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A novel pH-regulated, unusual 603 bp overlapping protein coding gene pop is encoded antisense to ompA in Escherichia coli O157:H7 (EHEC)

Antisense transcription is well known in bacteria. However, translation of antisense RNAs is typically not considered, as the implied overlapping coding at a DNA locus is assumed to be highly improbable. Therefore, such overlapping genes are systematically excluded in prokaryotic genome annotation. Here we report an exceptional 603 bp long open reading frame completely embedded in antisense to the gene of the outer membrane protein ompA. Ribosomal profiling revealed translation of the mRNA and the protein was detected in Western blots. A σ70 promoter, transcription start site, Shine-Dalgarno motif and rho-independent terminator were experimentally validated. A pH-dependent phenotype conferred by the protein was shown in competitive overexpression growth experiments of a translationally arrested mutant versus wild type. We designate this novel gene pop (pH-regulated overlapping protein-coding gene). Increasing evidence based on ribosome-profiling indicates translation of antisense RNA, suggesting that more overlapping genes of unknown function may exist in bacteria.


Introduction 29 30
Due to the nature of the genetic triplet code, six reading frames exist on the two strands of a 31 DNA molecule. Two genes encoded by two different reading frames at the same DNA locus 32 are called overlapping genes (OLGs). Most overlaps found in bacterial genomes are very 33 short; the majority are only 1 or 4 bp in same-strand orientation (Johnson and Chisholm,34 2004) and such overlaps seem to increase fitness (e. g. Saha et al., 2016). In our paper, 35 however, we focus on a non-trivial, extensively overlapping open reading frame (ORF) fully 36 embedded in antisense to an annotated gene. Non-trivially overlapping ORFs in alternative 37 reading frames of the coding strand were discovered in bacteriophage X174 by Barrell et 38 al. as early as 1976. Today, the existence of overlapping genes is accepted in viruses, 39 although the driving force for developing gene overlaps is still debated, with theories about 40 size constraint of the genome in the viral capsid, gene novelty, or evolutionary exploration 41 being discussed (Brandes andLinial, 2016, Chirico et al., 2010). 42 43 Very little work has been devoted to the exploration of overlapping genes in prokaryotes 44 (Ellis andBrown, 2003, Rogozin et al., 2002). As bacterial genomes are typically much larger 45 than those of viruses, the original hypothesis for the selection pressure associated with the 46 evolution of overlapping genes, namely, increasing the coding capacity in size-restricted 47 genomes (Normark et al., 1983), is not valid for prokaryotes. This may be one reason for the 48 lack of knowledge about these amazing gene constructs in bacteria. Furthermore, overlaps 49 of more than 30 amino acids (i.e., non-trivial overlaps) are systematically excluded in 50 genome annotations (Warren et al., 2010). Nevertheless, statistical analysis of bacterial 51 genomes showed that ORFs overlapping annotated genes in alternative reading frames are 52 longer than expected, leading to the hypothesis of a possible selection pressure due to 53 overlapping protein-coding genes (Mir et al., 2012). At least some evidence for functionality 54 of a few non-trivially overlapping genes has been presented (e. g. Balabanov et al., 2012, 55 Behrens et al., 2002). However, the task of functionally characterizing overlapping genes is 56 challenging. It is assumed that these genes originated by overprinting of existing, annotated 57 genes (Sabath et al., 2012) and may constitute an evolutionarily young part of the functional 58 genome of bacteria (Fellner et al., 2014, Fellner et al., 2015. As such, weak expression as 59 well as non-essential function rather than strong phenotypes are expected in general, and 60 sensitive methods are necessary to uncover phenotypic effects caused by the expression of 61 non-trivially overlapping genes. 62

63
The human pathogenic bacterium Escherichia coli O157:H7 (EHEC) and its genome are well 64 characterized, especially with respect to virulence and the associated diseases like 65 enterocolitis, diarrhea and hemolytic uremic syndrome (Betz et al., 2016, Lim et al., 2010 Stevens and Frankel, 2014). However, the coding capacity of EHEC's genome is likely to be 67 significantly underestimated, both regarding short intergenic genes (Hücker et  Additionally, using a variety of different next generation sequencing based methods (e.g., 71 RNAseq, Cappable-seq, ribosome profiling) evidence has accumulated for widespread 72 antisense transcription and probable translation of these RNAs (Conway et al., 2014, 73 Meydan et al., 2019). If true, this finding would be very significant. Nevertheless, the 74 specificity of the signals found in NGS is sometimes unclear since pervasive translation may 75 occur (Ingolia et al., 2014). To gather further evidence for an overlapping coding potential, 76 individual overlapping genes have to be characterized in detail. Such research is in its 77 infancy in bacteria even though, as noted, at least a few overlapping genes have been 78 defined in recent years. 79 Here, we report on a functional analysis of the unusually long, non-trivially overlapping gene 80 pop from E. coli O157:H7 strain EDL933, which is completely embedded in antisense to the 81 coding sequence of the conserved outer membrane protein ompA. GenBank accession CP008957) and has a length of 603 bp (Fig. 1, Fig. S1). It is completely 90 embedded in antisense to the coding sequence of the annotated, highly conserved outer 91 membrane protein gene ompA (1065 bp), which probably has multiple functions, among 92 which is a porine function and a local cell wall stabilizing action through interaction of OmpA 93 with TolR (Boags et al., 2019). pop is located in frame -1 with respect to ompA (Fig. 1 A). 94 Ribosome profiling of EHEC EDL933 revealed clear evidence of translation of this 95 overlapping gene in LB medium, which is reproducible across biological replicates (Fig. 2 A,  96 table S1) and EHEC strains (Fig. 2 A-C, see below). Expression of ompA is about 150 times 97 higher than pop, which is not surprising since OmpA is one of the most highly expressed plasmid and this single base substitution introduces a stop codon in the overlapping gene 148 ( Fig. 1 A) which is neutral in the mother frame of ompA. Thus, any difference in growth after 149 overexpression of either the intact or the mutated pop-ORF can be explained by the 150 presence or absence of a protein encoded by this overlapping gene. 151

152
The competition experiment was conducted in different stress conditions (Fig. 4 A). Altered 153 growth of cells overexpressing mutant or wild type sequences was detected in LB-based 154 media supplemented with different stressors, whereas plain LB medium did not have a 155 significant influence on the relative growth of mutant and wild type. For instance, addition of 156 the organic acids L-malic acid and malonic acid as stressors led to better growth of cells 157 containing the wild type plasmid compared to cells expressing the mutated sequence, thus, 158 the presence of pop is advantageous in this condition. Addition of the acidic substances 159 resulted in an initial pH shift from 7.4 to 5.8. Reversed proportions were detected when LB 160 was buffered with bicine to a pH of 8.7. However, LB adjusted to acidic (pH = 5.8) or near 161 neutral (pH = 7.4) milieu with the biologic buffers MES and MOPS, respectively, did not result 162 in significant growth differences. Nevertheless, a slightly increased proportion of wild type 163 cells can be seen for MES-buffering, which is in line with expectations based on results in the 164 other acidic conditions. In accordance with the growth advantage of the wild type in the 165 presence of malic acid (Fig. 4 A), the mRNA of pop was upregulated in the presence of this 166 acid (Fig. 4 B). 167 168 Next, a genomic knock-out for pop in E. coli O157:H7 EDL933 was constructed. As before, a 169 single base substitution led to a stop codon in pop, whereas amino acids in ompA remained 170 unchanged ( Fig. 1 A). We tested the mutant Δpop in several relevant stress conditions in 171 competitive growth against the wild type strain, but did not detect a significant difference in 172 growth in any condition (Fig. S2). 173 Based on the clear effect of overexpression, we propose that pop codes for a protein, as 175 mRNAs transcribed from the intact sequence and the translationally arrested variant differ in 176 one nucleotide only. Opposite overexpression phenotypes were found in alkaline buffered 177 and acidified media, so we propose a pH-dependent function. In line with this hypothesis, the 178 mRNA was increased in acidified conditions (Fig. 4 B).  Fig. 1 B, Fig. S1). Although the distance 191 between the transcriptional start site and -10 box of the promoter is not optimal (2 bp instead 192 of approx. 7 bp), promoter sequence activity was verified by means of a GFP-assay ( Fig. 5  193 A). We found a significantly enhanced fluorescence in cells harboring the plasmid containing 194 the putative promoter sequence compared to those with the empty vector in LB and bicine-195 buffered medium. The fluorescence signal of the promoter in the basic milieu (pH 8.7) was 196 strikingly higher, but this may result from GFP accumulation during longer incubation times 197 necessary in this medium (Miller et al., 2000). 198

199
Since the promoter activity for pop is weak compared to promoters of annotated genes, we 200 tested for polycistronic expression starting from the promoter of ycbG. Reverse transcription 201 PCR (RT-PCR) was performed to examine the transcript of pop (Fig. 5 B). No mRNA 202 spanning both genes was detectable, thus, we propose that pop is transcribed from the 203 tested promoter monocistronically. 204

205
A 120 bp long rho-independent terminator was predicted 295 bp downstream of the stop 206 codon of pop using FindTerm (Solovyev and Salamov, 2011). Hypothetical secondary 207 structures of 30-bp segments of this region were created with the tool Quickfold of Mfold 208 (Zuker, 2003). A stable stem loop structure (ΔG = -8.6 kcal/mol) within bases 35 to 78 of the 209 predicted terminator sequence was detected (Fig. 1 D). To verify the 3'-end of the mRNA 210 downstream of the hairpin structure, RT-PCRs were performed. We used reverse primers 211 binding either within the downstream ORFs or further downstream, beyond the secondary 212 structure. We observed that pop and the downstream ORFs are co-transcribed and 213 transcription is terminated just downstream of the predicted stem loop structure (Fig. 5 C). Since we detected an active promoter (Fig. 5 A) and phenotypes in competitive 223 overexpression experiments (Fig. 4), the coding capacity of pop was assessed using 224 Western blotting. pop was cloned in-frame with an SPA tag (7.7 kDa; following Zuker, 2003) 225 on a pBAD-based plasmid, and overexpressed in EHEC. SPA-tagged proteins were 226 visualized after separating whole cell lysates on tricine gels. The experiment was performed 227 in two different culture conditions (LB at pH 7.4 and bicine-buffered LB at pH 8.7; Fig. 6). 228 Besides the expected full-length protein (theoretically 30 kDa, detected approx. 34 kDa), 229 shorter products were immunostained (approx. 20 kDa and 24 kDa). The amount of the full 230 Pop-protein increases within the first 1.5 h after induction and decreases afterwards when 231 overexpressed in LB ( Fig. 6 A), pointing to an instability of the protein in this condition. In 232 contrast, in bicine-buffered medium, protein expression is somewhat higher at the beginning 233 and does not appear to decrease with time ( Fig. 6 B). A more detailed evaluation of the 234 smaller products revealed a constant increase in band intensities over time in LB medium, in 235 contrast to a constant signal in bicine. Summarizing, differential pop expression is detected 236 by Western blots when growth in LB and bicine is compared. of 4 -91 ) and the genomic sequence of the target organism Shigella sonnei showed that its 247 ompA homolog was not annotated due to ambiguous bases at its 5' end, which resulted in a 248 missing start codon for ompA in this case. Consequently, pop was 'allowed' to be predicted 249 ab initio as ompA had no obvious gene structure and was, therefore, rejected during 250 annotation. This result corroborates the known function of many algorithms like Glimmer, 251 Prodigal or Prokka, which systematically avoid annotation of long overlapping genes 252 In the ribosome profiling data, we identified three peak regions; a putative start codon of pop 306 could be contained in each of these (regions 1-3 in Fig. 2 A, Fig. S1). All regions are 307 covered with a substantial number of ribosomal profiling reads, and region 2 is covered best, 308 particularly in EHEC EDL933. We propose that translation for pop starts in region 2, 309 especially since a Shine-Dalgarno motif for ribosome binding was predicted and ribosome 310 profiling data across divergent strains point to a putative translation initiation site therein. As 311 mentioned, a nearby CTG is found downstream of the ribosome binding site, representing a 312 rare but sometimes used start codon for prokaryotes (Yamamoto et  Furthermore, a TTG start codon is present in region 1, representing the longest potential 316 ORF for pop. However, we could not find evidence for a TSS or SD-sequence, though the 317 latter is not obligatory for gene expression Pon, 2015, Moll et al., 2002). 318 Nevertheless, this TTG was the start codon predicted by Prodigal as the most probable one. 319 Although it has no transcription start site and bicistronic expression along with the upstream 320 gene was excluded in the tested condition, studies showed that read-through of a terminator 321 is common in bacteria in stress conditions (reviewed in Santangelo and Artsimovitch, 2011). 322 Therefore, this TTG start codon could serve as a stress-induced initiation site, which 323 depends on read-through of the transcription termination site of ycbG, thus making a TSS 324 unnecessary. To test this hypothesis, future studies using Term-Seq might unveil a 325 transcriptional regulation at the RNA level (Dar et al., 2016).

Creation of translationally arrested knock-out mutants 414
The genomic knock-out E. coli O157:H7 EDL933 pop was produced for subsequent 415 competitive growth experiments. Mutation fragments were amplified with primer pairs 1/4 and 416 2/3 and used in the subsequent overlap extension PCR with primers 3 and 4. The mutation 417 cassette pop was cloned in the plasmid pMRS101 using ApaI and SpeI (selection with 418 ampicillin). The plasmid pMRS101+pop was isolated and sequenced with primer 7 and 8. A 419 restriction digest with NotI was conducted to remove the high copy ori. The plasmid was re-420 ligated to the pir-protein dependent low copy plasmid pKNG101+pop, propagated into E. constructed. For the former construct, primers 9 and 10 were used. The latter construct was 439 created similar to the mutation cassette described in the previous section (primers for the 440 mutation fragments 1/9 and 2/10, primers for the mutation cassette: 9 and 10). Both PCR 441 fragments, either wild type or mutant, were cloned in the NcoI and PstI sites of pBAD/myc-442 HisC and plasmids were sequenced with primers 12 and 13. Each of the plasmids was 443 transformed in wild type E. coli O157:H7 EDL933 for subsequent competitive growth assays. were calculated. Statistically significant differences in the fluorescence of promoter construct 504 and empty plasmid or between promoter constructs in different growth conditions were 505 determined using the Welch two sample t-test (significant result for p-value ≤ 0.05). 506

RNA isolation 508
RNA was isolated from exponentially grown EHEC cultures (OD600 = 0.3) using Trizol 509 Reagent (Thermo Fisher Scientific). Cell pellets were resuspended in 600 µl cooled Trizol 510 and cells were disrupted with bead beating using a FastPrep (3-times at 6.5 ms -1 , 45 s, rest 511 5 min on ice between the runs). Cooled Chloroform (120 µl) was added, mixed vigorously 512 and incubated 5 min at room temperature. Phases were separated by centrifugation for 513 15 min (4 °C, 12000×g) and total RNA in the aqueous upper phase was precipitated with 514 isopropanol, NaOAc and glycogen (690 µl, 27 µl, and 1 µl, respectively) at -20 °C for 1 h. ribosomal and tRNAs were removed using bedtools (Quinlan and Hall, 2010). Read counts, 573 RPKMs, and coverage were then calculated with respect to the filtered BAM files, using 574 bedtools and a custom bash script. 575 Stalled-ribosome profiling data from the E. coli strain BL21 was obtained from Meydan et al. 576 (2019). The adapter sequence was predicted using DNApi.py (Tsuji and Weng, 2016), and 577 adapter trimming, alignment, and removal of rRNAs and tRNAs was conducted as described 578 above. The positions of all reads mapped to the forward strand were obtained using 579 SAMtools (Li et al., 2009) and the "bamtobed" tool from BamTools (Barnett et al., 2011). 580 Reads with predicted ribosomal p-sites within 30 nucleotides in each direction of an 581 annotated forward-strand gene start codon ("start region") were extracted. Weakly expressed 582 annotated genes with no single position (peak) represented by three or more reads, and also 583 with at least four reads situated within the start region, were found using a custom bash 584 script.

Acknowledgements. 594
We thank Romy Wecko for excellent technical assistance and Christopher Huptas for his 595 support in gene prediction. We thank Sarah Hücker for providing ribosomal profiling data for 596 the two biological replicates of E. coli O157:H7 Sakai. This work was supported in part by the 597 Deutsche Forschungsgemeinschaft (DFG) to S.S. (SCHE316/3-1,2,3). 598 599

Competing interests 600
The authors declare no competing interests.