1. Discovery of small proteins expressed from novel open reading frames in
Escherichia coli
Charles Allen, Stefan Bordonaro, Austin Gallegos, Jordan Lingo and Ryan McGuirk
Spring 2015 Molecular Biology Laboratory Class
Mentor: Dr. Joseph F. Sanchez
Department of Biological Sciences, Towson University, Towson, Maryland 21252
Abstract
Materials and Methods
Bioinformatics Analysis – candidate intragenic open reading frames (iORFs) were analyzed using online tools to assess potential for ribosomal binding, secondary structure, functional-domains and species conservation. Shown is a prediction of potential α-helices ( ) for rnpA’ using SABLE (
Mutagenesis using Site-directed Recombination – candidate iORF-SPA amplicons were electroporated into rec+ NM400 bacterial cells and selected for resistance to kanamycin. Putative recombinant strains were subjected to genomic screening PCR analysis followed by single-strand sequence verification.
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Growth Curve Analysis and Sample Processing – NM400-iORF-SPA recombinant strains were grown in LB media at 37°C with shaking. Absorbance (OD600) was monitored and samples were collected during exponential, stationary and death-phase (overnight) growth. Total protein lysates were generated by boiling in 4X protein loading dye supplemented with β-mercaptoethanol.
Immunoblot Analysis – Total protein lysates were fractionated by SDS PAGE using a Benchmark pre-stained protein marker (Life Technologies) as the reference. Negative control (MG1655) and positive control (cydX-SPA; ~12kDa) lysates were included. Fractionated samples were transferred to PVDF membrane, blocked in 10% milk and probed using a mouse monoclonal α-FLAG-AP (alkaline phosphatase) antibody (1:5000; Sigma). Single-step detection was performed using 1X LumiPhos (CDP-Star; Novex) substrate and exposure to film.
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Results
Discussion
This semester the Molecular Biology Laboratory Course students have successfully identified the utilization of five novel open reading frames in E. coli, adding to the growing list of genes that encode small proteins in prokaryotes.
As seen with previously identified small proteins, these gene products are likely to have specific functions within the cell and are likely to be regulated in response to specific stimuli and challenges to cellular homeostasis (VanOrsdel et al., 2013). For example, research investigating the small protein, CydX, has shown that this protein interacts with the well-known CydAB cytochrome bd oxidase complex and that loss of CydX is coupled with reduced oxidase activity (VanOrsdel et al., 2013).
We demonstrate for the three small proteins shown here that expression from the novel open reading frames is detectable during exponential growth and stationary phases in E. coli; however, rnpA’-SPA appears to be down-regulated during overnight/death phase. This supports the theory that genes are expressed during specific times in the life cycle of an organism and serves as the first clue as to the potential role these novel proteins play in vivo.
Due to the lack of efficient identification techniques for sORFs, it is very possible that numerous small genes are yet to be discovered within the genomes of many organisms. It is also plausible that small proteins play a role in previously characterized cellular mechanisms or could bridge unknown relationships between different cellular signaling cascades. The results of these experiments, and others like it, imply the need to re-evaluate how genomes are annotated and the role potential small proteins play in the inner workings of the cell.
In recent years the presence and importance of small proteins, proteins made up of amino acids, has become clear. Escherichia coli (E. coli), a well-studied model organism, contains thousands of un-explored short open reading frames (sORFs) that may code for small proteins of interest.  Identifying novel genes that are expressed in E. coli is important because it opens the door to the exploration of how their protein products function in both bacteria and perhaps higher organisms; however, the size and location (intragenic and intergenic) of the sORFs makes expression analysis difficult. Our study focused on analyzing intragenic open reading frames (iORFs) that reside within known functional genes in E. coli. iORFs that were both in-frame (f0) and out-of-frame (f1) or (f2) were found to express small proteins during different stages of bacterial growth. 1
Figure 1. rnpA’. The start triplet for the iORF rnpA’ is located in the 3’ region of the known gene, rnpA. rnpA’ is an f(2) frame-shift with respect to rnpA, resulting in a novel ORF sequence that does not correlate with other genes within the operon, including rnpA and yidC.
Figure 2. Expression of rnpA’-SPA fusion protein. a) Immunoblot analysis using α-FLAG reveals differential expression of rnpA’-SPA (~17kDa) in total cell lysates. Exponential and stationary phase conditions are shown in duplicate. b) rnpA’-SPA fusion protein expression is markedly down-regulated in the overnight (death phase) sample.
Background
For years, molecular biologists, bio-informaticians and biochemists have developed methods for discovering genes and analyzing the associated gene products. Indeed, many genomes have been sequenced and proteins identified, to date. However, the proteomes of several organisms, including E. coli, are not fully annotated because very small proteins (16-50 amino acids) have been largely ignored.
Being a prokaryotic organism, the E. coli genome is relatively simple and despite the presence of Group II Introns (Marcia et al., 2012), conventional thinking is loath to abandon the one gene-one protein hypothesis of old in prokaryotes. Our research focused specifically on intragenic open reading frames of annotated (parent) genes in E. coli. That is, open reading frames that have start codons downstream from the parental start and that may be in-frame, out-of-frame or altogether embedded within the parent gene. Past research on microsatellites highlights the presence of mono-, di-nucleotide repeats in the coding regions of genes, which alters expression. Therefore, the study of alternate reading frames is cogent, as they occur naturally (da Silva et al., 2000).
A diagnostic protocol that labels and detects small proteins in E. coli has recently been drafted (Hemm et al., 2008). Based on this principle, we utilized a multi-tiered bioinformatics approach to select iORFs that may code for protein products smaller than 50 amino acids. The collective effort presented here culminated in the true discovery of novel genes in E. coli. It creates the opportunity for future characterization studies and summarizes the opportunity for undergraduates to engage in primary, biomedical research as part of the curriculum.
dsbB-SPA
dsbB’-SPA
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References
Hemm, Matthew R. et al. “Small Membrane Proteins Found by Comparative Genomics and Ribosome Binding Site Models.” Molecular microbiology 70.6 (2008): 1487–1501. PMC. Web. 13 Apr
Marcia, Marco, and Anna Marie Pyle. “Visualizing Group II Intron Catalysis through the Stages of Splicing.” Cell (2012): 497–507. PMC. Web. 13 Apr
Baisnée, Pierre-François, et al. "Flexibility of the genetic code with respect to DNA structure."Bioinformatics 17.3 (2001):
da Silva, Elizabeth Fidalgo, and Linda J. Reha-Krantz. "Dinucleotide repeat expansion catalyzed by bacteriophage T4 DNA polymerase in vitro." Journal of Biological Chemistry  (2000):
Diamant, Eran et al. “Phylogeny and Strain Typing of Escherichia Coli, Inferred from Variation at Mononucleotide Repeat Loci.” Applied and Environmental Microbiology 70.4 (2004): 2464–2473. PMC. Web. 19 Apr
VanOrsdel, CE et al. “The Escherichia coli CydX protein is a member of the CydAB cytochrome bd oxidase complex and is required for cytochrome bd oxidase activity.” J Bacteriol (2013): 3640 –3650 . PMC. Web. 19 Apr
Figure 3. Expression of the f(0) dsbB’-SPA fusion protein. Immunoblot analysis using α-FLAG reveals expression of the smaller dsbB’-SPA protein. Also detected is expression from the parent gene, dsbB-SPA.
YbaM 205 Expo.
YbaM 205 Stat.
YbaM 194 Expo
YbaM 194 Stat.
Control
Figure 4. Expression of the f(2) ligB’-SPA fusion protein. Immunoblot analysis using α-FLAG reveals differential expression of ligB’-SPA in total cell lysates. Exponential and stationary phase conditions are shown in duplicate.
Acknowledgements
This research was funded by the Jess and Mildred Fisher College of Science and Mathematics undergraduate research grant, and a NSF research grant received by Dr. M. Hemm.