Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor

The mutant cIts genes from seven different λcIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/ or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25°C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures

Investigation of mechanical and thermal properties of the cetyltrimethylammonium bromide functionalized molybdenum disulfide (MoS2)/epoxy composites

Molybdenum disulfide (MoS2), a 2D layered material has been recognised as a new paradigm in the area of materials science due to its graphene like structure. Herein, we prepared functionalized MoS2 nanosheets through one-pot hydrothermal technique using cationic surfactant, cetyltrimethylammonium bromide (CTAB). The surfactant functionalized MoS2 (CTAB-MoS2) was subsequently dispersed in epoxy matrix at the loading level of 0.1–0.5 wt% to investigate the reinforcing competence of MoS2 on the mechanical and thermal properties of the composites. Fourier transform infrared spectroscopy, X-ray diffraction and field emission scanning electron microscopy (FE-SEM) were used to characterize the microstructure and morphology of the hydrothermally prepared pristine MoS2 and CTAB-MoS2. Dynamic mechanical and tensile properties were studied to comprehend the effect of functionalized MoS2 on the mechanical properties of the composites. At 0.2 wt% loading, the tensile strength and Young’s modulus was improved by ~23 and 26.7%, respectively, while ~83% improvement in storage modulus was recorded. Thermal stability of all the studied specimens were compared by selecting the temperatures at 10 and 50% weight losses which showed small decrease in onset degradation temperature

Cloning and sequencing analysis of the repressor gene of temperate mycobacteriophage L1

The wild-type and temperature-sensitive (ts) repressor genes were cloned from the temperate mycobacteriophage L1 and its mutant L1cIts391, respectively. A sequencing analysis revealed that the 131st proline residue of the wildtype repressor was changed to leucine in the ts mutant repressor. The 100% identity that was discovered between the two DNA regions of phages L1 and L5, carrying the same sets of genes including their repressor genes, strengthened the speculation that L1 is a minor variant of phage L5 or vice versa. A comparative analysis of the repressor proteins of different mycobacteriophages suggests that the mycobacteriophage-specific repressor proteins constitute a new family of repressors, which were possibly evolved from a common ancestor. Alignment of the mycobacteriophage-specific repressor proteins showed at least 7 blocks (designated I-VII) that carried 3-8 identical amino acid residues. The amino acid residues of blocks V, VI, and some residues downstream to block VI are crucial for the function of the L1 (or L5) repressor. Blocks I and II possibly form the turn and helix 2 regions of the HTH motif of the repressor. Block IV in the L1 repressor is part of the most charged region encompassing amino acid residues 72-92, which flanks the putative Nterminal basic (residues 1-71) and C-terminal acidic (residues 93-183) domains of L1 repressor

The mutation that makes Escherichia coli resistant to λ P gene-mediated host lethality is located within the DNA initiator gene dnaA of the bacterium

Earlier, we reported that the bacteriophage λ P gene product is lethal to Escherichia coli, and the E. coli rpl mutants are resistant to this λ P gene-mediated lethality. In this paper, we show that under the λ P gene-mediated lethal condition, the host DNA synthesis is inhibited at the initiation step. The rpl8 mutation maps around the 83 min position in the E. coli chromosome and is 94% linked with the dnaA gene. The rpl8 mutant gene has been cloned in a plasmid. This plasmid clone can protect the wild-type E. coli from λ P gene-mediated killing and complements E. coli dnaAts46 at 42° C. Also, starting with the wild-type dnaA gene in a plasmid, the rpl-like mutations have been isolated by in vitro mutagenesis. DNA sequencing data show that each of the rpl8, rpl12 and rpl14 mutations has changed a single base in the dnaA gene, which translates into the amino acid changes N313T, Y200N, and S246T respectively within the DnaA protein. These results have led us to conclude that the rpl mutations, which make E. coli resistant to λ P gene-mediated host lethality, are located within the DNA initiator gene dnaA of the host