History and Prospects of Drug Discovery and Development Collaboration between Industry and Academia.

Research collaborations and licensing deals are critical for the discovery and development of life-saving drugs. This practice has been ongoing since the inception of the pharmaceutical industry. The current process of drug discovery and development is complex, regulated, and highly regimented, having evolved over time. Academia excels in the discovery of fundamental scientific concepts and biological processes, while industry excels in translational science and product development. Potential for collaboration exists at every step of the drug discovery and development continuum. This perspective walks through such collaborative activities, provides examples, and offers tips for potential collaborations.

Genome of a Gut Strain of Bacillus subtilis.

Bacillus subtilis is a Gram-positive, rod-shaped, spore-forming bacterium. We present the genome sequence of an undomesticated strain, BSP1, isolated from poultry. The sequence of the BSP1 genome supports the view that B. subtilis has a biphasic lifestyle, cycling between the soil and the animal gastrointestinal tract, and it provides molecular-level insight into the adaptation of B. subtilis to life under laboratory conditions.

The origins of 168, W23, and other Bacillus subtilis legacy strains.

Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three--NCIB 3610T, PY79, and SMY--we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more "W23 islands" in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains.

Genes for small, noncoding RNAs under sporulation control in Bacillus subtilis.

The process of sporulation in the bacterium Bacillus subtilis is known to involve the programmed activation of several hundred protein-coding genes. Here we report the discovery of previously unrecognized genes under sporulation control that specify small, non-protein-coding RNAs (sRNAs). Genes for sRNAs were identified by transcriptional profiling with a microarray bearing probes for intergenic regions in the genome and by use of a comparative genomics algorithm that predicts regions of conserved RNA secondary structure. The gene for one such sRNA, SurA, which is located in the region between yndK and yndL, was induced at the start of development under the indirect control of the master regulator for entry into sporulation, Spo0A. The gene for a second sRNA, SurC, located in the region between dnaJ and dnaK, was switched on at a late stage of sporulation by the RNA polymerase sigma factor sigmaK, which directs gene transcription in the mother cell compartment of the developing sporangium. Finally, a third intergenic region, that between polC and ylxS, which specified several sRNAs, including two transcripts produced under the control of the forespore-specific sigma factor sigmaG and a third transcript generated by sigmaK, was identified. Our results indicate that the full repertoire of sporulation-specific gene expression involves the activation of multiple genes for small, noncoding RNAs.

Isolation and characterization of new thiamine-deregulated mutants of Bacillus subtilis.

In bacteria, thiamine pyrophosphate (TPP) is an essential cofactor that is synthesized de novo. Thiamine, however, is not an intermediate in the biosynthetic pathway but is salvaged from the environment and phosphorylated to TPP. We have isolated and characterized new mutants of Bacillus subtilis that deregulate thiamine biosynthesis and affect the export of thiamine products from the cell. Deletion of the ydiA gene, which shows significant similarity to the thiamine monophosphate kinase gene of Escherichia coli (thiL), did not generate the expected thiamine auxotroph but instead generated a thiamine bradytroph that grew to near-wild-type levels on minimal medium. From this DeltathiL deletion mutant, two additional ethyl methanesulfonate-induced mutants that derepressed the expression of a thiC-lacZ transcriptional reporter were isolated. One mutant, Tx1, contained a nonsense mutation within the B. subtilis yloS (thiN) gene that encodes a thiamine pyrophosphokinase, a result which confirmed that B. subtilis contains a single-step, yeast-like thiamine-to-TPP pathway in addition to the bacterial TPP de novo pathway. A second mutant, strain Tx26, was shown to contain two lesions. Genetic mapping and DNA sequencing indicated that the first mutation affected yuaJ, which encodes a thiamine permease. The second mutation was located within the ykoD cistron of the ykoFEDC operon, which putatively encodes the ATPase component of a unique thiamine-related ABC transporter. Genetic and microarray studies indicated that both the mutant yuaJ and ykoD genes were required for the derepression of thiamine-regulated genes. Moreover, the combination of the four mutations (the DeltathiL, thiN, yuaJ, and ykoD mutations) into a single strain significantly increased the production and excretion of thiamine products into the culture medium. These results are consistent with the proposed "riboswitch" mechanism of thiamine gene regulation (W. C. Winkler, A. Nahvi, and R. R. Breaker, Nature 419:952-956, 2002).

Small untranslated RNA antitoxin in Bacillus subtilis.

Toxin-antitoxin (TA) modules are pairs of genes in which one member encodes a toxin that is neutralized or whose synthesis is prevented by the action of the product of the second gene, an antitoxin, which is either protein or RNA. We now report the identification of a TA module in the chromosome of Bacillus subtilis in which the antitoxin is an antisense RNA. The antitoxin, which is called RatA (for RNA antitoxin A), is a small (222 nucleotides), untranslated RNA that blocks the accumulation of the mRNA for a toxic peptide TxpA (for toxic peptide A; formerly YqdB). The txpA and ratA genes are in convergent orientation and overlap by ca. 75 nucleotides, such that the 3' region of ratA is complementary to the 3' region of txpA. Deletion of ratA led to increased levels of txpA mRNA and lysis of the cells. Overexpression of txpA also caused cell lysis and death, a phenotype that was prevented by simultaneous overexpression of ratA. We propose that the ratA transcript is an antisense RNA that anneals to the 3' end of the txpA mRNA, thereby triggering its degradation.

Prevalence of occupational hand dermatitis in U.K. hairdressers.

Prevalence of hand dermatitis was investigated in 60 U.K. hairdressing salons. Exposure variables and individual characteristics were examined for associations with prevalence. 38.6% of respondents reported prevalent hand dermatitis and 72.7% of prevalent cases reported interdigital symptoms. Trainee hairdressers reported an increased risk of hand dermatitis, (RR = 2.95, 95% CI = 1.13-7.66), as did those with < or =2 years in the profession (RR = 4.91, 95% CI = 1.09-22.22). There was a positive association between prevalence and frequent use of protective measures (p = 0.04), suggesting that use of protective measures may be a reaction to incidence rather than a precaution. Wet work was significantly associated with prevalence, and RR was increased in those who performed more frequent wet work, i.e., trainee hairdressers. Prevalence was far higher than suggested by the surveillance schemes in the U.K.

Removing a bottleneck in the Bacillus subtilis biotin pathway: bioA utilizes lysine rather than S-adenosylmethionine as the amino donor in the KAPA-to-DAPA reaction.

In biotin biosynthesis, DAPA aminotransferase encoded by the bioA gene catalyzes the formation of the intermediate 7,8-diaminopelargonic acid (DAPA) from 7-keto-8-aminopelargonic acid (KAPA). DAPA aminotransferases from Escherichia coli, Serratia marcescens, and Bacillus sphaericus use S-adenosylmethionine (SAM) as the amino donor. Our observation that SAM is not an amino donor for B. subtilis DAPA aminotransferase led to a search for an alternative amino donor for this enzyme. Testing of 26 possible amino acids in a cell-free extract assay revealed that only l-lysine was able to dramatically stimulate the in vitro conversion of KAPA to DAPA by the B. subtilis DAPA aminotransferase. The K(m) for lysine and KAPA was estimated to be between 2 and 25 mM, which is significantly higher than the K(m) of purified E. coli BioA for SAM (0.15 mM). This higher requirement for lysine resulted in accumulation of KAPA during fermentation of B. subtilis biotin producing strains. However, this pathway bottleneck could be relieved by either addition of exogenous lysine to the medium or by introduction of lysine deregulated mutations into the production strains.

Thermodynamic and biophysical characterization of cytochrome P450 BioI from Bacillus subtilis.

Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin synthesis. Previous studies have indicated that BioI can hydroxylate fatty acids and may also perform an acyl bond cleavage reaction [Green, A. J., Rivers, S. L., Cheesman, M., Reid, G. A., Quaroni, L. G., Macdonald, I. D. G., Chapman, S. K., and Munro, A. W. (2001) J. Biol. Inorg. Chem. 6, 523-533. Stok, J. E., and De Voss, J. J. (2000) Arch. Biochem. Biophys. 384, 351-360]. Here we show novel binding features of P450 BioI--specifically that it binds steroids (including testosterone and progesterone) and polycyclic azole drugs with similar affinity to that for fatty acids (K(d) values in the range 0.1-160 microM). Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in this case. BioI as isolated from Escherichia coli is in a mixed heme iron spin state. Alteration of the pH of the buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content). Steroids containing a carbonyl group at the C(3) position induce a shift in heme iron spin-state equilibrium toward the low-spin form, whereas fatty acids produce a shift toward the high-spin form. Electron paramagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from optical titrations with steroids and fatty acids. Potentiometric studies demonstrate that the heme iron reduction potential becomes progressively more positive as the proportion of high-spin heme iron increases (potential for low-spin BioI = -330 +/- 1 mV; for BioI as purified from E. coli (mixed-spin) = 228 +/- 2 mV; for palmitoleic acid-bound BioI = -199 +/- 2 mV). Extraction of bound substrate-like molecule from purified BioI indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the BioI protein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonance Raman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotrope guanidinium chloride. Molecular modeling of the BioI structure indicates that a disulfide bond is present between Cys250 and Cys275. Calorimetry shows that structural stability of the protein was altered by addition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure.