PR Promoter Research Articles

Establishment of Highly Efficient Plant Regeneration, Callus Transformation and Analysis of Botrytis cinerea-Responsive PR Promoters in Lilium brownii var. viridulum.

Lilium brownii var. viridulum, commonly called Longya lily, is a well-known flower and vegetable plant in China that has poor tolerance to Botrytis fungal disease. The molecularimprovement has mainly been restricted to an efficient regeneration and transformation system. In this study, the highly efficient regeneration of Longya lily was established through the optimization of embryogenic callus, adventitious shoot and rooting induction. The major factors influencing transformation (antibiotics, Agrobacterium concentration, infection time, suspension solution and coculture medium) were examined. The expression responses of PR promoters (ZmPR4 and BjCHI1) to B. cinerea were assessed in transgenic calli. The results showed that Murashige and Skoog (MS) medium with 1.0 mg·L-1 picloram (PIC) and 0.2 mg·L-1 1-naphthaleneacetic acid (NAA) under light conditions and MS with 0.5 mg·L-1 6-benzylaminopurine (6-BA) and 1.0 mg·L-1 NAA under darkness were optimal for embryogenic callus induction (64.67% rate) and proliferation (3.96 coefficient). Callus inoculation into MS containing 2.0 mg·L-1 thidiazuron (TDZ), 0.4 mg·L-1 NAA, 1.0 mg·L-1 TDZ and 0.5 mg·L-1 NAA led to shooting induction (92.22 of rate) and proliferation (3.28 of coefficient) promotion, respectively. The rooting rate reached 99.00% on MS with 0.3 mg·L-1 NAA. Moreover, a transformation rate of 65.56% was achieved by soaking the callus in Agrobacterium at an OD600 of 0.4 for 10 min in modified MS without NH4NO3 as the suspension solution and coculture medium before selecting 75 mg·L-1 hygromycin and 300 mg·L-1 cefotaxime. Only the BjCHI1 promoter was obviously expressed in transgenic calli. These results could facilitate the generation of Longya lily transgenic plants with improved B. cinerea resistance.

Tunable Temperature-Sensitive Transcriptional Activation Based on Lambda Repressor.

Temperature is a versatile input signal for the control of engineered cellular functions. Sharp induction of gene expression with heat has been established using bacteria- and phage-derived temperature-sensitive transcriptional repressors with tunable switching temperatures. However, few temperature-sensitive transcriptional activators have been reported that enable direct gene induction with cooling. Such activators would expand the application space for temperature control. In this technical note, we show that temperature-dependent versions of the Lambda phage repressor CI can serve as tunable cold-actuated transactivators. Natively, CI serves as both a repressor and activator of transcription. Previously, thermolabile mutants of CI, known as the TcI family, were used to repress the cognate promoters PR and PL. We hypothesized that TcI mutants can also serve as temperature-sensitive activators of transcription at CI’s natural PRM promoter, creating cold-inducible operons with a tunable response to temperature. Indeed, we demonstrate temperature-responsive activation by two variants of TcI with set points at 35.5 and 38.5 °C in E. coli. In addition, we show that TcI can serve as both an activator and a repressor of different genes in the same genetic circuit, leading to opposite thermal responses. Transcriptional activation by TcI expands the toolbox for control of cellular function using globally or locally applied thermal inputs.

Bacteriophage λ RexA and RexB functions assist the transition from lysogeny to lytic growth.

The CI and Cro repressors of bacteriophage λ create a bistable switch between lysogenic and lytic growth. In λ lysogens, CI repressor expressed from the PRM promoter blocks expression of the lytic promoters PL and PR to allow stable maintenance of the lysogenic state. When lysogens are induced, CI repressor is inactivated and Cro repressor is expressed from the lytic PR promoter. Cro repressor blocks PRM transcription and CI repressor synthesis to ensure that the lytic state proceeds. RexA and RexB proteins, like CI, are expressed from the PRM promoter in λ lysogens; RexB is also expressed from a second promoter, PLIT , embedded in rexA. Here we show that RexA binds CI repressor and assists the transition from lysogenic to lytic growth, using both intact lysogens and defective prophages with reporter genes under the control of the lytic PL and PR promoters. Once lytic growth begins, if the bistable switch does return to the immune state, RexA expression lessens the probability that it will remain there, thus stabilizing the lytic state and activation of the lyticPL andPR promoters. RexB modulates the effect of RexA and may also help establish phage DNA replication as lytic growth ensues.

Characteristics and performance of the Co–CeO2 catalyst as a function of the promoter (La, Pr, and Zr) used in high temperature water gas shift reaction

The catalysts used to facilitate the water gas shift reaction (WGSR) are generally harmful to the environment. Therefore, catalysts that have high activity and stability in WGSR and do not pollute the environment need to be fabricated. Herein, three promoters (La, Pr, and Zr) are added into Co–CeO2 (CoCe) catalyst to improve catalytic performance in a high temperature WGSR to produce high-purity hydrogen from waste-derived synthesis gas. Various techniques are employed to confirm the changes in the properties that affect the catalytic performance. The catalytic reaction is performed at a high gas hourly space velocity to screen the performance of the promoted CoCe catalysts. The CoCeZr catalyst shows the highest CO conversion (XCO = 88% at 450 °C) due to its high Co dispersion and oxygen vacancy resulting from the addition of Zr to the CoCe catalyst; thus, it is most suitable for use in high temperature WGSR.

A method for the transient inhibition of toxicity of secretory recombinant proteins, exemplified by bacterial alkaline phosphatase. Novel protocol for problematic DNA termini dephosphorylation

Genes encoding proteins ‘toxic' to recombinant host are difficult for cloning/expression and recombinant clones are unstable. Even tightly controlled inducible T7-lac, PBAD, PL, PR promoters are not totally silent in an uninduced state and thus not adequate for highly toxic proteins. An innovative approach to engineering and expression of the gene, encoding bacterial alkaline phosphatase (BAP) is proposed. The native precursor enzyme contains a signal peptide at the N-terminus and is secreted to the Escherichia coli (E. coli) periplasm. The signal peptide is then removed that allows oxidation and formation of active dimers. To decrease toxicity of the bap gene, its secretion leader coding section was replaced with a N-terminal His6-tag. The gene was expressed in E. coli in a PBAD vector, resulting in the accumulation of soluble His6-BAP in the cytoplasm. The His6-BAP was neutral to the cells, as no maturation was possible in the reducing cytoplasm. The purified homogenous protein was further reactivated in a redox buffer containing the protein structure stabilizing cofactors. The His6-BAP exhibited high activity. A dephosphorylation protocol for all types of DNA termini was developed.The method appears well suited for the industrial production of BAP and can be applied to other problematic proteins.•Efficient toxic gene expression•Novel approach to toxic gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme•Scaled-up production of ultrapure BAP•Improved protocol for all types of DNA termini dephosphorylation

Differential induction of Shiga toxin in environmental Escherichia coli O145:H28 strains carrying the same genotype as the outbreak strains

Shiga toxin-producing Escherichia coli (STEC) O145 is a major serotype associated with severe human disease. Production of Shiga toxins (Stxs), especially Stx2a, is thought to be correlated with STEC virulence. Since stx genes are located in prophages genomes, induction of prophages is required for effective Stxs production. Here, we investigated the production of Stxs in 12 environmental STEC O145:H28 strains under stresses STEC encounter in natural habitats and performed comparative analysis with two O145:H28 clinical strains, one linked to a 2010 U.S. lettuce-associated outbreak (RM13514) and the other linked to a 2007 Belgium ice cream-associated outbreak (RM13516). Similar to the outbreak strains, all environmental strains belong to Sequence Type (ST)-78 using the EcMLST typing scheme. Although all Stx1a-prophages were grouped together, variations in Stx1a production were observed prior to or following the inductions. Among all stx2a positive environmental strains, only the Stx2a-prophage in cattle isolate RM9154-C1 was clustered with the Stx2a-prophages in RM13514, the Stx2a-phage induced from a STEC O104:H4 strain linked to the 2011 outbreak of enterohemorrhagic infection in Germany, and the Stx2a-prophage in STEC O157:H7 strain EDL933, a prototype of enterohemorrhagic E. coli. Furthermore, the Stx2a-prophage in RM9154-C1 shared the same chromosomal insertion site and carried the same antiterminator Q gene and the late promoter PR' as the Stx2a-prophage in RM13514. Following mitomycin C or enrofloxacin treatment, the production of Stx2a in RM9154-C1 was the highest among all environmental strains tested. In contrast, following acid challenge and recovery, the production of Stx2a in RM9154-C1 was the lowest among all the environmental strains tested, at a level comparable to the clinical strains. A significant increase in Stx2a production was detected in all strains when exposed to H2O2, although the induction fold was much lower than those by other inducers. This low-efficiency induction of Stx-prophages by H2O2, a natural inducer of Stx-prophages, supports the hypothesis of bacterial altruism in controlling Stxs production, a strategy that assures the survival of the STEC population as a whole by sacrificing a small fraction of cells for Stxs production and release. Differential induction of Stxs among strains carrying nearly identical Stx-prophages suggests a role of host bacteria in regulating Stxs production. Our study revealed diverse Stx-prophages in STEC O145:H28 strains that were genotypically indistinguishable. Identification of a cattle isolate harboring a Stx2a-prophage associated with high virulence supports the premise that cattle, a natural reservoir of STEC, serve as a source of hypervirulent STEC strains.

Repression of the lysogenic PR promoter in bacteriophage TP901-1 through binding of a CI-MOR complex to a composite OM-OR operator

A functional genetic switch from the lactococcal bacteriophage TP901-1, deciding which of two divergently transcribing promoters becomes most active and allows this bi-stable decision to be inherited in future generations requires a DNA region of less than 1 kb. The fragment encodes two repressors, CI and MOR, transcribed from the PR and PL promoters respectively. CI can repress the transcription of the mor gene at three operator sites (OR, OL, and OD), leading to the immune state. Repression of the cI gene, leading to the lytic (anti-immune) state, requires interaction between CI and MOR by an unknown mechanism, but involving a CI:MOR complex. A consensus for putative MOR binding sites (OM sites), and a common topology of three OM sites adjacent to the OR motif was here identified in diverse phage switches that encode CI and MOR homologs, in a search for DNA sequences similar to the TP901-1 switch. The OR site and all putative OM sites are important for establishment of the anti-immune repression of PR, and a putative DNA binding motif in MOR is needed for establishment of the anti-immune state. Direct evidence for binding between CI and MOR is here shown by pull-down experiments, chemical crosslinking, and size exclusion chromatography. The results are consistent with two possible models for establishment of the anti-immune repression of cI expression at the PR promoter.

From Quorum Sensing to Positional Sensing

With Quorum Sensing (QS) bacteria adapt their physiology and metabolism when they reach a certain density and activate genes relevant for their nutrient metabolism, survival and virulence. Genes controlled by QS have relevance for the pathogenic behavior of bacteria including adherence, secretion, toxin production, immune subversion, biofilm formation and resistance to both antimicrobial and biological/chemical stressors. Most studies assess QS by growing bacteria to various densities in liquid culture. Here, we report that when bacteria are grown on agar plates at various densities, in addition to QS, positional sensing (PS) comes into play. Genes controlled by PS can be studied by cloning their promoter in front of an autonomous bioluminescent cassette (luxCDABE) and measuring their time‐ and position‐dependent expression in biofilms by using an in vivo imaging system (IVIS). The Pseudomonas putida XylR/XylS intergenic region controlling genes relevant for the metabolism and degradation of xylene and related organic molecules was cloned in front of the autonomous luciferase cassette from Photorhabdus luminescence, with the sigma 54 controlled Ps1 promoter regulating the a luxCDABE genes and the Pr promoter regulating the XylR gene. Interestingly, when E. coli carrying this construct were grown to various density on agar plates, bioluminescence was not only expressed in a density‐ and time‐dependent, but also position‐dependent manner. Such positional cues may be mediated by signaling molecules in response to cell density, growth stage, and nutrient availability. Overexpression of the sigma factor 54 (NtrA) from Pseudomonas putida modified the density‐ and time‐dependent response suggesting a regulatory role in QS and PS. The use of bioluminescence in QS and PS promises to reveal important regulatory circuits and communication systems relevant for bacterial normal physiology, pathogenicity and virulence.

Elements in the λ immunity region regulate phage development: beyond the 'Genetic Switch'.

Genetic elements in the bacteriophage λ immunity region contribute to stable maintenance and synchronous induction of the integrated Escherichia coli prophage. There is a bistable switch between lysogenic and lytic growth that is orchestrated by the CI and Cro repressors acting on the lytic (PL and PR ) and lysogenic (PRM ) promoters, referred to as the Genetic Switch. Other less well-characterized elements in the phage immunity region include the PLIT promoter and the immunity terminator, TIMM . The PLIT promoter is repressed by the bacterial LexA protein in λ lysogens. LexA repressor, like the λ CI repressor, is inactivated during the SOS response to DNA damage, and this regulation ensures that the PLIT promoter and the lytic PL and PR promoters are synchronously activated. Proper RexA and RexB protein levels are critical for the switch from lysogeny to lytic growth. Mutation of PLIT reduces RexB levels relative to RexA, compromising cellular energetics and causing a 10-fold reduction in lytic phage yield. The RexA and RexB proteins interact with themselves and each other in a bacterial two-hybrid system. We also find that the transcription terminator, TIMM , is a Rho-independent, intrinsic terminator. Inactivation of TIMM has minimal effect on λ lysogenization or prophage induction.

Comparative Genomics and Characterization of the Late Promoter pR' from Shiga Toxin Prophages in Escherichia coli.

Shiga-toxin producing Escherichia coli (STEC) causes human illness ranging from mild diarrhea to death. The bacteriophage encoded stx genes are located in the late transcription region, downstream of the antiterminator Q. The transcription of the stx genes is directly under the control of the late promoter pR’, thus the sequence diversity of the region between Q and stx, here termed the pR’ region, may affect Stx toxin production. Here, we compared the gene structure of the pR’ region and the stx subtypes of nineteen STECs. The sequence alignment and phylogenetic analysis suggested that the pR’ region tends to be more heterogeneous than the promoter itself, even if the prophages harbor the same stx subtype. Furthermore, we established and validated transcriptional fusions of the pR’ region to the DsRed reporter gene using mitomycin C (MMC) induction. Finally, these constructs were transformed into native and non-native strains and examined with flow cytometry. The results showed that induction levels changed when pR’ regions were placed under different regulatory systems. Moreover, not every stx gene could be induced in its native host bacteria. In addition to the functional genes, the diversity of the pR’ region plays an important role in determining the level of toxin induction.

C/EBPβ LIP and c-Jun synergize to regulate expression of the murine progesterone receptor.

CCAAT/enhancer binding protein β (C/EBPβ) is required for murine mammary ductal morphogenesis and alveologenesis. Progesterone is critical for proliferation and alveologenesis in adult mammary glands, and there is a similar requirement for progesterone receptor isoform B (PRB) in alveologenesis. We examined C/EBPβ regulation of PR expression. All three C/EBPβ isoforms, including typically inhibitory LIP, transactivated the PR promoter. LIP, particularly, strongly synergized with c-Jun to drive PR transcription. Endogenous C/EBPβ and c-Jun stimulated a PR promoter-reporter and these two factors showed promoter occupancy on the endogenous PR gene. Additionally, LIP overexpression elevated endogenous PR protein expression. In pregnancy, both PRB and the relative abundance of LIP among C/EBPβ isoforms increase. Consistent with a role in PRB expression, in vivo C/EBPβ and PR isoform A expression showed mutually exclusive localization in mammary epithelium, while C/EBPβ and PRB largely co-localized. We suggest a critical role for C/EBPβ, particularly LIP, in PRB expression.

Hydrogen Production through Steam Reforming of Diesel over Highly Efficient Promoted Ni/γ-Al2O3 Catalysts Containing Lanthanide Series (La, Ce, Eu, Pr, and Gd) Promoters

The wet incipient impregnation technique was used for the synthesis of nonpromoted and lanthanide series-promoted (La, Ce, Eu, Pr, and Gd) Ni/γ-Al2O3 catalysts. The physicochemical properties of th...

Improved ethanol productivity from lignocellulosic hydrolysates by Escherichia coli with regulated glucose utilization

BackgroundLignocellulosic ethanol could offer a sustainable source to meet the increasing worldwide demand for fuel. However, efficient and simultaneous metabolism of all types of sugars in lignocellulosic hydrolysates by ethanol-producing strains is still a challenge.ResultsAn engineered strain Escherichia coli B0013-2021HPA with regulated glucose utilization, which could use all monosaccharides in lignocellulosic hydrolysates except glucose for cell growth and glucose for ethanol production, was constructed. In E. coli B0013-2021HPA, pta-ackA, ldhA and pflB were deleted to block the formation of acetate, lactate and formate and additional three mutations at glk, ptsG and manZ generated to block the glucose uptake and catabolism, followed by the replacement of the wild-type frdA locus with the ptsG expression cassette under the control of the temperature-inducible λ pR and pL promoters, and the final introduction of pEtac-PA carrying Zymomonas mobilis pdc and adhB for the ethanol pathway. B0013-2021HPA was able to utilize almost all xylose, galactose and arabinose but not glucose for cell propagation at 34 °C and converted all sugars to ethanol at 42 °C under oxygen-limited fermentation conditions.ConclusionsEngineered E. coli strain with regulated glucose utilization showed efficient metabolism of mixed sugars in lignocellulosic hydrolysates and thus higher productivity of ethanol production.

Optimal bioprocess design through a gene regulatory network – Growth kinetic hybrid model: Towards replacing Monod kinetics

Currently, design and optimisation of biotechnological bioprocesses is performed either through exhaustive experimentation and/or with the use of empirical, unstructured growth kinetics models. Whereas, elaborate systems biology approaches have been recently explored, mixed-substrate utilisation is predominantly ignored despite its significance in enhancing bioprocess performance. Herein, bioprocess optimisation for an industrially-relevant bioremediation process involving a mixture of highly toxic substrates, m-xylene and toluene, was achieved through application of a novel experimental-modelling gene regulatory network – growth kinetic (GRN-GK) hybrid framework. The GRN model described the TOL and ortho-cleavage pathways in Pseudomonas putida mt-2 and captured the transcriptional kinetics expression patterns of the promoters. The GRN model informed the formulation of the growth kinetics model replacing the empirical and unstructured Monod kinetics. The GRN-GK framework's predictive capability and potential as a systematic optimal bioprocess design tool, was demonstrated by effectively predicting bioprocess performance, which was in agreement with experimental values, when compared to four commonly used models that deviated significantly from the experimental values. Significantly, a fed-batch biodegradation process was designed and optimised through the model-based control of TOL Pr promoter expression resulting in 61% and 60% enhanced pollutant removal and biomass formation, respectively, compared to the batch process. This provides strong evidence of model-based bioprocess optimisation at the gene level, rendering the GRN-GK framework as a novel and applicable approach to optimal bioprocess design. Finally, model analysis using global sensitivity analysis (GSA) suggests an alternative, systematic approach for model-driven strain modification for synthetic biology and metabolic engineering applications.

Production of RNase III-knockout, auxotrophic Edwardsiella tarda mutant for delivery of long double-stranded RNA and evaluation of its immunostimulatory potential

The artificially synthesized polyinosinic-polycytidylic acid (poly IC) has been widely used to induce type I IFN responses in various vertebrates including fish. However, as poly IC is too expensive to use in aquaculture, the development of another economical long dsRNA producing method is needed to practically use long dsRNAs in aquaculture farms for the control of infectious diseases. In the present study, to produce long dsRNAs economically, we developed a novel long dsRNA production system based on the RNase III gene deleted auxotrophic mutant E. tarda (ΔalrΔrncΔasd E. tarda) and a long dsRNA-producing vector that was equipped with two modified λ phage PR promoters arranged in a head-to-head fashion. As the present genetically engineered E. tarda cannot live without supplementation of d-alanine and DAP, environmental and medicinal risks are minimized. Olive flounder (Paralichthys olivaceus) fingerlings administered the long dsRNA-producing auxotrophic E. tarda mutant (Δalr ΔrncΔasd E. tarda) showed significantly higher expressions of TLR22, Mx1, and ISG15 genes, indicating a potential to increase type I interferon responses.

YMC-2011, a Temperate Phage of Streptococcus salivarius 57.I.

Streptococcus salivarius is an abundant isolate of the oral cavity. The genome of S. salivarius 57.I consists of a 2-Mb chromosome and a 40,758-bp circular molecule, designated YMC-2011. Annotation of YMC-2011 revealed 55 open reading frames, most of them associated with phage production, although plaque formation is not observed in S. salivarius 57.I after lytic induction using mitomycin C. Results from Southern hybridization and quantitative real-time PCR confirmed that YMC-2011 exists extrachromosomally, with an estimated copy number of 3 to 4. Phage particles were isolated from the supernatant of mitomycin C-treated S. salivarius 57.I cultures, and transmission electron microscopic examination indicated that YMC-2011 belongs to the Siphoviridae family. Phylogenetic analysis suggests that phage YMC-2011 and the cos-type phages of Streptococcus thermophilus originated from a common ancestor. An extended -10 element (p L ) and a σ70-like promoter (p R ) were mapped 5' to Ssal_phage00013 (encoding a CI-like repressor) and Ssal_phage00014 (encoding a hypothetical protein), respectively, using 5' rapid amplification of cDNA ends, indicating that YMC-2011 transcribes at least two mRNAs in opposite orientations. Studies using promoter-chloramphenicol acetyltransferase reporter gene fusions revealed that p R , but not p L , was sensitive to mitomycin C induction, suggesting that the switch from lysogenic growth to lytic growth was controlled mainly by the activity of these two promoters. In conclusion, a lysogenic state is maintained in S. salivarius 57.I, presumably by the repression of genes encoding proteins for lytic growth.IMPORTANCE The movement of mobile genetic elements such as bacteriophages and the establishment of lysogens may have profound effects on the balance of microbial ecology where lysogenic bacteria reside. The discovery of phage YMC-2011 from Streptococcus salivarius 57.I suggests that YMC-2011 and Streptococcus thermophilus-infecting phages share an ancestor. Although S. salivarius and S. thermophilus are close phylogenetically, S. salivarius is a natural inhabitant of the human mouth, whereas S. thermophilus is commonly found in the mammary mucosa of bovine species. Thus, the identification of YMC-2011 suggests that horizontal gene transfer via phage infection could take place between species from different ecological niches.

Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in Escherichia coli.

To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose. The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the PR promoter and kanamycin resistance gene (PR-kan R ) at 30°C, but have no effect on PR-kan R gene at 37°C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~107 cfu per µg of genomic DNA, with no empty vectors detected. Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.

Optimization of phage λ promoter strength for synthetic small regulatory RNA-based metabolic engineering

Synthetic small regulatory RNAs (sRNAs) are gene-silencing tools that can be used to tune gene expression in prokaryotes. A recent study by our group proposed rational design principles, introduced a regulatory system that may be used to implement synthetic sRNAs, and showed their utility in metabolic engineering. The regulatory system employed the strong phage λ PR promoter to tightly control synthetic sRNA production. Here, we fine-tuned the strength of the PR promoter via mutagenesis in order to optimize the level of synthetic sRNAs while maintaining the ability of the promoter to be regulated by CI proteins. Five mutant promoters of different strengths, ranging from 24 to 87% of that of the wild-type PR promoter, were identified and confirmed to be repressed by CI proteins. A mutated promoter with only 40% of the original strength still produced enough synthetic sRNAs to inhibit the translation of the target mRNA to ~10% of the original level. As a practical application, we tested our promoters as drivers for a synthetic anti-murE sRNA, which was used to adjust the production of cadaverine. As the promoter strength decreased, the cadaverine titer first increased and then dropped. A mutated promoter with 39% of the original strength achieved the improved cadaverine titer of 2.15 g/L. The mutant promoters developed in this study should prove useful for tuning the expression levels of synthetic sRNAs for metabolic engineering.

Modulation of Lactobacillus casei bacteriophage A2 lytic/lysogenic cycles by binding of Gp25 to the early lytic mRNA.

The genetic switch of Lactobacillus casei bacteriophage A2 is regulated by the CI protein, which represses the early lytic promoter PR and Cro that abolishes expression from the lysogenic promoter PL . Lysogens contain equivalent cI and cro-gp25 mRNA concentrations, i.e., CI only partially represses P(R), predicting a lytic cycle dominance. However, A2 generates stable lysogens. This may be due to Gp25 binding to the cro-gp25 mRNA between the ribosomal binding site and the cro start codon, which abolishes its translation. Upon lytic cycle induction, CI is partially degraded, cro-gp25 mRNA levels increase, and Cro accumulates, launching viral progeny production. The concomitant concentration increase of Gp25 restricts cro mRNA translation, which, together with the low but detectable levels of CI late during the lytic cycle, promotes reentry of part of the cell population into the lysogenic cycle, thus explaining the low proportion of L. casei lysogens that become lysed (∼ 1%). A2 shares its genetic switch structure with many other Firmicutes phages. The data presented may constitute a model of how these phages make the decision for lysis versus lysogeny.

An Unusual Phage Repressor Encoded by Mycobacteriophage BPs.

Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection—lysogenic or lytic growth—as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33–34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.