Isolation Of Regulatory Mutants Research Articles

Biochemical screening approach to identify regulatory DNA-binding proteins

The interaction between transcription factors, promoter elements and RNA polymerase is crucial to bacterial adaptation to physical and chemical changes in the environment. Transcription factor discovery has strongly relied on the isolation of regulatory mutants followed by biochemical confirmation. However, highly pleiotropic regulatory mutations can often be deleterious, unstable and difficult to select, or could directly or indirectly affect gene expression at multiple levels. Here, we describe a biochemical approach to identify trans-acting regulatory proteins independent of their cellular function that could be used as an alternative to genetic screens. The method consists in (i) incubating bacterial lysates with an immobilized DNA encompassing a promoter as defined by RNA-Seq data (ii) pull down of bound proteins and (iii) liquid chromatography tandem mass spectrometry (LC/MS/MS). We tested the usefulness of this approach by identifying proteins binding to the Vibrio cholerae rpoS promoter that drives the expression of the general stress response regulator RpoS. The approach identified several proteins binding to the rpoS promoter that included the factor for inversion stimulation (FIS), and the master quorum sensing regulator HapR. Binding of both purified proteins to the rpoS promoter was confirmed by electrophoresis mobility shift assays. The role of fis and hapR on rpoS expression was examined in strains containing a chromosomally-integrated rpoS-lacZ fusion. Deletion of fis had little effect while HapR appeared to enhance rpoS expression.

Isolation of regulatory mutants in photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 and partial complementation of a PrrB mutant by the HupT histidine-kinase

The photosynthetic bacterium Rhodobacter sphaeroides responds to the transition from aerobiosis to anaerobic photosynthesis by increasing the expression of the photosynthesis genes. Mutants have been isolated based on their inability, following such a transition, to increase transcription of the puc operon encoding the apoproteins of the light-harvesting complex II. Mutant D5, a representative of one mutant class, described here, although remaining photosynthetically competent, produced only low levels of the photosynthetic spectral complexes. Complementation analysis revealed that either the gene for the photosynthesis response regulator prrA or the gene encoding its cognate sensor kinase, prrB, was capable of rescuing this mutant. However, partial complementation of this mutant was achieved by placing in trans additional copies of other defined genes from the cosmid library of R. sphaeroides. We describe this effect in detail, attributable to the hupT gene, which has been proposed to encode a histidine-kinase for the hydrogen uptake system in Rhodobacter capsulatus. The effect of HupT on the expression of the photosynthesis genes was mediated through PrrA and independent of a functioning hydrogen uptake system. Thus, we raise the possibility that HupT can participate in phosphorylation of the heterologous response regulator PrrA by so-called cross-talk and therefore partially compensate for the defect in the mutant described. The observation of cross-talk, together with the complementation analysis, allowed us to assign the original mutation to the prrB gene; this was confirmed by DNA sequencing. Analysis of cross-talk in the wild-type, prrB and prrA genetic backgrounds suggested that besides kinase activity, PrrB may possess phosphatase activity toward PrrA. We also report the cloning, organization and structure of some of the hup genes from R. sphaeroides and construction of a Hup- strain.

Use oflysC-lacZfusion for the isolation of regulatory mutants in the lysine biosynthetic pathway

Use of<i>lysC-lacZ</i>fusion for the isolation of regulatory mutants in the lysine biosynthetic pathway

Use of lysC-lacZ fusion for the isolation of regulatory mutants in the lysine biosynthetic pathway

Use of lysC-lacZ fusion for the isolation of regulatory mutants in the lysine biosynthetic pathway

The effect of a non-metabolizable analog on mandelate catabolism in Pseudomonas putida.

DL-2,3,4,5,6-pentafluoromandelic acid (PFM) specifically inhibits the growth of Pseudomonas putida (ATCC 12633) on medium containing mandelate as sole carbon and energy source by competitive inhibition of mandelate dehydrogenase. PFM is not metabolized and is neither an inducer of the mandelate catabolic enzymes nor an antagonist of induction. Mutants resistant to the inhibitory effects of PFM (PFMr) were isolated; most prove to be superinducible, i.e. synthesize corrdinately the mandelate-specific catabolic enzymes at elevated levels following induction. In at least one case the PFMr mutation maps very near the structural genes that encode the enzymes functional in the first two steps of mandelate catabolism. It is reasoned that the PFMr mutation is of the promotor type. Resistance to substrate analogs such as PFM offers a general method for isolation of regulatory mutants in catabolic metabolism.

Isolation of regulatory mutants of Pseudomonas acidovorans by use of amino acid analogs.

Mutants resistance to various combinations of threonine, lysine and/or their analogs were obtained and characterized in Pseudomonas acidovorans. In particular, mutants resistant to aminoethylcysteine had a dihydrodipicolinate synthetase insensitive to lysine inhibition whereas mutants resistant to threonine plus a low concentration of aminoethylcysteine had a feedback-insensitive aspartokinase.

Regulation of Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium : Isolation of Regulatory Mutants

5',5',5'-Trifluoro-dl-leucine inhibited the activity of alpha-isopropylmalate synthetase (the initial enzyme unique to leucine biosynthesis) as well as the growth of Salmonella typhimurium. Mutants of S. typhimurium resistant to the analogue were isolated and characterized. In most cases, they overproduced and excreted leucine or leucine, valine, and isoleucine as a result of an alteration in the regulation of branched-chain amino acid biosynthesis. Biochemical and genetic tests allowed the mutants to be grouped into three classes: I, a moderately large group (13%) which had high, constitutive leucine biosynthetic enzyme levels and mutant sites linked to the leucine operon (operator constitutive); II, a single mutant in which the mutant site was linked to the leucine operon and in which alpha-isopropylmalate synthetase was not inhibited by leucine (feedback negative); III, a majority type which had constitutive levels of leucine, valine, and isoleucine biosynthetic enzymes and mutant sites unlinked to the leucine operon. Mutants of class I provide important evidence for the concept of an operon organization of genes involved in leucine biosynthesis. The properties of class III mutants indicate that there is some element involved in regulation which is common to the three pathways.