Effector Overlap between the lac and mel Operons of Escherichia coli: Induction of the mel Operon with ß-Galactosides.

The lac (lactose) operon (which processes ß-galactosides) and the mel (melibiose) operon (which processes α-galactosides) of Escherichia coli have a close historical connection. A number of shared substrates and effectors of the permeases and regulatory proteins have been reported over the years. Until now, ß-thiogalactosides like TMG (methyl-ß-d-thiogalactopyranoside) and IPTG (isopropyl-ß-d-thiogalactopyranoside) have not generally been considered to be inducers of the mel operon. The same is true for ß-galactosides such as lactose [ß-d-galactopyranosyl-(1→4)-d-glucose], which is a substrate but is not itself an inducer of the lac operon. This report shows that all three sugars can induce the mel operon significantly when they are accumulated in the cell by Lac permease. Strong induction by ß-thiogalactosides is observed in the presence of Lac permease, and strong induction by lactose (more than 200-fold) is observed in the absence of ß-galactosidase. This finding calls for reevaluation of TMG uptake experiments as assays for Lac permease that were performed with mel+ strains.IMPORTANCE The typical textbook picture of bacterial operons is that of stand-alone units of genetic information that perform, in a regulated manner, well-defined cellular functions. Less attention is given to the extensive interactions that can be found between operons. Well-described examples of such interactions are the effector molecules shared by the lac and mel operons. Here, we show that this set has to be extended to include ß-galactosides, which have been, until now, considered not to effect the expression of the mel operon. That they can be inducers of the mel operon as well as the lac operon has not been noted in decades of research because of the Escherichia coli genetic background used in previous studies.

Principles for Developing Benchmark Criteria for Staff Training in Responsible Gambling.

One approach to minimizing the negative consequences of excessive gambling is staff training to reduce the rate of the development of new cases of harm or disorder within their customers. The primary goal of the present study was to assess suitable benchmark criteria for the training of gambling employees at casinos and lottery retailers. The study utilised the Delphi Method, a survey with one qualitative and two quantitative phases. A total of 21 invited international experts in the responsible gambling field participated in all three phases. A total of 75 performance indicators were outlined and assigned to six categories: (1) criteria of content, (2) modelling, (3) qualification of trainer, (4) framework conditions, (5) sustainability and (6) statistical indicators. Nine of the 75 indicators were rated as very important by 90 % or more of the experts. Unanimous support for importance was given to indicators such as (1) comprehensibility and (2) concrete action-guidance for handling with problem gamblers, Additionally, the study examined the implementation of benchmarking, when it should be conducted, and who should be responsible. Results indicated that benchmarking should be conducted every 1-2 years regularly and that one institution should be clearly defined and primarily responsible for benchmarking. The results of the present study provide the basis for developing a benchmarking for staff training in responsible gambling.

Protein distributions from a stochastic model of the lac operon of E. coli with DNA looping: analytical solution and comparison with experiments.

Although noisy gene expression is widely accepted, its mechanisms are subjects of debate, stimulated largely by single-molecule experiments. This work is concerned with one such study, in which Choi et al., 2008, obtained real-time data and distributions of Lac permease in E. coli. They observed small and large protein bursts in strains with and without auxiliary operators. They also estimated the size and frequency of these bursts, but these were based on a stochastic model of a constitutive promoter. Here, we formulate and solve a stochastic model accounting for the existence of auxiliary operators and DNA loops. We find that DNA loop formation is so fast that small bursts are averaged out, making it impossible to extract their size and frequency from the data. In contrast, we can extract not only the size and frequency of the large bursts, but also the fraction of proteins derived from them. Finally, the proteins follow not the negative binomial distribution, but a mixture of two distributions, which reflect the existence of proteins derived from small and large bursts.

Imidacloprid does not induce Cyp genes involved in insecticide resistance of a mutant Drosophila melanogaster line.

Certain xenobiotics have the capacity to induce the expression of genes involved in various biological phenomena, including insecticide resistance. The induction potential of different chemicals, among them different insecticides, has been documented for a number of insect species. In this study, we have analyzed the induction potential of Imidacloprid, a widely used member of the neonicotinoid insecticide family. Genes Cyp6g1 and Cyp6a2, known to be involved in the resistance of mutant Drosophila melanogaster line MiT[W⁻]3R2 to Imidacloprid and DDT were included in the analyzed sample. We find that Imidacloprid does not induce expression of the analyzed genes.

Use of mutagenesis, genetic mapping and next generation transcriptomics to investigate insecticide resistance mechanisms.

Insecticide resistance is a worldwide problem with major impact on agriculture and human health. Understanding the underlying molecular mechanisms is crucial for the management of the phenomenon; however, this information often comes late with respect to the implementation of efficient counter-measures, particularly in the case of metabolism-based resistance mechanisms. We employed a genome-wide insertional mutagenesis screen to Drosophila melanogaster, using a Minos-based construct, and retrieved a line (MiT[w(-)]3R2) resistant to the neonicotinoid insecticide Imidacloprid. Biochemical and bioassay data indicated that resistance was due to increased P450 detoxification. Deep sequencing transcriptomic analysis revealed substantial over- and under-representation of 357 transcripts in the resistant line, including statistically significant changes in mixed function oxidases, peptidases and cuticular proteins. Three P450 genes (Cyp4p2, Cyp6a2 and Cyp6g1) located on the 2R chromosome, are highly up-regulated in mutant flies compared to susceptible Drosophila. One of them (Cyp6g1) has been already described as a major factor for Imidacloprid resistance, which validated the approach. Elevated expression of the Cyp4p2 was not previously documented in Drosophila lines resistant to neonicotinoids. In silico analysis using the Drosophila reference genome failed to detect transcription binding factors or microRNAs associated with the over-expressed Cyp genes. The resistant line did not contain a Minos insertion in its chromosomes, suggesting a hit-and-run event, i.e. an insertion of the transposable element, followed by an excision which caused the mutation. Genetic mapping placed the resistance locus to the right arm of the second chromosome, within a ∼1 Mb region, where the highly up-regulated Cyp6g1 gene is located. The nature of the unknown mutation that causes resistance is discussed on the basis of these results.

Regulation of Wolbachia ankyrin domain encoding genes in Drosophila gonads.

The maternally inherited obligatory intracellular bacterium Wolbachia is a reproductive parasite of many insect species. Wolbachia evades the host immune system, uses the mitotic apparatus to ensure infection of daughter cells, migrates through the host to the gonads and causes reproductive phenotypes, most commonly cytoplasmic incompatibility (CI), i.e. incompatibility of sperm from infected males and eggs from uninfected females. Due to the interconnected facts that Wolbachia is not ex vivo culturable and that no established transformation system exists, virtually nothing is known about Wolbachia-host interactions at the macromolecular level. Intriguingly, the Wolbachia genome codes for an unusually high number of ankyrin repeat (ANK) proteins. ANKs mediate protein-protein interactions in many different contexts. More common in eukaryotes, they also occur in prokaryotes. Some intracellular pathogenic bacteria export ANK effector proteins to the host cytoplasm. This makes the Wolbachia ANK genes candidates for mediating interactions with host cells. We quantified expression of ANK genes of Wolbachia strain wMel in adult gonads and detected host sex-specific regulation of two wMel ANK genes in the gonads in two different backgrounds. Regulation was tissue-specific and independent of host background. We further analyzed expression of their homologues in strains wAu and wRi and found regulation only in wAu. Regulation was tissue-specific and there was no correlation between regulation of these genes and the ability of a strain to induce CI.

Comment on "A bacterium that can grow by using arsenic instead of phosphorus".

Wolfe-Simon et al. (Research Articles, 3 June 2011, p. 1163; published online 2 December 2010) reported that a naturally occurring bacterium, strain GFAJ-1, can substitute arsenic for phosphorus in its biomolecules. However, straightforward experiments to support this claim, including density gradient centrifugation of DNA assumed to contain arsenic, were either not performed or not presented. As a result, the authors' conclusions remain uncertain.

Wolbachia prophage DNA adenine methyltransferase genes in different Drosophila-Wolbachia associations.

Wolbachia is an obligatory intracellular bacterium which often manipulates the reproduction of its insect and isopod hosts. In contrast, Wolbachia is an essential symbiont in filarial nematodes. Lately, Wolbachia has been implicated in genomic imprinting of host DNA through cytosine methylation. The importance of DNA methylation in cell fate and biology calls for in depth studying of putative methylation-related genes. We present a molecular and phylogenetic analysis of a putative DNA adenine methyltransferase encoded by a prophage in the Wolbachia genome. Two slightly different copies of the gene, met1 and met2, exhibit a different distribution over various Wolbachia strains. The met2 gene is present in the majority of strains, in wAu, however, it contains a frameshift caused by a 2 bp deletion. Phylogenetic analysis of the met2 DNA sequences suggests a long association of the gene with the Wolbachia host strains. In addition, our analysis provides evidence for previously unnoticed multiple infections, the detection of which is critical for the molecular elucidation of modification and/or rescue mechanism of cytoplasmic incompatibility.

High local concentration: a fundamental strategy of life.

Local increase in concentration is a basic principle of transcriptional control. Closer inspection reveals that it is a major force governing all interactions within and between proteins and DNA. Local increase in concentration acts on all levels of organization of living matter. The structures and functions of two central molecules of life-the linear polymers DNA and protein-are particularly illuminating examples. Local increase in concentration leads to cooperative or competitive interactions between molecules. It is an important principle of life.

Feedback regulation of Lac repressor expression in Escherichia coli.

Negative feedback regulation, mediated through repressor binding site O3, which overlaps the lacI gene, could explain the robustness of the weak expression of Lac repressor. Significant autorepression of Lac repressor has never been ruled out. In the work presented here, the degree of autoregulation of Lac repressor was determined. It is negligible.

The DNA transposon Minos as a tool for transgenesis and functional genomic analysis in vertebrates and invertebrates.

Transposons are powerful tools for conducting genetic manipulation and functional studies in organisms that are of scientific, economic, or medical interest. Minos, a member of the Tc1/mariner family of DNA transposons, exhibits a low insertional bias and transposes with high frequency in vertebrates and invertebrates. Its use as a tool for transgenesis and genome analysis of rather different animal species is described.

"Cold-sensitive" mutants of the Lac repressor.

Thirteen of more than 4,000 single-amino-acid-replacement mutants of the Lac repressor, generated by suppression of amber nonsense mutants, were characterized as having a cold-sensitive phenotype. However, when expressed as missense mutations, none of the replacements cause cold sensitivity, implicating the suppression mechanism as being responsible for this phenotype.

Induction of the lac promoter in the absence of DNA loops and the stoichiometry of induction.

In vivo induction of the Escherichia coli lactose operon as a function of inducer concentration generates a sigmoidal curve, indicating a non-linear response. Suggested explanations for this dependence include a 2:1 inducer-repressor stoichiometry of induction, which is the currently accepted view. It is, however, known for decades that, in vitro, operator binding as a function of inducer concentration is not sigmoidal. This discrepancy between in vivo and in vitro data has so far not been resolved. We demonstrate that the in vivo non-linearity of induction is due to cooperative repression of the wild-type lac operon through DNA loop formation. In the absence of DNA loops, in vivo induction curves are hyperbolic. In the light of this result, we re-address the question of functional molecular inducer-repressor stoichiometry in induction of the lac operon.

Minos as a genetic and genomic tool in Drosophila melanogaster.

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 5'-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient "jump-starting" scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and approximately 55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila.