Mycobacteriophages are infectious particles that infect mycobacteria, and little is known about cis-regulatory elements that control their gene expression. In phage genomes, cis-regulatory elements commonly precede a series of genes that are expressed as an operon. JacoRen57 is a cluster AB mycobacteriophage that possesses forward and reverse genes with non-coding gaps interspersed throughout its genome. We assayed one of the gap regions of JacoRen57 (40644-40974 bps) for regulatory activity in the downstream direction when present in its host, Mycobacterium smegmatis, by cloning the region into pLO86, a vector containing the mCherry reporter gene. The putative regulatory region induced the expression of mCherry in vivo, indicating the presence of a promoter in this region of the JacoRen57 genome. Utilizing 5’ deletions analysis, we identified promoter and repressor elements within this regulatory region. We are conducting further experiments to understand the characteristics of the repressor region and which sigma factor/s binds to this promoter

Almail, Ali

Heeg, Elizabeth

Noordewier, Byron

Tolsma, Sara S

English

NWCommons: Institutional Repository of Northwestern College

cAli Almail, Elizabeth Heeg , Byron Noordewier, Sara S. TolsmaNorthwestern College, Department of Biology, Orange City, IA 51041AbstractMycobacteriophages are infectious particles that infect mycobacteria, and little is knownabout cis-regulatory elements that control their gene expression. In phage genomes, cis-regulatory elements commonly precede a series of genes that are expressed as an operon.JacoRen57 is a cluster AB mycobacteriophage that possesses forward and reverse genes withnon-coding gaps interspersed throughout its genome. We assayed one of the gap regions ofJacoRen57 (40644-40974 bps) for regulatory activity in the downstream direction whenpresent in its host, Mycobacterium smegmatis, by cloning the region into pLO86 – a vectorcontaining the mCherry reporter gene. The putative regulatory region induced the expressionof mCherry in vivo, indicating the presence of a promoter in this region of the JacoRen57genome. Utilizing 5’ deletion analysis, we identified transcriptional repressor and activatorelements within this regulatory region. We are conducting further experiments to understandthe characteristics of the transcriptional repressor region and which sigma factor(s) bind(s) tothis regulatory region.IntroductionMaterials and MethodsReferences Almail,A., Dorhout,K.E., Johnson,J., Jorgensen,H.J., Tolsma,S., Garlena,R.A., Russell,D.A., Pope,W.H., Jacobs-Sera,D. and, & Hatfull,G.F. (2019). Mycobacterium phage JacoRen57, complete genome (1547005285). NCBI Nucleotide Database. http://www.ncbi.nlm.nih.gov/nuccore/MK279840.1Clokie, M. R., Millard, A. D., Letarov, A. V., & Heaphy, S. (2011). Phages in nature. Bacteriophage, 1(1), 31–45. https://doi.org/10.4161/bact.1.1.14942Manganelli, R., Dubnau, E., Tyagi, S., Kramer, F. R., & Smith, I. (1999). Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis. Molecular Microbiology, 31(2), 715–724. https://doi.org/10.1046/j.1365-2958.1999.01212.xManganelli, Riccardo, Proveddi, R., Rodrigue, S., Beaucher, J., Gaudreau, L., & Smith, I. (2004). σ Factors and Global Gene Regulation in Mycobacterium tuberculosis. Journal of Bacteriology, 186(4), 895–902. https://doi.org/10.1128/JB.186.4.895-902.2004Murphy, K. C. (2012). Phage recombinases and their applications. Advances in Virus Research, 83, 367–414. https://doi.org/10.1016/B978-0-12-394438-2.00008-6Oldfield, L. M., & Hatfull, G. F. (2014). Mutational analysis of the mycobacteriophage BPs promoter PR reveals context-dependent sequences for mycobacterial gene expression. Journal of Bacteriology, 196(20), 3589–3597. https://doi.org/10.1128/JB.01801-14Phagesdb. (2021). The Actinobacteriophage Database | Phage JacoRen57. https://phagesdb.org/phages/JacoRen57/Valero-Rello, A., López-Sanz, M., Quevedo-Olmos, A., Sorokin, A., & Ayora, S. (2017). Molecular Mechanisms That Contribute to Horizontal Transfer of Plasmids by the Bacteriophage SPP1. Frontiers in Microbiology, 8. https://doi.org/10.3389/fmicb.2017.01816ResultsFigure 1 (a): Diagram of the 5’ deletion mutants of the transcriptional regulatory region of JacoRen57. The dashed regions represent deleted segments. (b): mCherry fluorescence ratios of M. smegmatiscontaining pLo86 constructs with the 5’ deletion mutant inserts. We had biological triplicates for each clone. Different letters indicate significant difference between clones (p<0.05). Error bars represent standard deviation. 5’ Deletion Assays:We constructed 5’ deletion mutants to identify elements of the regulatory region that modulate gene expression. We found that the deletion of bases 57-152 resulted in a significant increase of the fluorescence ratio of mCherry compared to pLo86_51 and pLo86_51 Δ1, indicating increased mCherry expression (Figure 1 ). We also found that the the deletion of bases 153-202 resulted in a significant decrease in the fluorescence of mCherry  compared to pLo86_51 Δ2, which indicates a decrease in mCherry expression (Figure 1 ). DiscussionFigure 4: Proposed model of the transcriptional regulatory region. This model contains a repressor region (57-153 bp), a promoter element (153-203 bp), and another promoter element (203-249). The possible hypothetical binding site of sigE in promoter element 1 and sigG in promoter element 2 are listed. To identify the transcriptional regulatory elements within this region, we constructed5’ deletion mutants . We successfully constructed four deletion mutants, as we obtainedthe expected band sizes from PCR confirmation (Figure 2). We used the constructs toconduct reporter gene assays to identify which segments of the transcriptional regulatoryregion modulated reporter gene expression. We found that:1. The deletion of bases 57-152 resulted in a significant increase in thefluorescence ratio of mCherry compared to all other clones, indicating theincrease in mCherry gene expression. This supports the presence of atranscriptional repressor element (Figure 4).2. The deletion of bases 153-202 in pLo86_51Δ2.5 resulted in a significant decreasein fluorescence ratio of mCherry compared to pLo86_51Δ2, indicating a decreasein mCherry expression. This supports the model that there are promoterelements within 153-202 bp and within 203-249 bp, as 249-282 bp did notmodulate mCherry gene expression (Figure 1 and 4).To narrow down which sigma factors interact with the transcriptional regulatoryregion, we conducted the heat shock assays. We found that 45°C treatment resulted in asignificant decrease in the fluorescence ratio of mCherry in pLo86_51 and pLo86_51 Δ2(Figure 3). This led us to hypothesize which sigma factors may be interacting with ourregulatory region based on studies by Manganelli et al. (1999) that measured which sigmafactors were downregulated in M. tuberculosis due to heat shock. The closest consensusbinding sequence present in our regulatory region is that of sigG (Figure 4). Interestingly,SDS treatment at 45°C resulted in an increased ratio of mCherry fluorescence (Figure 3).This may support some synergistic interactions between two sigma factors that modulategene expression independently. We had previously found a sigE consensus binding site inthe transcriptional region that had a larger spacing between the -35 and -10 elementsthan the published consensus in M. tuberculosis (Figure 4), and we hypothesize that thiscould be the synergistic sigma factor. We do not suspect that the transcriptional repressorregion is necessary for this synergistic activity, as the absence of the repressor elementresulted in a significant increase with combined SDS and heat shock treatments. We planon conducting this assay with the recently constructed pLo86_51Δ2.5.The 5’ deletion assay data supports the model that there is a transcriptional repressorregion and two promoter elements. Furthermore, sequence comparison to publishedsigma factor binding sites in M. tuberculosis support the presence of two promoterelements that bind different sigma factors (Figure 4). We hypothesize that promoterelement 1 binds sigE and promoter element 2 binds sigG (Figure 4). We are in the processof conducting site-directed mutagenesis to confirm if the hypothesized cis-actingelements of sigE and sigG modulate gene expression. We also plan to send the remainingconstructs for sequencing to confirm the absence of mutations (note: we used a high-fidelity DNA polymerase to construct all expression constructs).These results present interesting implications for phage gene expression regulation.This transcriptional region precedes an operon of three overlapping genes, one of which isa RecA -like DNA recombinase (NCBI:MK279840.1, 2019). Given that horizontal genetransfer is common between the host and phage genome, it is possible that thisregulatory region resulted from multiple horizontal gene transfer events that lateraccumulated mutations (Valero-Rello, 2017). These regulatory regions were possiblymaintained since neither of these consensus sequences may represent ideal binding sitesfor sigma factors, so maintaining both sigma factor binding sites resulted in higher levelsof gene expression than is possible with only one promoter element, which is supportedby our data and also provides alternative modes of gene expression regulation that arecontext dependent (Figure 1). Further research is needed to understand how this fits intomodels of phage evolution, to explore the binding site and transcpiriotnal repressorprotein that binds to the transcriptional repressor element, and why RecA–like DNArecombinase expression would need to be regulated by such a regulatory region.PCR to Confirm 5’ Deletion Mutants:We designed primers that annealed to plasmid sequences outside the transcriptional regulatory region to confirm the 5’ deletion mutations. If the 5’ deletion mutants were successful, we expected the following band sizes: pLo86=  234 bp, pLo86_51= 516 bp, pLo86_51Δ1= 460 bp, pLo86_51Δ2= 361 bp, pLo86_51Δ2.5= 314 bp, pLo86_51Δ3= 268 bp. We obtained the expected band sizes for each respective deletion (Figure 2). Figure 2: (a) 1% agarose gel of PCR products to confirm the 5’ deletion mutations of pLo86_51, pLo86_51Δ1, pLo86_51Δ2, and pLo86_51Δ3. (b) 1% agarose gel of PCR products from colony verification to confirm the 5’ deletion mutation of pLo86_51Δ2.5. In both (a) and (b), we used the NEB 1kB plus ladder.cAcknowledgementsI would like to thank Dr. Tolsma, Dr. Heeg, and Dr. Noordewier for your wisdom and supervision. I would like to thank the Hatfull Lab at the University of Pittsburgh for the provision of the pLO86 and pLO87. Support for this project was provided by Northwestern College and Howard Hughes Medical Institute . I would also like to thank the directors of the NWC Honors Program Dr. Feenstra and Dr. Vonder Bruegge for the research opportunity.Mycobacteriophages are viruses that infect bacteria from the genus Mycobacteriumthrough the lytic and lysogenic cycles (Clokie et al., 2011). JacoRen57 is a Siphoviridaemorphotype, lytic, and cluster AB mycobacteriophage that infects Mycobacterium smegmatismc2155 (Phagesdb, 2021). Its double stranded DNA genome is 70,300 bps, consisting of 73genes, 33 of which are forward genes followed by 16 reverse genes and 24 forward genesfrom the right to left arm of its genome (NCBI:MK279840.1, 2019).The second transition from reverse to forward genes (40644-40974 bp) possesses a 331bp region with minimal coding potential. The gene directly downstream of this gap is a RecA-like DNA recombinase, which functions in phage DNA replication, genome packaging,generation of genomic diversity, and dsDNA repair (NCBI:MK279840.1, 2019; Murphy, 2012).There are two genes proceeding the RecA-like DNA recombinase that overlap with each other,suggesting the expression of the three genes in the same operon (NCBI:MK279840.1, 2019).Utilizing online promoter predictor softwires (PePPER, BPROM, Neural Network PromoterPredictor), we found matches putative matches to promoter sequences.Regulatory regions in prokaryotes and phages follow an operon model, where aregulatory region precedes a series of genes. A regulatory region typically consists of apromoter and a repressor/operator region Additionally, various sigma factor consensussequences have been characterized in Mycobacterium tuberculosis, which is in the samegenus as M. smegmatis. Many of the characterized sigma factor sites are responsive to cellstress signals (Manganelli et al., 2004).To assess regulatory activity of a DNA sequence, fluorescent reporter genes constructs,pLO86 and pLO87, were used to assess promoter activity of the JacoRen57 putative regulatoryregion which we refer to as 51. pLo86 lacks a promoter region, but pLo87 possesses an hsp60promoter (Oldfield et al., 2014). These plasmids encode the mCherry gene, a reporter genethat codes for a pink fluorescent protein which is observable under visible light conditions. Ifthe cloned sequence functions as a promoter in vivo, then the mCherry gene will betranscribed and translated to produce mCherry. However, if the cloned sequence does notfunction as a promoter in vivo, then mCherry will not be expressed.In this study we utilized reporter gene assays with pLO86 and pLO87 to assess the activityof the putative regulatory region (40644-40974 bp) in JacoRen57. We demonstrate that theputative regulatory region consists of three elements, a repressor region and two promoterelements that function in the downstream direction. We are in the process of conductingmore experiments to elucidate the trans-acting elements of this regulatory sequence.Figure 3 : We exposed M. smegmatis to shock treatments to narrow down the possible interacting sigma factors. Each of the clones in (a), (b), and (c) were treated with 0.05% SDS at 37°C,45°C, and 0.05% SDS at 45°C. The experiment was replicated sixfold for each clone. Results are graphed on a log scale of a ratio of the fluorescence levels of mCherry at the treatmentcondition over the control. Values above 1 indicate an increase in fluorescence ratios, while values below 1 indicate a decrease in fluorescence ratios. * indicates a significant difference fromthe no treatment control at 37°C (p<0.05). The error bars represent percent deviation from the sample’s mean ratio.Shock Assays:Given what we know about sigma factor expression in M. tuberculosis under different conditions (Manganelli et al., 1999), we attempted to expose M. smegmatis with pLo86_51 andpLo86_51 Δ2 to shock treatments to narrow down which sigma factors bind to the transcriptional regulatory region. M. smegmatis containing pLo87 served as an experimental control toconfirm if the observed mCherry fluorescence ratio changes were unique to our transcriptional regulatory region. This experiment assumes that increased sigma factor expression can lead toincreased reporter gene expression. We found that heat shock at 45°C significantly decreased the fluorescence ratio of mCherry compared to a non-treatment control in both pLo86_51 andpLo86_51 Δ2, indicating a decrease in reporter gene expression (Figure 3). In both these clones, we found that SDS treatment at 45°C significantly increased the fluorescence ratio of mCherrysignificantly, indicating an increase in mCherry gene expression (Figure 3). None of the treatments resulted in significant differences between the mCherry fluorescence ratios in M. smegmatistransformed with pLo87 (Figure 3).Cloning the Transcriptional Regulatory Region into pLo86 Construction of 5’ Deletion Mutants Shock AssayscQR Code to Access Detailed Materialsand Methods

Exploring a Transcriptional Regulatory Region in Mycobacteriophage JacoRen57

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Exploring a Transcriptional Regulatory Region in Mycobacteriophage JacoRen57

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