Effect of LacI (lac repressor) on transcription in Salmonella
ABSTRACT: Salmonella is an important enteric pathogen that causes a spectrum of diseases varying from mild gastroenteritis to life threatening typhoid fever. Salmonella does not have lac operon. However, E. Coli, Salmonella’s close relative, has lac operon. Being an enteric pathogen like E. coli, Salmonella will also benefit from lac operon. Then, why Salmonella has lost lac operon?. To address this question, lacI, an important component of lac operon was expressed in Salmonella via pTrc99A plasmid. As a control, pTrc99A without lacI was also expressed in Salmonella. The effect of LacI on the transcription profile of Salmonella was analyzed using microarray technique. Overall design: Overall design The total RNA was isolated from Salmonella using ‘RNeasy Mini Kit’ (QIAGEN). Organism used: Salmonella enterica serovar Typhimurium LT2 * Slides: Agilent’s arrays (8x15k) AMADID: NO: 17809 * Starting material: Salmonella RNA in nuclease-free water * RNA Samples used: Wild type (WT), WT + pTrc99A+LacI (PTRC), WT + pTrc99A-LacI(Delta lac) * Labeling kit: MessageAmp™ II-Bacteria RNA Amplification Kit from Ambion Cat # AM1790 * Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA * Total RNA and cRNA Purification Kit: Qiagen’s RNeasy minikit Cat#74104 * Hybridization Kit: Agilent’s In situ Hybridzation kit 5184-3568 * RNA quality was checked using Bioanalyzer. The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Two arrays were used with mutant and rescue experiments and dye swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from both replicates.
INSTRUMENT(S): Indian Institute of Science, Salmonella Typhimurium 15K array version 1
Project description:Salmonella is an important enteric pathogen that causes a spectrum of diseases varying from mild gastroenteritis to life threatening typhoid fever. Salmonella does not have lac operon. However, E. Coli, Salmonella’s close relative, has lac operon. Being an enteric pathogen like E. coli, Salmonella will also benefit from lac operon. Then, why Salmonella has lost lac operon?. To address this question, lacI, an important component of lac operon was expressed in Salmonella via pTrc99A plasmid. As a control, pTrc99A without lacI was also expressed in Salmonella. The effect of LacI on the transcription profile of Salmonella was analyzed using microarray technique. Overall design The total RNA was isolated from Salmonella using ‘RNeasy Mini Kit’ (QIAGEN). Organism used: Salmonella enterica serovar Typhimurium LT2 * Slides: Agilent’s arrays (8x15k) AMADID: NO: 17809 * Starting material: Salmonella RNA in nuclease-free water * RNA Samples used: Wild type (WT), WT + pTrc99A+LacI (PTRC), WT + pTrc99A-LacI(Delta lac) * Labeling kit: MessageAmp™ II-Bacteria RNA Amplification Kit from Ambion Cat # AM1790 * Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA * Total RNA and cRNA Purification Kit: Qiagen’s RNeasy minikit Cat#74104 * Hybridization Kit: Agilent’s In situ Hybridzation kit 5184-3568 * RNA quality was checked using Bioanalyzer. The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Two arrays were used with mutant and rescue experiments and dye swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from both replicates.
Project description:Classical bacteriological characteristics of Salmonella enterica indicate that the members of this species are unable to utilize lactose as a carbon source. However, lactose-fermenting (Lac+) strains of several Salmonella serovars have been isolated from different foodborne outbreaks as well as different geographical regions worldwide. In the present study, we sequenced the genomes of 13 Lac?+?S. enterica isolates and characterized the lac region, comparing it to the lac region in other enteric bacterial species.Genetic analysis of the lac operons in the S. enterica genomes revealed that they all contain intact lacI, lacZ, and lacY genes. However, lacA was truncated in all of the S. enterica subsp. enterica isolates, encoding a 56 amino acid peptide rather than the full length 220 amino acid LacA protein. Molecular analyses of the 13 isolates revealed that the lac operon resided on a plasmid in some strains and in others was integrated into the bacterial chromosome. In most cases, an insertion sequence flanked at least one end of the operon. Interestingly, the S. enterica Montevideo and S. enterica Senftenberg isolates were found to harbor a plasmid with a high degree of sequence similarity to a plasmid from Klebsiella pneumoniae strain NK29 that also harbors the lac operon. In addition, two S. enterica Tennessee isolates carried two copies of the lac operon. Phylogenetic analysis based on lacIZY gene sequences determines distinct clusters, and reveals a greater correlation between lacIZY sequence and flanking organization than with either bacterial species or genomic location.Our results indicate that the lac region is highly mobile among Enterobacteriaceae and demonstrate that the Lac?+?S. enterica subsp. enterica serovars acquired the lac region through parallel events. The acquisition of the lac operon by several S. enterica serovars may be indicative of environmental adaptation by these bacteria.
Project description:Escherichia coli lac repressor (LacI) is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. This tetrameric protein specifically binds to the O1, O2 and O3 operators of the lac operon and forms a DNA loop to repress transcription from the adjacent lac promoter. In this article, we demonstrate that upon binding to the O1 and O2 operators at their native positions LacI constrains three (-) supercoils within the 401-bp DNA loop of the lac promoter and forms a topological barrier. The stability of LacI-mediated DNA topological barriers is directly proportional to its DNA binding affinity. However, we find that DNA supercoiling modulates the basal expression from the lac operon in E. coli. Our results are consistent with the hypothesis that LacI functions as a topological barrier to constrain free, unconstrained (-) supercoils within the 401-bp DNA loop of the lac promoter. These constrained (-) supercoils enhance LacI's DNA-binding affinity and thereby the repression of the promoter. Thus, LacI binding is superhelically modulated to control the expression of lacZYA in the lac operon under varying growth conditions.
Project description:The genus Salmonella includes many pathogens of great medical and veterinary importance. Bacteria belonging to this genus are very closely related to those belonging to the genus Escherichia. lacZYA operon and lacI are present in Escherichia coli, but not in Salmonella enterica. It has been proposed that Salmonella has lost lacZYA operon and lacI during evolution. In this study, we have investigated the physiological and evolutionary significance of the absence of lacI in Salmonella enterica. Using murine model of typhoid fever, we show that the expression of LacI causes a remarkable reduction in the virulence of Salmonella enterica. LacI also suppresses the ability of Salmonella enterica to proliferate inside murine macrophages. Microarray analysis revealed that LacI interferes with the expression of virulence genes of Salmonella pathogenicity island 2. This effect was confirmed by RT-PCR and Western blot analysis. Interestingly, we found that SBG0326 of Salmonella bongori is homologous to lacI of Escherichia coli. Salmonella bongori is the only other species of the genus Salmonella and it lacks the virulence genes of Salmonella pathogenicity island 2. Overall, our results demonstrate that LacI is an antivirulence factor of Salmonella enterica and suggest that absence of lacI has facilitated the acquisition of virulence genes of Salmonella pathogenicity island 2 in Salmonella enterica making it a successful systemic pathogen.
Project description:BACKGROUND:The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two ?70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences. RESULTS:We showed that, in genome-integrated E. coli expression systems that used ?70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacIQ, on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. CONCLUSIONS:Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.
Project description:The Escherichia coli lac operon is regulated by a positive feedback loop whose potential to generate an all-or-none response in single cells has been a paradigm for bistable gene expression. However, so far bistable lac induction has only been observed using gratuitous inducers, raising the question about the biological relevance of bistable lac induction in the natural setting with lactose as the inducer. In fact, the existing experimental evidence points to a graded rather than an all-or-none response in the natural lactose uptake system. In contrast, predictions based on computational models of the lactose uptake pathway remain controversial. Although some argue in favor of bistability, others argue against it. Here, we reinvestigate lac operon expression in single cells using a combined experimental/modeling approach. To this end, we parameterize a well-supported mathematical model using transient measurements of LacZ activity upon induction with different amounts of lactose. The resulting model predicts a monostable induction curve for the wild-type system, but indicates that overexpression of the LacI repressor would drive the system into the bistable regime. Both predictions were confirmed experimentally supporting the view that the wild-type lac induction circuit generates a graded response rather than bistability. More interestingly, we find that the lac induction curve exhibits a pronounced maximum at intermediate lactose concentrations. Supported by our data, a model-based analysis suggests that the nonmonotonic response results from saturation of the LacI repressor at low inducer concentrations and dilution of Lac enzymes due to an increased growth rate beyond the saturation point. We speculate that the observed maximum in the lac expression level helps to save cellular resources by limiting Lac enzyme expression at high inducer concentrations.
Project description:In this study the repressor of Escherichia coli lac operon, LacI, has been engineered for altered effector specificity. A LacI saturation mutagenesis library was subjected to Fluorescence Activated Cell Sorting (FACS) dual screening. Mutant LacI-L5 was selected and it is specifically induced by lactulose but not by other disaccharides tested (lactose, epilactose, maltose, sucrose, cellobiose and melibiose). LacI-L5 has been successfully used to construct a whole-cell lactulose biosensor which was then applied in directed evolution of cellobiose 2-epimerase (C2E) for elevated lactulose production. The mutant C2E enzyme with ~32-fold enhanced expression level was selected, demonstrating the high efficiency of the lactulose biosensor. LacI-L5 can also be used as a novel regulatory tool. This work explores the potential of engineering LacI for customized molecular biosensors which can be applied in practice.
Project description:The lac repressor protein (LacI) efficiently represses transcription of the lac operon in Escherichia coli by binding to two distant operator sites on the bacterial DNA and causing the intervening DNA to form a loop. We employed single-molecule tethered particle motion to observe LacI-mediated loop formation and breakdown in DNA constructs that incorporate optimized operator binding sites and intrinsic curvature favorable to loop formation. Previous bulk competition assays indirectly measured the loop lifetimes in these optimized DNA constructs as being on the order of days; however, we measured these same lifetimes to be on the order of minutes for both looped and unlooped states. In a range of single-molecule DNA competition experiments, we found that the resistance of the LacI-DNA complex to competitive binding is a function of both the operator strength and the interoperator sequence. To explain these findings, we present what we believe to be a new kinetic model of loop formation and DNA competition. In this proposed new model, we hypothesize a new unlooped state in which the unbound DNA-binding domain of the LacI protein interacts nonspecifically with nonoperator DNA adjacent to the operator site at which the second LacI DNA-binding domain is bound.
Project description:The lac operon provides cells with the ability to switch from glucose to lactose metabolism precisely when necessary. This metabolic switch is mediated by the lac repressor (LacI), which in the absence of lactose binds to the operator DNA sequence to inhibit transcription. Allosteric rearrangements triggered by binding of the lactose isomer allolactose to the core domain of the repressor impede DNA binding and lift repression. In Nature, the ability to detect and respond to environmental conditions comes at the cost of the encoded enzymes being constitutively expressed at low levels. The readily-switched regulation provided by LacI has resulted in its widespread use for protein overexpression, and its applications in molecular biology represent early examples of synthetic biology. However, the leakiness of LacI that is essential for the natural function of the lac operon leads to an increased energetic burden, and potentially toxicity, in heterologous protein production.Analysis of the features that confer promiscuity to the inducer-binding site of LacI identified tryptophan 220 as a target for saturation mutagenesis. We found that phenylalanine (similarly to tryptophan) affords a functional repressor that is still responsive to IPTG. Characterisation of the W220F mutant, LacIWF, by measuring the time dependence of GFP production at different IPTG concentrations and at various incubation temperatures showed a 10-fold reduction in leakiness and no decrease in GFP production. Cells harbouring a cytotoxic protein under regulatory control of LacIWF showed no decrease in viability in the early phases of cell growth. Changes in responsiveness to IPTG observed in vivo are supported by the thermal shift assay behaviour of purified LacIWF with IPTG and operator DNA.In LacI, long-range communications are responsible for the transmission of the signal from the inducer binding site to the DNA binding domain and our results are consistent with the involvement of position 220 in modulating these. The mutation of this single tryptophan residue to phenylalanine generated an enhanced repressor with a 10-fold decrease in leakiness. By minimising the energetic burden and cytotoxicity caused by leakiness, LacIWF constitutes a useful switch for protein overproduction and synthetic biology.
Project description:Gene expression regulation is a fundamental biological process which deploys specific sets of genomic information depending on physiological or environmental conditions. Several transcription factors (including lac repressor, LacI) are present in the cell at very low copy number and increase their local concentration by binding to multiple sites on DNA and looping the intervening sequence. In this work, we employ single-molecule manipulation to experimentally address the role of DNA supercoiling in the dynamics and stability of LacI-mediated DNA looping. We performed measurements over a range of degrees of supercoiling between -0.026 and +0.026, in the absence of axial stretching forces. A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed. Our experiments also provide evidence for multiple structural conformations of the LacI-DNA complex, depending on torsional constraints. The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon. In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes.