Natural genetic transformation in Haemophilus influenzae involves DNA binding, uptake, translocation, and recombination. In this study, we cloned and sequenced a 3.8-kbp H. influenzae DNA segment capable of complementing in trans the transformation defect of an H. influenzae strain carrying the tfo-37 mutation. We used subcloning, deletion analysis, and in vivo protein labeling experiments to more precisely define the gene required for efficient DNA transformation on the cloned DNA. A novel gene, which we called dprA ؉ , was shown to encode a 41.6-kDa polypeptide that was required for efficient chromosomal but not plasmid DNA transformation. Analysis of the deduced amino acid sequence of DprA suggested that it may be an inner membrane protein, which is consistent with its apparent role in DNA processing during transformation. Four other open reading frames (ORFs) on the cloned DNA segment were identified. Two ORFs were homologous to the phosphofructokinase A (pfkA) and alpha-isopropyl malate synthase (leuA) genes of Escherichia coli and Salmonella typhimurium, respectively. Homologs for the two other ORFs could not be identified.Natural genetic transformation in bacteria is a complex genetically programmed process involving DNA binding, uptake, translocation, and recombination. Haemophilus influenzae is a gram-negative bacterium that in growing cultures can be induced to become competent for transformation by a temporary shift to anaerobic conditions, by physiological change occurring during the late-log phase growth, or by a transfer of cells to a nutrient-poor chemically defined medium such as MIV (10).Only recently has the molecular cloning of transformation genes allowed identification of several structural and regulatory components of the transformation apparatus (4-6, 16, 17, 26, 39). To identify the genes involved in transformation, Tomb and coworkers (35) performed minitransposon mutagenesis using H. influenzae chromosomal DNA and isolated 24 mutant strains that were defective in transformation. These mutant strains were characterized for DNA binding and uptake and for their transformability with H. influenzae chromosomal DNA. Among the 24 strains analyzed, only 2 bound and took up radiolabeled DNA in a manner similar to that of the wild type but transformed at frequencies less than 0.1% of that of the wild type. This suggested that the two strains were defective in events occurring subsequent to DNA uptake, i.e., DNA processing. This processing may involve DNA translocation and/or recombination.In this study, we examine the nature of the defect in one of the two DNA uptake-proficient mutant strains, JG37. The mutated locus derived from strain JG37 was cloned and used to isolate the wild-type locus. The DNA sequence of a 3.8-kbp portion of the locus was determined and shown to encode several polypeptides. At least one of these, DprA, is essential for efficient chromosomal DNA transformation but is not required for plasmid transformation. MATERIALS AND METHODSBacterial strains, plasmids, and culture cond...

We have developed a xylose-dependent expression system for tight and modulated expression of cloned genes in Bacillus subtilis. The expression system is contained on plasmid pSWEET for integration at the amyE locus of B. subtilis and incorporates components of the well-characterized, divergently transcribed xylose utilization operon. The system contains the xylose repressor encoded by xylR, the promoter and 5 portion of xylA containing an optimized catabolite-responsive element, and intergenic xyl operator sequences. We have rigorously compared this expression system to the isopropyl-␤-D-thiogalactopyranoside-induced spac system using a thermostable ␤-galactosidase reporter (BgaB) and found the xyl promoter-operator to have a greater capacity for modulated expression, a higher induction/repression ratio (279-fold for the xyl system versus 24-fold with the spac promoter), and lower levels of expression in the absence of an inducer. We have used this system to probe an essential function in wall teichoic acid biosynthesis in B. subtilis. Expression of the teichoic acid biosynthesis gene tagD, encoding glycerol-3-phosphate cytidylyltransferase, from the xylose-based expression system integrated at amyE exhibited xylose-dependent complementation of the temperature-sensitive mutant tag-12 when grown at the nonpermissive temperature. Plasmid pSWEET thus provides a robust new expression system for conditional complementation in B. subtilis.

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We have developed a xylose-dependent expression system for tight and modulated expression of cloned genes in Bacillus subtilis. The expression system is contained on plasmid pSWEET for integration at the amyE locus of B. subtilis and incorporates components of the well-characterized, divergently transcribed xylose utilization operon. The system contains the xylose repressor encoded by xylR, the promoter and 5 portion of xylA containing an optimized catabolite-responsive element, and intergenic xyl operator sequences. We have rigorously compared this expression system to the isopropyl-␤-D-thiogalactopyranoside-induced spac system using a thermostable ␤-galactosidase reporter (BgaB) and found the xyl promoter-operator to have a greater capacity for modulated expression, a higher induction/repression ratio (279-fold for the xyl system versus 24-fold with the spac promoter), and lower levels of expression in the absence of an inducer. We have used this system to probe an essential function in wall teichoic acid biosynthesis in B. subtilis. Expression of the teichoic acid biosynthesis gene tagD, encoding glycerol-3-phosphate cytidylyltransferase, from the xylose-based expression system integrated at amyE exhibited xylose-dependent complementation of the temperature-sensitive mutant tag-12 when grown at the nonpermissive temperature. Plasmid pSWEET thus provides a robust new expression system for conditional complementation in B. subtilis.

Background: LEE011 is an oral small molecule inhibitor with highly specific nanomolar inhibitory activity against CDK4/cyclinD1 and CDK6/cylinD3 complexes. Preclinical studies with LEE011 have demonstrated robust anti-tumor activity in several tumor models with functional retinoblastoma protein (pRb). Methods: The primary objective this phase 1 dose escalation study was to determine the maximum tolerated dose (MTD) based on cycle 1 dose-limiting toxicities (DLTs) and establish the recommended dose for expansion (RDE) of LEE011 in patients (pts) with pRb-positive advanced solid tumors or lymphomas. Using a Bayesian logistic regression model with overdose control principle, escalating doses of LEE011 were administered daily for 21 days of 28-day cycles. Following MTD determination, a continuous dosing schedule was also evaluated. Evaluation of safety and pharmacokinetics (PK) from all pts contributed to defining the RDE. Results: As of June 28, 2013, a total of 70 pts were treated in the escalation phase at doses from 50-1200 mg. DLTs are described in the table. MTD was defined at 900 mg on a 21 of 28-day dosing schedule. Additional patients were treated at 750 mg and 600 mg on the current schedule and at 600 mg on a continuous schedule to define the RDE and sustain optimal long-term dosing. Following review of PK and safety data, including AEs observed beyond cycle 1, the planned RDE is 600 mg for 21 days of a 28-day treatment cycle. Common adverse events (AEs) were gastrointestinal (nausea 40%, diarrhea 30%) and hematologic (anemia 43%, neutropenia 40%); the majority of AEs were grades 1/2 and reversible. Asymptomatic QTc prolongation was observed at the higher doses. LEE011 is absorbed orally with Tmax ≈ 4 hours and effective t1/2 ≈24 hours. Dose-dependent increases in exposure were observed. Preliminary evidence of activity was observed including stable disease for ≥ 6 cycles in 10/70 pts (14%) and 1 confirmed partial response in a pt with ER-positive breast cancer. Preliminary tumor pharmacodynamic data shows evidence of modulation at doses of 600 mg and above. Conclusions: LEE011, a highly selective inhibitor of CDK4/6, exhibited an acceptable safety profile, dose dependent PK, and preliminary clinical activity. Enrollment will continue in the expansion phase. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A276. Citation Format: Jeffrey R. Infante, Geoffrey I. Shapiro, Petronella O. Witteveen, John F. Gerecitano, Vincent Ribrag, Rashmi Chugh, Abhijit Chakraborty, Alessandro Matano, Xumei Zhao, Sudha Parasuraman, Philippe A. Cassier. Phase 1 multicenter, open label, dose-escalation study of LEE011, an oral inhibitor of cyclin-dependent kinase 4/6, in patients with advanced solid tumors or lymphomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A276.