throughput Screens in Antibacterial Discovery.

Antimicrobial resistance is one of the greatest global health challenges today. For over 3 decades, antibacterial discovery research and development have been focused on cell-based and target-based high-throughput assays. Target-based screens use diagnostic enzymatic reactions to look for molecules that can bind directly to and inhibit the target. Target-based screens are applied only to proteins that can be successfully expressed and purified and the activity of which can be effectively measured using a biochemical assay. Often the molecules found in these in vitro screens are not active in cells due to poor permeability or efflux. On the other hand, cell-based screens use whole cells and look for growth inhibition. These screens give higher numbers of hits than target-based assays and can simultaneously test many targets of one process or pathway in their physiological context. Both strategies have advantages and disadvantages when used separately. In the past 15 years, our increasing knowledge of bacterial physiology has led to the development of innovative and sophisticated technologies to perform high-throughput screening combining these two strategies and thus minimizing their disadvantages. In this review, we discuss recent examples of high-throughput approaches that used both target-based and whole-cell screening to find new antibacterials, the new insights they have provided, and how this knowledge can be applied to other in vivo-validated targets to develop new antimicrobials.

Natural Transformation in a Classical-Biotype Vibrio cholerae Strain.

Vibrio cholerae causes the gastrointestinal illness cholera, which spreads throughout the globe in large pandemics. The current pandemic is caused by O1 El Tor biotype strains, whereas previous pandemics were caused by O1 classical biotype strains. El Tor V. cholerae is noted for its ability to acquire exogenous DNA through chitin-induced natural transformation, which has been exploited for genetic manipulation of El Tor strains in the laboratory. In contrast, the prototypical classical strain O395 lacks this ability, which was suspected to be due to a mutation in the regulatory gene hapR HapR and the regulator TfoX control expression of a third competence regulator, QstR. We found that artificial induction of both TfoX and QstR in the presence of HapR in O395 was required for efficient DNA uptake. However, natural transformation in the classical strain is still orders of magnitude below that of an El Tor strain. O395 expressing HapR could also undergo natural transformation after growth on chitin, which could be increased by artificial induction of TfoX and/or QstR. A plasmid that expresses both TfoX and QstR was created that allowed for consistent DNA uptake in O395 carrying a hapR plasmid. This technique was also used to facilitate cotransformation into O395 of unmarked DNA (ΔlacZ, ΔflaA, ΔflgG) for multiplex genome editing by natural transformation (MuGENT). These results demonstrate that the classical biotype O395 strain is functionally capable of DNA uptake, which allows for the rapid genetic manipulation of its genome.IMPORTANCE Natural transformation (uptake of exogenous DNA) in Vibrio cholerae has contributed to the evolution of these human pathogens. Classical biotype V. cholerae strains were responsible for the first six cholera pandemics but were replaced by El Tor biotype V. cholerae in the current pandemic. This study demonstrates that classical V. cholerae is functionally capable of natural transformation, but inactivation of the transformation regulator HapR and inherent levels of transformation that are lower than those of El Tor V. cholerae suggest that the classical biotype may be less able to utilize natural transformation for horizontal gene transfer.

New cloning vectors to facilitate quick allelic exchange in gram-negative bacteria.

New cloning vectors have been developed with features to enhance quick allelic exchange in gram-negative bacteria. The conditionally replicative R6K and transfer origins facilitate conjugation and chromosomal integration into a variety of bacterial species, whereas the sacB gene provides counterselection for allelic exchange. The vectors have incorporated the lacZ alpha fragment with an enhanced multicloning site for easy blue/white screening and priming sites identified for efficient in vivo assembly or other DNA assembly cloning techniques. Different antibiotic resistance markers allow versatility for use with different bacteria, and transformation into an Escherichia coli strain capable of conjugation enables a quick method for allelic exchange. As a proof of principle, the authors used these vectors to inactivate genes in Vibrio cholerae and Salmonella typhimurium.