Approximation of circle number π by spread-plating Escherichia coli.

Ideally, microbial spread plating results in randomly distributed colonies on the agar surface. This can be seen as a Monte Carlo simulation and enables probabilistic approximation of circle number π. We perform π approximation in a microbiology undergraduate course to awaken the students' ambition for a good spread plating technique.

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli.

Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD'2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell's replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the "steric gate" residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical "nick translation". In vitro, pol I displaces 1-3 nucleotides of the RNA/DNA hybrid and through its 5'→3' (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I's strand displacement and FLAP-exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the "nick-translation" activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes.

Regulatory systems controlling motility and gene transfer agent production and release in Rhodobacter capsulatus.

Production of the gene transfer agent of Rhodobacter capsulatus, RcGTA, is dependent upon several cellular regulatory systems, including a putative phosphorelay involving the CtrA and CckA proteins. These proteins are also involved in flagellar motility in R. capsulatus. The interactions of proteins in this system are best understood in Caulobacter crescentus where CtrA is activated by phosphorylation by the CckA-ChpT phosphorelay. CtrA~P activity is further controlled by SciP, which represses ctrA transcription and CtrA activation of transcription. We show that R. capsulatus chpT and cckA mutants both have greatly reduced motility and RcGTA activity. Unlike the ctrA mutant where RcGTA gene transcription is absent, the decrease in RcGTA activity is because of reduced release of RcGTA from the cells. The sciP mutant is not affected for RcGTA production but our results support the C. crescentus model of SciP repression of flagellar motility genes. We show that both unphosphorylated and phosphorylated CtrA can activate RcGTA gene expression, while CtrA~P seems to be required for release of the particle and expression of motility genes. This has led us to a new model of how this regulatory system controls motility and production of RcGTA in R. capsulatus.

Red/ET recombination with chimeric oligonucleotides allows rapid generation of BAC transgenes harboring full-length or truncated huntingtin cDNA.

Huntington's disease (HD) is a fatal neurodegenerative disorder that is caused by a CAG repeat expansion encoding a polyglutamine tract in the huntingtin (htt) gene. None of the existing HD mouse models recapitulate the exact disease symptoms and course as it is seen in humans and the generation of further HD disease models is challenging because of the size and complexity of the htt gene locus. Starting from a single substrate plasmid harboring human htt cDNA comprising 98 glutamine (Q) residues, we applied Red/ET recombination to generate four BDNF-BAC transgenes harboring full-length or truncated (N171) htt cDNA comprising 98 or 15 Q residues. BDNF (brain-derived neurotrophic factor) is expressed in the cortical neurons projecting to the striatal medium spiny neurons, and was used to direct htt transgene expression to investigate the contribution of these cell types to HD.

Modification of the genome of Rhodobacter sphaeroides and construction of synthetic operons.

The α-proteobacterium Rhodobacter sphaeroides is an exemplary model organism for the creation and study of novel protein expression systems, especially membrane protein complexes that harvest light energy to yield electrical energy. Advantages of this organism include a sequenced genome, tools for genetic engineering, a well-characterized metabolism, and a large membrane surface area when grown under hypoxic or anoxic conditions. This chapter provides a framework for the utilization of R. sphaeroides as a model organism for membrane protein expression, highlighting key advantages and shortcomings. Procedures covered in this chapter include the creation of chromosomal gene deletions, disruptions, and replacements, as well as the construction of a synthetic operon using a model promoter to induce expression of modified photosynthetic reaction center proteins for structural and functional analysis.

Bacteriophytochrome-dependent regulation of light-harvesting complexes in Rhodopseudomonas palustris anaerobic cultures.

Bacteriophytochromes (Bphs) are photoreceptors that help bacteria sense changes in light wavelength and intensity. Bphs contain a linear tetrapyrrole chromophore that, upon absorption of red or far-red light, undergoes a cis-trans isomerization that leads to a conformational change in the holoprotein. The conformation and type of Bph affects the expression of genes. The linear tetrapyrrole bound by Bphs is thought to come from O(2)-dependent cleavage of heme by a heme oxygenase. We have discovered that the absence of O(2) does not inhibit the normal function of two Bphs in the regulation of Rhodopseudomonas palustris light-harvesting complexes. These observations imply that: (i) a linear tetrapyrrole can be made anaerobically, either through anaerobic heme cleavage by a novel enzyme or directly from the heme precursor hydroxymethylbilane without ring cleavage; or (ii) that Bph-dependent signal transduction does not require a chromophore.

Site-directed mutagenesis of multi-copy-number plasmids: Red/ET recombination and unique restriction site elimination.

Existing methods for site-directed plasmid mutagenesis are restrained by the small spectrum of modifications that can be introduced by mutagenic primers and the amplicon size limitations of in vitro DNA synthesis. As demonstrated here, the combined use of Red/ET recombination and unique restriction site elimination enables extensive manipulation regardless of plasmid size and DNA sequence elements. First, a selectable marker is PCR-amplified with synthetic primers attaching 50-bp homology target flanks for Red/ET recombination and an arbitrary restriction site absent in the substrate plasmid. The resulting cassette is co-electroporated with substrate plasmids in Red/ET-proficient Escherichia coli cells. Following isolation of recombinant plasmids, linear nonselectable DNA replaces the cassette and introduces the desired mutation(s) in a second Red/ET recombination step. Upon selective digestion of parental plasmids and retransformation, a 38% mutation efficiency was achieved using a synthetic 97-nucleotide oligonucleotide to cure a 17-bp deletion within lacZalpha of pUC19 (2,686 bp). A PCR fragment was used with similar efficiency to co-replace mouse Cdkn1b codons 9 and 76 in gene-targeting vector pGTC (13,083 bp).