I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

Plasmids for independently tunable, low-noise expression of two genes

Some microbiology experiments and biotechnology applications can be improved if it is possible to tune the expression of two different genes at the same time with cell-to-cell variation at or below the level of genes constitutively expressed from the chromosome (the “extrinsic noise limit”). This was recently achieved for a single gene by exploiting negative autoregulation by the tetracycline repressor (TetR) and bicistronic gene expression to reduce gene expression noise. We report new plasmids that use the same principles to achieve simultaneous, low-noise expression for two genes. The TetR system was moved to a compatible plasmid backbone, and a system based on the lac repressor (LacI) was found to also exhibit gene expression noise below the extrinsic noise limit. We characterize gene expression mean and noise across the range of induction levels for these plasmids, apply the LacI system to tune expression for single-molecule mRNA detection in two different growth conditions, and show that two plasmids can be co-transformed to independently tune expression of two different genes.

In Vivo DNA Protection by Relaxed-Specificity SinI DNA Methyltransferase Variants

ABSTRACT The SinI DNA methyltransferase, a component of the SinI restriction-modification system, recognizes the sequence GG(A/T)CC and methylates the inner cytosine to produce 5-methylcytosine. Previously isolated relaxed-specificity mutants of the enzyme also methylate, at a lower rate, GG(G/C)CC sites. In this work we tested the capacity of the mutant enzymes to function in vivo as the counterpart of a restriction endonuclease, which can cleave either site. The viability of Escherichia coli cells carrying recombinant plasmids with the mutant methyltransferase genes and expressing the GGNCC-specific Sau96I restriction endonuclease from a compatible plasmid was investigated. The sau96IR gene on the latter plasmid was transcribed from the araBAD promoter, allowing tightly controlled expression of the endonuclease. In the presence of low concentrations of the inducer arabinose, cells synthesizing the N172S or the V173L mutant enzyme displayed increased plating efficiency relative to cells producing the wild-type methyltransferase, indicating enhanced protection of the cell DNA against the Sau96I endonuclease. Nevertheless, this protection was not sufficient to support long-term survival in the presence of the inducer, which is consistent with incomplete methylation of GG(G/C)CC sites in plasmid DNA purified from the N172S and V173L mutants. Elevated DNA ligase activity was shown to further increase viability of cells producing the V173L variant and Sau96I endonuclease.

Effect of the CopB Auxiliary Replication Control System on Stability of Maintenance of Par+ Plasmid R1

ABSTRACT Plasmid R1 is a low-copy-number plasmid that is present at a level of about four or five copies per average cell. The copy number is controlled posttranscriptionally at the level of synthesis of the rate-limiting initiator protein RepA. In addition to this, R1 has an auxiliary system that derepresses a second promoter at low copy numbers, leading to increased repA mRNA synthesis. This promoter is normally switched off by a constitutively synthesized plasmid-encoded repressor protein, CopB; in cells with low copy numbers, the concentration of CopB is low and the promoter is derepressed. Here we show that the rate of loss of a Par+ derivative of the basic replicon of R1 increased about sevenfold when the cells contained a high concentration of the CopB protein formed from a compatible plasmid.

The merG Gene Product Is Involved in Phenylmercury Resistance in Pseudomonas Strain K-62

ABSTRACT The physiological function of a new gene, hereby designatedmerG, located between merA and merBon the broad-spectrum mer operon of Pseudomonasstrain K-62 plasmid pMR26 was investigated. The 654-bp merGgene encodes a protein with a canonical leader sequence at its N terminus. The processing of the signal peptide of this protein was dose-dependently inhibited by sodium azide, a potent inhibitor of protein export. These results suggest that the mature MerG protein (ca. 20 kDa) may be located in the periplasm. Deletion of themerG gene from the broad-spectrum mer operon of pMR26 had no effect on the inorganic mercury resistance phenotype, but rendered the bacterium more sensitive to phenylmercury than its isogenic wild-type strain. Escherichia coli cells bearing pMU29, which carries a deletion of the merG gene, took up significantly more phenylmercury than the bacteria with the intact plasmid pMRA17. When the merG gene in a compatible plasmid was transformed into the E. coli strain carrying pMU29, the high uptake of and high sensitivity to phenylmercury were almost completely restored to their original levels. These results demonstrate that the merG gene is involved in phenylmercury resistance, presumably by reducing in-cell permeability to phenylmercury.

Mutational analysis of a C-dependent late promoter of bacteriophage Mu.

Late transcription of bacteriophage Mu initiates at four promoters, P(lys), PI, PP and Pmom, and requires the Mu C protein and the host RNA polymerase. Promoter-containing DNA fragments extending approximately 200 bp upstream and downstream of the 5' starts of the lys, I and P transcripts were cloned into a multicopy lacZ-expression plasmid. Promoter activity, assayed by beta-galactosidase expression, was determined under two different conditions: (1) with C provided from a compatible plasmid in the absence of other Mu factors and (2) with C provided from an induced Mu prophage. beta-galactosidase activities were greatest for P(lys), intermediate for PI, and lowest for PP. Similar analysis of plasmids containing nested sets of deletions removing 5' or 3' sequences of P(lys) demonstrated that a 68-bp region was sufficient for full activity. Point mutations were generated within the 68-bp region by mutagenic oligonucleotide-directed PCR (Mod-PCR). Properties of the lys promoter mutants indicated that, in addition to the -10 region, a 19-bp region from -52 to -34 containing the C footprint is required for C-dependent promoter activity.

Dislodgement and thymineless elimination of N-group plasmids

SUMMARYThymineless strains ofEscherichia coliC600 were constructred harbouring both an R factor of the N incompatibility group (R46 or R447b) and a compatible plasmid (Plac-of the A-C group or the Iα plasmid R62), which contained a segment of N group DNA. Selection was made for the transferred plasmid and dislodgement phenomena were manifest either as loss of an entire plasmid or as deletions of a region of plasmid DNA. Even after the two R factors had become established as separate replicons, the N group R factor but not the other plasmid exhibited instability.Thymine starvation of strain C600thy(R447b/R62) increased the elimination rate of the N group plasmid R447b but no elimination of R62 was observed. However, thymine starvation of strain C600thy(R46/Plac-) not only increased the rate of elimination of R46 but also increased the rate of loss of Plac-. There was no detectable increase in nuclease activity in unstarved R46/Plac-strains and it is concluded that dislodgement of R46 from these strains is not due to induction of the nuclease that has been proposed to be responsible for the elimination of N group plasmids during thymine starvation.Two variants of Plac-were isolated. These did not dislodge R46 from unstarved R46/Plac-strains and were not lost during thymine starvation even though thymineless elimination of R46 occurred at normal frequency.