Expression of the ubiE gene of Geobacillus stearothermophilus V in Escherichia coli K-12 mediates the evolution of selenium compounds into the headspace of selenite- and selenate-amended cultures.

The ubiE gene of Geobacillus stearothermophilus V, with its own promoter, was cloned and introduced into Escherichia coli. The cloned gene complemented the ubiE gene deficiency of E. coli AN70. In addition, the expression of this gene in E. coli JM109 resulted in the evolution of volatile selenium compounds when these cells were grown in selenite- or selenate-amended media. These compounds were dimethyl selenide and dimethyl diselenide.

Isolation of bacteriophage T4 baseplate proteins P7 and P8 and in vitro formation of the P10/P7/P8 assembly intermediate.

Two bacteriophage T4 proteins, P7 and P8, which are components of the phage baseplate have been purified to apparent homogeneity. P7 and P8 are the protein products of T4 genes 7 and 8. A plasmid has been constructed which contains approximately 5 kilobases of T4 DNA, including genes 7 and 8, under the control of the tac promoter. Induction of Escherichia coli W3110iQ cells containing this plasmid resulted in the production of functional P7 and P8. Standard protein isolation procedures were used to purify both P7 and P8 from extracts of induced cells. In T4-infected cells, these two proteins and P10 interact in a strictly ordered sequential manner (P10 + P7----P10/P7,P10/P7 + P8----P10/P7/P8) to form an intermediate in the baseplate assembly pathway. The three purified proteins assembled in vitro to form a limited number of oligomeric species, as determined by nondenaturing gel electrophoresis. P10 and P7 interacted in vitro to form two assemblies with distinct electrophoretic mobilities, both containing P10 and P7. Addition of P8 to this mixture resulted in the disappearance of both P10/P7 species and the appearance of a single new assembly with a different electrophoretic mobility. These interactions occurred without the addition of any catalyst or cofactors. Isolated P11 appeared to add as predicted to the in vitro-formed complexes without affecting the formation of the two P10/P7 or the single P10/P7/P8 intermediates. Interactions between P7 and P8 in the absence of P10 or interactions between P10 and P8 in the absence of P7 could not be detected. These data indicate that purified P10, P7, and P8 interact in vitro in a manner completely in accord with the published assembly pathway and thus establish a system for further study of the regulation of the formation of this assembly intermediate in vitro.

Isolation and characterization of precursors in bacteriophage T4 baseplate assembly. III. The carboxyl termini of protein P11 are required for assembly activity.

The assembly activity and electrophoretic mobility of a T4 bacteriophage baseplate protein, P11, have been found to be affected by digestion with the proteases trypsin, subtilisin and carboxypeptidase Y. Analysis of the trypsin limit-digestion product of P11 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and size analysis by high performance liquid chromatography indicate that there is a decrease of approximately 5000 in the molecular weight of the P11 molecule or a loss of 2500 in Mr from each of the gp11 subunits of the dimer. During protease treatment P11 demonstrates a time-dependent loss in the ability to interact with the baseplate protein P10 to form the P(10/11) complex, the first assembly intermediate of the T4 baseplate 1/6th arm. Similar treatments of the P(10/11) complex indicate that P11 in the complex is not affected by these proteases. Concomitant with the loss of assembly activity is a change in the electrophoretic mobility of P11 on non-denaturing polyacrylamide gels from a single band to a series of more mobile bands suggesting sequential loss of positive charge. P11 assembly activity is completely lost after removal of the first positive charge. These results suggest that the carboxyl termini of the two gp11 subunits of the P11 molecule are involved in the interaction of P11 with P10 to form the P(10/11) complex. Analysis of the portion of gp11 removed by carboxypeptidase Y demonstrates that there are up to 13 aliphatic and aromatic carboxyl-terminal amino acids.

Isolation and characterization of precursors in bacteriophage T4 baseplate assembly. II. Purification of the protein products of genes 10 and 11 and the in vitro formation of the P(10/11) complex.

Two bacteriophage T4 proteins which are precursors to the phage baseplate have been purified to homogeneity. These proteins, P10 and P11, are components of the P(10/11) complex, which is the first intermediate in the assembly of T4 baseplate 1/6th arms. Each protein was isolated from cells infected with a T4 amber mutant defective in the production of the other protein. Thus these purified proteins have never been assembled into the P(10/11) complex in vivo. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and the ability of these proteins to block the phage killing activity of specific antisera were used to monitor the purification steps. Sedimentation equilibrium experiments reveal a molecular weight of 188,000 g/mol for P10 and 60,000 g/mol for P11. These data together with the previously determined molecular weights of the gene 10 and gene 11 polypeptide chains (King & Mykolajewycz, 1973) and the in vivo assembled P(10/11) complex (Berget & King, 1978b) are consistent with P10 being a dimer of the product of gene 10, P11 being a dimer of the product of gene 11, and P(10/11) being a tetramer containing one of each of these dimers. Purified P10 and P11 are active in assembly because they complement 10- and 11- defective extracts, respectively, to form viable bacteriophage in vitro. Furthermore, these proteins assemble in vitro to form a protein structure identical to the P(10/11) complex formed in vivo as determined by non-denaturing gel electrophoresis. This P(10/11) complex formed in vitro complements 10-/11- defective extracts to form viable phage. The overall extent of this in vitro assembly reaction is not affected by NaCl to 1.5 M or 2% Triton X-100. The reaction is, however, prevented by the denaturing effects of urea and sodium dodecyl sulfate.

Use of RNA polymerase as an enzymatic probe of nucleosomal structure.

Nucleosomes prepared from human placental nuclei and Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyl transferase EC.2.7.7.6) form stable initiation complexes. This property is utilized as a probe of nucleosome structure. RNA polymerase initiation has been studied on purified nucleosomes, nucleosome cores, and nucleosomal DNA. The affinity of E. coli RNA polymerase for both nucleosome cores and monomers was 5-6 fold less than found for nucleosomal DNA. No difference in apparent initiation Km was found between cores and mononucleosomes. This suggests that initiation does not preferentially occur on the DNA tails of nucleosomes. Once initiated and allowed to form nascent RNA, these complexes are very stable to ionic strength changes. Under conditions in which free enzyme is inactivated with rifampicin, the enzyme in the complex retains activity as demonstrated by its ability to transcribe and reinitiate on both nucleosomes and free DNA. These complexes can be well resolved from free nucleosomes on preparative polyacrylamide gels and both can be eluted from gels for analysis of proteins and DNA sequence complexity. Studies using (125I) labelled nucleosomes show that histones are retained in the initiation complex, and are not dissociated by the enzyme during initiation.