Jean-François Bretagne
email: jf.bretagne@gmail.com

MD Aurore Carlo

MD Christine Piette

BS Chloé Rousseau

MD Mathilde Cosson

Astrid Lievre

MD Jean-François Bretagne

MD, PhD Astrid Lièvre

J.-F Bretagne

Significant decrease in interval colorectal cancer incidence after implementing immunochemical testing in a multiple-round guaiac-based screening programme

Keywords: interval colorectal cancer, colorectal cancer screening, faecal immunochemical test, guaiac faecal occult blood test

Significant decrease in interval colorectal cancer incidence after implementing immunochemical testing in a multiple-round guaiac-based screening programme

INTRODUCTION

Colorectal cancer (CRC) remains the second leading cause of cancer death worldwide, despite the declining mortality rates in North America and Northwest Europe. [START_REF] Arnold | Global Burden of 5 Major Types of Gastrointestinal Cancer[END_REF][START_REF] Sung | Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF] In the latter areas, screening programmes have been widely implemented, but death can occur when patients are not screened or inadequately followed up or when the screening test has failed. [START_REF] Doubeni | Modifiable Failures in the Colorectal Cancer Screening Process and Their Association With Risk of Death[END_REF] One of the ways in which failure is expressed is through the emergence of interval colorectal cancers (In-CRC); In-CRC has been defined internationally as CRC diagnosed after a screening or surveillance examination in which no cancer was detected but before the next recommended examination date. [START_REF] Sanduleanu | Definition and taxonomy of interval colorectal cancers: a proposal for standardising nomenclature[END_REF] For screening with a biennial faecal occult blood test (FOBT), In-CRC is defined as a cancer detected after a negative screening test and before the next scheduled invitation two years later. [START_REF] Sanduleanu | Definition and taxonomy of interval colorectal cancers: a proposal for standardising nomenclature[END_REF] The rate of occurrence is strongly correlated with the sensitivity of the screening test used and reflects the quality of the screening programme. [START_REF] Moss | European guidelines for quality assurance in colorectal cancer screening and diagnosis. First Edition -Evaluation and interpretation of screening outcomes[END_REF] Faecal immunochemical testing (FIT), compared with guaiac-FOBT (gFOBT), has been shown to increase participation and to increase cancer detection in randomized controlled trials and population-based screening programmes. [START_REF] Tinmouth | Faecal immunochemical tests versus guaiac faecal occult blood tests: what clinicians and colorectal cancer screening programme organisers need to know[END_REF][START_REF] Robertson | Recommendations on Fecal Immunochemical Testing to Screen for Colorectal Neoplasia: A Consensus Statement by the US Multi-Society Task Force on Colorectal Cancer[END_REF][START_REF] Senore | Performance of colorectal cancer screening in the European Union Member States: data from the second European screening report[END_REF] The evidence that FIT decreases the incidence of In-CRC compared to gFOBT is based on comparison of studies where a single test was used or where the analysis of gFOBT and FIT arms was undertaken. [START_REF] Wieten | Incidence of faecal occult blood test interval cancers in population-based colorectal cancer screening: a systematic review and meta-analysis[END_REF] However, little is known about the benefits of implementing FIT in an already established gFOBT-based screening programme, especially in regard to the incidence and characteristics of In-CRCs, which are data that may be important in the context of test switching in countries where gFOBT is still offered as the first-line screening test. [START_REF] Senore | Performance of colorectal cancer screening in the European Union Member States: data from the second European screening report[END_REF][START_REF] Cardoso | Utilisation of Colorectal Cancer Screening Tests in European Countries by Type of Screening Offer: Results from the European Health Interview Survey[END_REF] Therefore, the aim of the present study was to retrospectively evaluate the benefits of switching from gFOBT to FIT in a long-term, 6-round, gFOBT-based screening programme. We have previously reported on the increased participation and diagnostic yield of such a switch, [START_REF] Bretagne | Switching from guaiac to immunochemical faecal occult blood test increases participation and diagnostic yield of colorectal cancer screening[END_REF] but in this study, we sought to evaluate the incidence rates and characteristics of In-CRCs recorded after one FIT-based round and to compare them with those observed after each of the previous two gFOBT-based rounds.

POPULATION AND METHODS

The screening programme and study population

In 2003, the French national screening programme based on a biennial gFOBT (Hemoccult II;

Beckman Coulter Inc., Villepinte, France) was fully implemented in 'Ille-et-Vilaine' in western France.

CRC screening was proposed for individuals aged between 50 and 74 years with an average risk of CRC and no contraindications for colonoscopy. Individuals with a personal or family history of CRC or adenoma, individuals with inflammatory bowel disease, and individuals who had undergone total colonoscopy in the previous five years were excluded from the mass screening programme. The target population of the CRC screening programme was defined as the population aged 50 to 74 years minus the excluded individuals.

The transition from gFOBT to FIT occurred in 'Ille-et-Vilaine' in May 2015; the FIT selected by the French Health Authorities was the OC-Sensor™ test (Eiken, Tokyo, Japan). Thus, six rounds of gFOBTbased screening and one round of FIT-based screening were performed between 2003 and 2017.

gFOBT was defined as positive when one to six square(s) were positive, and a cutoff value of 30 µg of haemoglobin per gram of faeces (i.e., 150 ng of haemoglobin per milliliter of buffer) was used for FIT positivity. All positive individuals were recommended to undergo a colonoscopy, while those with negative tests were informed to participate again every two years until they were 74 years old. More details on the screening process data have been reported previously. [START_REF] Bretagne | Switching from guaiac to immunochemical faecal occult blood test increases participation and diagnostic yield of colorectal cancer screening[END_REF][START_REF] Piette | Additional mailing phase for FIT after a medical offer phase: The best way to improve compliance with colorectal cancer screening in France[END_REF] Because the 6th gFOBT-based round was somewhat shortened due to national public health decisions related to test switching, we also chose to include the 5th round in the present study for comparison with the 7th FIT-based round.

Data collection and outcome measures

The screening centre (ADECI 35) was responsible for recording exclusions, inviting the target population, and reminding them to enroll in the study if they did not participate after an initial letter.

The centre also prospectively recorded all data regarding participation, FOBT results, and if positive, colonoscopy compliance and findings. Lesions found during colonoscopy and the corresponding treatment were prospectively recorded in the database, and these data were submitted by the gastroenterologists. Since the implementation of CRC screening in 2003, all pathology laboratories in the district and adjacent districts were required to send the details (first name, last name, date of birth) of all patients 50 years of age and older diagnosed with CRC to the screening centre, allowing the screening centre to cross-reference all CRCs diagnosed in patients with a negative test in 'Ille-et-Vilaine'. An In-CRC was defined as a CRC that was noted clinically within two years in any one of the individuals with a negative test, gFOBT or FIT. Details of In-CRC patients and their tumours (location, stage) were collected retrospectively from general practitioners, gastroenterologists and surgeons.

The age of patients with screen-detected CRCs (SD-CRCs) and In-CRCs was classified into 3 categories: <60 yrs, 60-69 yrs, and ≥70 yrs. Tumours were categorized according to the TNM staging system from stage 0 to IV, according to the UICC/AJCC classification. [START_REF]The TNM classification of malignant tumors, 8th edn[END_REF] Stage 0 tumours corresponded to neoplasms with intramucosal infiltration, while intraepithelial tumours were excluded from the present study. The location of tumours was classified into 3 categories: proximal for tumours located beyond the splenic flexure, distal for those located between the rectosigmoid junction and the splenic flexure, and the rectum.

The main outcomes were the cumulative 2-yr incidence of In-CRCs, the In-CRC rate and the sensitivity of the test used, gFOBT or FIT. The cumulative incidence of In-CRCs was expressed as the number of

In-CRCs per 100 000 persons-years among those with a negative test. The In-CRC rate was calculated by dividing the number of In-CRCs by the sum of SD-CRCs and In-CRCs while FOBT sensitivity was calculated by dividing the number of SD-CRCs by the sum of SD-CRCs and In-CRCs.

The secondary outcomes were a comparison of the characteristics of In-CRCs between the 3 rounds and with those of SD-CRCs. Moreover, we aimed to assess the consequences of lowering the threshold of positivity on FIT performance.

Statistical analysis

Quantitative The rate ratios and corresponding 95% CIs were used to express the association between the studied factors and the In-CRC cumulative incidence. We sought to determine the sensitivity of the FIT if the screening programme had adopted a lower test positivity threshold of 20 and 10 µg/g, so that we could compare our results with those of screening programmes that had adopted such thresholds.

The number of individuals needed to undergo colonoscopy was also determined to find one more cancer at these latter thresholds. All analyses were performed using the statistical software programme SAS, version 9.4 (SAS Institute, Inc., Cary, North Carlina, USA). A test was interpreted as significant if the p value was <0.05. All authors had access to the study data and reviewed and approved the final manuscript.

RESULTS

Population of screen-detected and interval cancers

The main characteristics of the 3 rounds considered in the analyses are given in Table 1. A total of 279 041 tests were performed by 156 186 individuals in rounds 5, 6 and 7. There were 612 cases of SD-CRC and 209 cases of In-CRC, for a total of 821 cases of CRC. The comparative characteristics of the two types of cancers are given in Table 2. When compared with SD-CRCs, In-CRCs were more likely to be proximal and rectal tumours, were less likely to be distal tumours, and were more often advanced cancers (stage III/IV). In addition, among patients with In-CRC, the proportion of women was increased, and the proportion of individuals participating for the first time was less than that among SD-CRC patients (Table 2).

Cumulative incidence of interval cancers

The number of In-CRCs was 99, 62 and 48 in rounds 5, 6 and 7, respectively, corresponding to cumulative 2-year incidence rates of 55. except for the first screening category in the fifth round (Table 5). The gFOBT sensitivity estimates did not differ between the two gFOBT-based rounds, except for the distal location of CRCs (data not shown). After excluding stage 0, CRCs from the total population did not change the comparative results of FIT and gFOBT sensitivities, as shown by the odds ratios of 6.9 [95% CI, 4.4-10.9] and 7.5

[95% CI, 4.5-12.6] for rounds 5 and 6, respectively.

Characteristics of interval cancers compared between gFOBT-and FIT-based rounds

As expected because most participants at each round had already been tested in the previous rounds, the age of patients at the time of testing and In-CRC diagnosis, as well as the screening rank, were higher in the FIT-based round than in the previous two rounds (Table 6). However, there were no significant differences between the 3 rounds in the proportion of never-screened versus previously screened participants, in the time between negative testing and In-CRC diagnosis, in the proportion of In-CRCs diagnosed in the first or second year after negative testing, or in the distribution of cancer location and stage (Table 6).

Consequences of lowering the cutoff value for FIT positivity

If a lower threshold of positivity had been chosen by the programme, it can be presumed that 8 cases of In-CRC at the threshold of 20 µg/g and another 15 at the threshold of 10 µg/g would have been detected at an earlier stage. At the threshold of 20 µg/g FIT, sensitivity would have increased from

DISCUSSION

The primary objective of the present study was to evaluate the benefit of switching from gFOBT to FIT in a long-term, 6-round, gFOBT-based screening programme on the In-CRC incidence rate. We previously reported that the switch from gFOBT to FIT was responsible for a fivefold increase in the detection of cancer, an increased participation of nearly 20%, a higher test positivity rate, and a higher positive predictive value for cancer; [START_REF] Bretagne | Switching from guaiac to immunochemical faecal occult blood test increases participation and diagnostic yield of colorectal cancer screening[END_REF] these results are consistent with those from other screening centres in France. [START_REF] Koïvogui | Detection of Colorectal Neoplasia in a Cohort Before and After the Change of Faecal Occult Blood Test in a French Colorectal Cancer Screening Program[END_REF][START_REF] Vitellius | Contribution of the OC Sensor® immunoassay in comparison to the Hemoccult II® guaiac-test in organized colorectal cancer screening[END_REF] In England, a pilot study with FIT within the established national screening programme also reported markedly improved participation and neoplasia detection, but In-CRCs were not assessed in that study. [START_REF] Moss | Increased uptake and improved outcomes of bowel cancer screening with a faecal immunochemical test: results from a pilot study within the national screening programme in England[END_REF] Thus, a decrease in the occurrence of In-CRC after switching was expected, but this remains to be proven in a mature programme. 100 000 person-years for FIT and gFOBT, respectively. [START_REF] Wieten | Incidence of faecal occult blood test interval cancers in population-based colorectal cancer screening: a systematic review and meta-analysis[END_REF] Of note, our findings for FIT were also very similar to those recently reported in Italy based on a population followed for 13 years with FIT repeated every two years (1.87 per 10 000 person-years). [START_REF] Zorzi | Interval colorectal cancers after negative faecal immunochemical test in a 13-year screening programme[END_REF] To our knowledge, the only previous study that reported a benefit of switching from gFOBT to FIT on the incidence of In-CRC within a screening programme is a Belgian study, in which the incidence rates for gFOBT and FIT In-CRCs were higher at 21.3 and 4.9 per 10 000 person-years, respectively. [START_REF] Guo | Benefits of switching from guaiac-based faecal occult blood to faecal immunochemical testing: experience from the Wallonia-Brussels colorectal cancer screening programme[END_REF] However, whereas in the Belgian study the comparison of both tests was made on the occasion of a first screening, our study is more consistent with real life, with a large majority of people already screened among the participants at each round analyzed here. In the Belgian study, [START_REF] Guo | Benefits of switching from guaiac-based faecal occult blood to faecal immunochemical testing: experience from the Wallonia-Brussels colorectal cancer screening programme[END_REF] the incidence rate ratio between FIT-and gFOBT In-CRCs was 0.23 versus 0.36, respectively, in the meta-analysis that analyzed three studies that compared gFOBT and FIT in two arms. [START_REF] Wieten | Incidence of faecal occult blood test interval cancers in population-based colorectal cancer screening: a systematic review and meta-analysis[END_REF] In the present study, the incidence rate ratio between FITand gFOBT In-CRCs was 0.38 [95% CI, 0.27-0.54] when taking round 5 as the reference in the multivariable analysis. Factors significantly associated with the In-CRC incidence rate in our study, other than the test, were increasing age, sex (men over women), and rank of screening (subsequent over first screening). However, when analyzing the FIT-based round separately, the only significant factors were increasing age, season for screening, and faecal haemoglobin level. The role of aging has already been demonstrated. [START_REF] Wieten | Incidence of faecal occult blood test interval cancers in population-based colorectal cancer screening: a systematic review and meta-analysis[END_REF][START_REF] De Klerk | Participant-Related Risk Factors for False-Positive and False-Negative Fecal Immunochemical Tests in Colorectal Cancer Screening: Systematic Review and Meta-Analysis[END_REF][START_REF] Wong | Factors associated with false-positive and false-negative fecal immunochemical test results for colorectal cancer screening[END_REF][START_REF] Stegeman | Risk factors for false positive and for false negative test results in screening with fecal occult blood testing: Risk factors for false positive and false negative test[END_REF] Seasonal variations in the In-CRC risk were reported in a Korean study with different FITs, including the OC-Sensor™ test at a cut-off of 20 µg/g, with an odds ratio of 1.31 for the summer months, highlighting the role of temperature on haemoglobin stability. [START_REF] Cha | Risk of Interval Cancer in Fecal Immunochemical Test Screening Significantly Higher During the Summer Months: Results from the National Cancer Screening Programme in Korea[END_REF] We have to be careful when interpreting our results because of the very wide confidence intervals. Other studies conducted in France, such as ours (data not shown), have not shown seasonal variations in the rate of FIT positivity, but the rate of In-CRC has not been investigated in these studies. [START_REF] Chausserie | Seasonal variations do not affect the superiority of fecal immunochemical tests over guaiac tests for colorectal cancer screening: Seasonal variation in FIT performance[END_REF][START_REF] Dancourt | Influence of sample return time and ambient temperature on the performance of an immunochemical faecal occult blood test with a new buffer for colorectal cancer screening[END_REF] Unlike the cumulative incidence rate, which was calculated from negative tests, the calculation of the In-CRC rate depended on the number of SD-CRCs and consequently on the test positivity rate, its positive predictive value for CRC diagnosis and the colonoscopy compliance. In the present study, the In-CRC rate was quite similar in the two gFOBT-based rounds (44.0%, 44.6%), i.e., approximately 4-fold higher than in the FIT-based round (10.5%). In our gFOBT-based screening programme that started in 2003, the In-CRC rate was 33% in the first round and 40% in the second round (data not shown). Very similar data were recorded thereafter, highlighting the role that the switch from gFOBT to FIT plays in the dramatic decrease reported here. Because the sensitivity of the screening test is an inverse function of the interval cancer rate, the age-and sex-adjusted sensitivity estimates in this study were 54.5% and 55.4% for gFOBT and 88.6% for FIT. In two recent metaanalyses, FIT sensitivity estimates for CRC detection ranged from 69% to 80% and 77% to 94% depending on the positivity threshold. [START_REF] Selby | Effect of Sex, Age, and Positivity Threshold on Fecal Immunochemical Test Accuracy: A Systematic Review and Metaanalysis[END_REF][START_REF] Imperiale | Performance Characteristics of Fecal Immunochemical Tests for Colorectal Cancer and Advanced Adenomatous Polyps: A Systematic Review and Meta-analysis[END_REF] Corresponding values at the cutoff of 15 µg/g were 84% and 90.5% in two recent studies from Belgium and the Netherlands. [START_REF] Guo | Benefits of switching from guaiac-based faecal occult blood to faecal immunochemical testing: experience from the Wallonia-Brussels colorectal cancer screening programme[END_REF][START_REF] Toes-Zoutendijk | Incidence of Interval Colorectal Cancer After Negative Results From First-Round Fecal Immunochemical Screening Tests, by Cutoff Value and Participant Sex and Age[END_REF] In the present study, we showed that lowering the positivity threshold from 30 to 10 µg/g faeces would have transformed 48% of In-CRCs (23 cases) into as many SD-CRCs and thus increased the sensitivity of FIT for CRC by 5%. Nevertheless, detection of these 23 cases would have required more than 5000 colonoscopies, i.e., more than what was needed to detect the 409 cancers at a positivity threshold of 30 µg/g, which seems disproportionate and validates a posteriori the choice of the 30 µg/g threshold made by the French programme. As expected, the present study demonstrates a higher sensitivity of FIT on gFOBT regardless of age, sex, screening rank, season for screening, and cancer location. Regarding the sensitivity of the tests according to cancer location, which has been the subject of controversy in the literature, our results are in agreement with those of two recent meta-analyses that concluded that gFOBT and FIT have better diagnostic performance for detecting cancers in the distal colon than in the proximal colon. [START_REF] Hirai | Systematic review with meta-analysis: faecal occult blood tests show lower colorectal cancer detection rates in the proximal colon in colonoscopy-verified diagnostic studies[END_REF][START_REF] Lu | Diagnostic Accuracy of Fecal Occult Blood Tests For Detecting Proximal Versus Distal Colorectal Neoplasia: A Systematic Review And Meta-Analysis[END_REF] Not surprisingly, comparison of In-CRC characteristics between the 3 rounds of this study showed no significant difference in the distribution of cancer location and a predominance of proximal and rectal location. The only significant difference between participant characteristics with In-CRCs was the higher age of participants at the time of testing and CRC diagnosis of In-CRCs after FIT, simply related to the aging of a population that has already participated in previous gFOBT-based rounds, as also evidenced by the higher screening rank. When analyzing participant and tumour characteristics according to In-and SD-CRCs, In-CRCs occurred more often in women, were less frequent after a first screening, had more frequent proximal and rectal locations and had more frequent advanced stages, in agreement with gFOBT and FIT literature data. [START_REF] Steele | Interval cancers in a FOBT-based colorectal cancer population screening programme: implications for stage, gender and tumour site[END_REF][START_REF] Van Der Vlugt | Interval Colorectal Cancer Incidence Among Subjects Undergoing Multiple Rounds of Fecal Immunochemical Testing[END_REF][START_REF] Van De Veerdonk | Occurrence and characteristics of faecal immunochemical screen-detected cancers vs non-screen-detected cancers: Results from a Flemish colorectal cancer screening programme[END_REF][START_REF] Vicentini | Impact of screening programme using the fecal immunochemical test on stage of colorectal cancer: Results from the IMPATTO study[END_REF] Our study has several limitations. First, it was a retrospective study, but nevertheless, missing data were limited to 5.6% and 0.7% of the population for cancer stage and location, respectively.

Second, our region does not have a cancer registry, which could theoretically lead to missing some

In-CRCs in the event of medical care outside the region or emigration. However, if such a problem exists, it is certainly marginal, as is its net effect on the comparison between tests. Third, it has recently been stated that the test sensitivity should be measured with the proportional In-CRC rate method rather than the In-CRC proportion method, which means that the incidence rate of In-CRCs in population-based screening programmes should be analyzed in relation to the expected incidence rate of CRC in background populations. [START_REF] Zorzi | Interval colorectal cancers after negative faecal immunochemical test in a 13-year screening programme[END_REF][START_REF] Rossi | Incidence of interval cancers in faecal immunochemical test colorectal screening programmes in Italy[END_REF][START_REF] Mancini | Proportional incidence of interval colorectal cancer in a large population-based faecal immunochemical test screening programme[END_REF] However, in the recent study by Zorzi et al, [START_REF] Zorzi | Interval colorectal cancers after negative faecal immunochemical test in a 13-year screening programme[END_REF] only small differences were shown between the FIT sensitivity for cancer detection measured by either method (86.9% versus 83.9%). Fourth, our study did not take into account potential CRCs occurring in positive individuals who did not undergo colonoscopy (less than 7% in each of the 3 rounds) or the cancers diagnosed after colonoscopy, also called post-colonoscopy In-CRCs. Thus, the sensitivity measured in our study was the sensitivity of the test, not the sensitivity of the programme. Fifth, we could be criticized for including intramucosal cancers (stage 0 CRC), which are noninvasive cancers, in this study. However, it is standard practice in France to count them among SD-CRCs. [START_REF] Bordet | Reappraisal of the characteristics, management, and prognosis of intramucosal colorectal cancers and their comparison with T1 carcinomas[END_REF] In any case, the analysis excluding stage 0 CRCs from this study showed no change in the test sensitivity results. Sixth, in searching risk factors for In-CRC among negatives, we were unable to examine the burden of participant factors such as smoking, diabetes, hypertension and obesity, which were previously identified in a meta-analysis as increasing the risk of false-negative FIT, because dietary or lifestyle patterns of participants in our study were not recorded. [START_REF] De Klerk | Participant-Related Risk Factors for False-Positive and False-Negative Fecal Immunochemical Tests in Colorectal Cancer Screening: Systematic Review and Meta-Analysis[END_REF] To conclude, this population-based study demonstrated a dramatic decrease in the cumulative incidence rate of In-CRCs after switching from gFOBT to FIT within an established mature CRC screening programme. Our study demonstrates the higher sensitivity of FIT compared to gFOBT regardless of age, sex, screening rank (except first screening), season for screening and cancer location. These data provide an additional incentive for countries still using gFOBT to switch to FIT.

Table 5 .

 5 FIT sensitivity was significantly higher than that of gFOBT as measured in the two previous rounds, regardless of the variable analyzed, including sex, age, season for screening, tumor location, and rank of screening,

	10 and 19 µg/g and 275.6 [95% CI, 137.8-551.1] per 100 000 person-years for a haemoglobin level between 20
	and 29 µg/g, contrasting with the rate of 11.6 [95% CI, 7.8-17.2] per 100 000 person-years for cutoff values
	below 10 µg/g.
	Interval cancer rate and test sensitivity estimates
	The In-CRC rate was 44.0% and 44.6% in the two gFOBT-based rounds and 10.5% in the FIT-based
	round (Table 1), reflecting test sensitivities for CRC detection of 56.0%, 55.4% and 89.5%,

7 (95% CI, 45.3 -68.5), 42.4 (95% CI, 32.6-55.2), and 15.8 (95% CI, 10.9-22.8) per 100 000 person-years after adjusting for sex and age. By multivariate analysis (Poisson regression), increasing age, sex (men versus women), screening rank (subsequent versus first screening), and the test used (gFOBT versus FIT) were the factors significantly associated with the incidence rate of In-CRCs among those with negative testing (Table

3

). Thus, the FIT/gFOBT incidence rate ratio was 0.38 [95% CI, 0.27-0.54] using round 5 as the reference. By analyzing the FITbased round separately by multivariate analysis, the 3 factors significantly associated with an increase in the incidence rate of In-CRCs were increasing age, the season for testing (spring/summer versus automn/winter), and the faecal haemoglobin level (Table

3

). The cumulative 2-year incidence rates of In-CRCs were 198.44 [95% CI, 119.6-329.2] per 100 000 person-years for a haemoglobin level between respectively (P<0.0001). Test sensitivity estimates after adjusting for sex and age, rank of screening, season for testing, and tumor location are given in Table

4

. The results of the logistic regression analysis comparing the sensitivity of FIT to that of gFOBT are given in

  Our study showed that the cumulative 2-year incidence rate of In-CRC, adjusted for sex and age, was 15.8 [95% CI, 10.9-22.8] per 100 000 person-years for the FIT and 55.7 [95% CI, 45.3-68.5] and 42.4 [95% CI, 32.6-55.2] per 100 000 person-years for the gFOBT. These values are very similar to those reported in a meta-analysis restricted to high-quality studies, with 15 (95% CI, 8-30) and 55 (95% CI, 35-87) per

Table 1 .

 1 Main characteristics of the populations of the two gFOBT-based rounds(5, 6) and the FITbased round(7)

	Round 5	Round 6	Round 7
	gFOBT	gFOBT	FIT
	2010-2012	2012-2014	2015-2017

Table 2 .

 2 Comparison of participant and tumour characteristics among subjects with screen-detected cancer or interval cancer

		Screen-detected	Interval cancers
		cancers
	Variable	n=612	n=209	P
	Round, n (%)	612 (0)	209 (0)	<0.0001
	Round 5	126 (20.6%)	99 (47.4%)
	Round 6	77 (12.6%)	62 (29.7%)
	Round 7	409 (66.8%)	48 (23.0%)
	Sex, n	612 (0)	209 (0)	0.0030
	Women	219 (35.8%)	99 (47.4%)
	Men	393 (64.2%)	110 (52.6%)
	Age at screening, yrs	611 (1)	209 (0)	0.6110
	mean ± SD	64.4 ± 6.7	64.2 ± 6.4
	Age at screening, yrs	611 (1)	209 (0)	0.9615
	<60	164 (26.8%)	57 (27.3%)
	60-69	302 (49.4%)	101 (48.3%)
	≥70	145 (23.7%)	51 (24.4%)
	Age at diagnosis, yrs	607 (5)	209 (0)	0.3062
	mean ± SD	64.7 ± 6.7	65.2 ± 6.4
	Time between screening and	608 (4)	209 (0)	<0.0001
	diagnosis, months
	median (IQR)	1.8 (1.3; 2.7)	13.2 (6.1; 17.9)
	Time between screening test	608 (4)	209 (0)	<0.0001
	result and diagnosis of
	colorectal cancer
	1-12 months	592 (97.4%)	95 (45.5%)
	13-24 months	16 (2.6%)	114 (54.5%)
	Screening rank	612 (0)	209 (0)	0.0070
	median (IQR)	5 (4; 7)	5 (4; 6)
	Screening rank (category)	612 (0)	209 (0)	0.0292
	First	47 (7.7%)	7 (3.3%)
	Subsequent	565 (92.3%)	202 (96.7%)
	Season for screening	612 (0)	209 (0)	0.2061
	Spring	148 (24.2%)	59 (28.2%)
	Summer	139 (22.7%)	48 (23.0%)
	Automn	176 (28.8%)	45 (21.5%)
	Winter	149 (24.3%)	57 (27.3%)
	Tumour location	610 (2)	205 (4)	0.0001
	Distal	292 (47.9%)	62 (30.2%)
	Proximal	164 (26.9%)	74 (36.1%)

Table 4 .

 4 Test sensitivity for cancer detection in the 3 rounds according to age and sex and other variables adjusted for age and sex (rank of screening, season for screening, tumour location)

		Round 5	Round 6	Round 7
	Variables	gFOBT	gFOBT	FIT
		mean [95% CI]	mean [95% CI]	mean [95% CI]
	Sex
		62.9%
	Men

Table 5 .

 5 Logistic regression comparison of FIT versus gFOBT sensitivity by participant and tumour characteristics (with Tukey adjustment) [3.14-20.39] 19.26 [7.10-52.23] <0.0001 <0.0001

			OR [95% CI]	OR [95% CI]	P	P
	Population	N	round 5 (gFOBT)	round 6 (gFOBT)	round 5	round 6
	Total population	821	6.69 [4.49-9.97]	6.86 [4.38-10.74] <0.0001 <0.0001
	Total population, age-and sex-adjusted	820	6.70 [4.48-10.01]	7.01 [4.46-11.03]
	Sex
	Men	503	7.03 [3.59-13.76]	9.68 [4.68-20.01] <0.0001 <0.0001
	Men, age-adjusted	503	7.22 [4.11-12.69]	9.86 [5.36-18.14]
	Women	317	6.57 [3.27-13.22]	4.48 [1.97-10.22] <0.0001 <0.0001
	Women, age-adjusted	317	6.63 [3.69-11.90]	4.46 [2.24-8.90]
	Age
	< 60 years	221 10.00 [3.48 -28.77] 19.76 [6.34-61.60] <0.0001 <0.0001
	< 60 years, sex-adjusted	221 13.23 [5.17-33.84] 23.23 [8.57-62.96]
	60-69 years	403	8.96 [4.45-18.05]	5.20 [2.31-11.69] <0.0001 <0.0001
	60-69 years, sex-adjusted	403	8.77 [4.88-15.76]	5.22 [2.65-10.27]
	≥ 70 years	196	2.96 [1.18-7.42]	4.71 [1.66-13.40]	0.0157	0.0015
	≥ 70 years, sex-adjusted	196	2.95 [1.37-6.39]	4.82 [2.00-11.62]
	Rank of screening
	First screening	54 11.00 [0.52-234.29] 16.50 [1.02-266.88] 0.1571	0.0479
	First screening, age-and sex-adjusted	54 10.84 [0.82-143.39] 15.68 [1.44-171.18]
	Subsequent screening	767	6.47 [3.98-10.50]	6.72 [3.86-11.70] <0.0001 <0.0001
	Subsequent screening, age-and sex-	766	6.41 [4.27-9.64]	6.78 [4.25-10.80]
	adjusted
	Season for screening
	Spring	207	5.86 [2.40-14.30]	3.75 [1.33-10.59] <0.0001	0.0081
	Spring, age-and sex-adjusted	206	5.67 [2.67-12.04]	3.53 [1.47-8.50]
	Summer	187	4.48 [1.65-12.21] 11.92 [3.89-36.55] 0.0013 <0.0001
	Sommer, age-and sex-adjusted	187	4.38 [1.87-10.25] 12.97 [4.98-33.79]
	Automn	221	8.27 [3.02-22.62]	8.48 [2.70-26.65] <0.0001 <0.0001
	Automn, age-and sex-adjusted	221	8.61 [3.64-20.39]	9.39 [3.50-25.17]
	Winter	206	8.45 [3.17-22.54]	6.87 [2.33-20.32] <0.0001 <0.0001
	Winter, age-and sex-adjusted	206	8.41 [3.70-19.11]	6.86 [2.77-17.00]
	Tumour location
	Distal colon	354	8.00

Table 6 .

 6 Comparison of participant characteristics with interval cancer and of tumour characteristics between the 3 rounds

		Round 5 a	Round 6	Round 7 c
		gFOBT	gFOBT	FIT
	Variables	n=99	n=62	n=48	P	P a,b	P a,c	P b,c
	Sex	99 (0)	62 (0)	48 (0)	0.1420
	Women	50 (50.5%)	23 (37.1%)	26 (54.2%)
	Men	49 (49.5%)	39 (62.9%)	22 (45.8%)
	Age at screening, yrs	99 (0)	62 (0)	48 (0)	0.0153	1.0000	0.0442	0.0192
	mean ± SD	63.7 ± 5.8	63.1 ± 7.0	66.5 ± 6.5
	Age at cancer diagnosis, yrs (SD)	99 (0)	62 (0)	48 (0)	0.0128	1.0000	0.0359	0.0166
	mean ± SD	64.7 ± 5.8	64.1 ± 6.9	67.5 ± 6.5
	Time between screening and cancer	99 (0)	62 (0)	48 (0)	0.6111
	diagnosis, months
	median (IQR)	13.0 (6.1; 17.5)	13.0 (5.8; 19.6)	14.9 (6.1; 18.0)
	Time between screening and cancer	99 (0)	62 (0)	48 (0)	0.6460
	diagnosis (category)
	0-11 months	47 (47.5%)	29 (46.8%)	19 (39.6%)
	12-24 months	52 (52.5%)	33 (53.2%)	29 (60.4%)
	Rank of screening, n (SD)	99 (0)	62 (0)	48 (0)	<0.0001	0.0219	<0.0001	<0.0001
	median (IQR)	5.0 (4.0; 5.0)	6.0 (3.0; 6.0)	7.0 (5.0; 7.0)
	Rank of screening (category)	99 (0)	62 (0)	48 (0)	0.2799
	First screening	2 (2.0%)	4 (6.5%)	1 (2.1%)
	Subsequent screening	97 (98.0%)	58 (93.5%)	47 (97.9%)
	Season for screening	99 (0)	62 (0)	48 (0)	0.4291

ACKNOWLEDGEMENTS

The authors acknowledge the contributions of the general practitioners, gastroenterologists and surgeons in the district of Ille-et-Vilaine. They also acknowledge the staff in charge of the database at the screening centre, particularly Samuel Foucrit, a computer scientist.

ADDITIONAL INFORMATION • • AUTHORS' CONTRIBUTIONS

Conception and design (JFB, CP, MC, AL); analysis and interpretation of the data (CR, JFB, AL, AC, CP, MC); drafting of the article (JFB, AL); all of the authors approved the final manuscript.

• ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The colorectal cancer screening programme was declared and approved by the CNIL (Commission Nationale de l'Informatique et des Libertés) on August 30, 2002 (no. 812571). This research was approved by the CCTIRS (Comité Consultatif pour le Traitement de l'Information en Matière de Recherche dans le Domaine de la Santé).

• CONSENT FOR PUBLICATION

Not applicable

• DATA AVAILABILITY

The authors state that the data from their study are available on request from the Biostatistics Department of the University Hospital Center of Rennes. chloe.rousseau@chu-rennes.fr

• COMPETING INTERESTS

The authors declare no conflict of interest.

• FUNDING INFORMATION

The authors received no specific funding for this work.


Maxence Lejars
email: maxence.lejars@md.tsukuba.ac.jp

Joël Caillet

Maude Guillier

Eliane Jacqueline A Plumbridge

Eliane Hajnsdorf
email: eliane.hajnsdorf@ibpc.fr

Regulatory interplay between RNase III and asRNAs in E. coli; the case of AsflhD and the master regulator of motility, flhDC

Keywords: asRNAs, RNase III, transcriptional attenuation, flhD, motility, phoP, E. coli

come

Importance

The importance of asRNAs in the regulation of gene expression has long been underestimated.

Here, we confirm that asRNAs can be part of layered regulatory networks since some are found opposite to genes encoding global regulators. In particular, we show how an antisense RNA (AsflhD) to the gene expressing a transcription factor serving as the primary regulator of bacterial swimming motility (FlhD 4 C 2 ) is involved in the transcriptional attenuation of flhD, which in turn impacts the expression of other genes of the motility cascade. The role of AsflhD highlights the importance of discrete fine-tuning mechanisms in the control of complex regulatory networks.

Introduction

In eukaryotes, hundreds of RNA-binding proteins (RBPs) and multiple classes of regulatory RNAs are involved in the complex regulation of gene expression (splicing, editing...). In bacteria, the relative scarcity of RBPs and regulatory RNAs, led to the supposition that they provided only accessory contributions to the major bacterial gene regulatory mechanisms. An important obstacle in deciphering regulatory networks is their multi-component nature and the existence of "missing links" between regulators and their targets. These intermediates can have both positive and negative impacts on gene expression, leading to compensatory effects upon removal of one of them [START_REF] Keseler | The EcoCyc database: reflecting new knowledge about Escherichia coli K-12[END_REF][START_REF] Theodorou | Involvement of AtoSC two-component system in Escherichia coli flagellar regulon[END_REF][START_REF] Lemke | DksA and ppGpp Directly Regulate Transcription of the Escherichia coli Flagellar Cascade[END_REF][START_REF] Mizushima | Decrease in expression of the master operon of flagellin synthesis in a dnaA46 mutant of Escherichia coli[END_REF][START_REF] Lay | A complex network of small non-coding RNAs regulate motility in Escherichia coli[END_REF][START_REF] Yakhnin | CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage[END_REF]. Hence, the fine-tuning of gene expression is far from fully understood in many if not most cases.

Many bacterial small RNAs (sRNAs) are regulators that base-pair with RNA, with their genes located in trans to their targets and acting by short, imperfect regions of base-pairing. This property allows them to act on multiple targets. In contrast, the genes for antisense RNAs (asRNAs) are located in cis to their complementary target and thus have, in most cases, a single dedicated target. Fewer asRNAs have been described as compared to sRNAs probably because of their high lability, their low conservation among species and because they were usually considered the products of pervasive transcription arising from leaky terminators [START_REF] Lejars | The world of asRNAs in Gram-negative and Gram-positive bacteria[END_REF][START_REF] Hör | Trans-Acting Small RNAs and Their Effects on Gene Expression in Escherichia coli and Salmonella enterica[END_REF][START_REF] Dornenburg | Widespread Antisense Transcription in Escherichia coli[END_REF][START_REF] Wade | Pervasive transcription: illuminating the dark matter of bacterial transcriptomes[END_REF].

Initially, asRNAs were identified on mobile genetic elements (prophages and plasmids), with their only purpose to control their replication and partition. The importance of asRNAs was later demonstrated to extend to almost all kinds of biological processes [START_REF] Lejars | Physiological roles of antisense RNAs in prokaryotes[END_REF], as in the case of type I toxin-antitoxin systems, involved in persistence, in which the toxin mRNA is neutralized by an asRNA that induces degradation and/or translation inhibition [START_REF] Masachis | Type I Toxin-Antitoxin Systems: Regulating Toxin Expression via Shine-Dalgarno Sequence Sequestration and Small RNA Binding[END_REF]. Furthermore, the double-strand-specific RNase III has been known to be an important player in asRNA regulation, as in the case of the regulation of plasmid copy number and toxin-antitoxin systems [START_REF] Malmgren | Antisense RNA Control of Plasmid R1 Replication. The dominant product of the antisense RNA-mRNA binding is not a full RNA duplex[END_REF][START_REF] Darfeuille | An antisense RNA inhibits translation by competing with standby ribosomes[END_REF].

The mechanisms of action of asRNAs are diverse. They can negatively regulate transcription by interference due to the collision of two converging RNA polymerases or by attenuation due, in some cases, to the stabilization of a terminator structure in the mRNA upon binding of the asRNA [START_REF] André | Sbox and T-box riboswitches and antisense RNA control a sulfur metabolic operon of Clostridium acetobutylicum[END_REF][START_REF] Giangrossi | A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri[END_REF].

However, despite complete complementarity, the interaction of asRNA and its target requires, in some cases, formation of an intermediate called "kissing complex" [START_REF] Malmgren | Antisense RNA Control of Plasmid R1 Replication. The dominant product of the antisense RNA-mRNA binding is not a full RNA duplex[END_REF][START_REF] Kolb | An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment[END_REF]. These interactions can have negative or positive consequences on gene expression since they induce modifications to the RNA secondary structure and/or physically interfere with the activity of other regulators [START_REF] Opdyke | GadY, a Small-RNA Regulator of Acid Response Genes in Escherichia coli[END_REF][START_REF] Opdyke | RNase III Participates in GadY-Dependent Cleavage of the gadX-gadW mRNA[END_REF].

Very often, the mechanism by which a specific asRNA regulates its target remains unclear due to the impossibility to modify the sequence of the asRNA independently of its target.

Various approaches have been used to enrich the E. coli transcriptome for double-stranded RNAs, which are the presumed intermediates in asRNA regulation. Studies using inhibition of Rhodependent transcription termination demonstrated that pervasive antisense transcription is common in almost all loci in E. coli [START_REF] Peters | Rho and NusG suppress pervasive antisense transcription in Escherichia coli[END_REF]. In 2010, one thousand asRNAs were identified suggesting their importance in the control of gene expression [START_REF] Dornenburg | Widespread Antisense Transcription in Escherichia coli[END_REF]. Another study focusing on the mapping of transcriptional units highlighted the presence of 498 asRNAs mostly from overlapping untranslated regions (UTRs) within convergent or divergent operons [START_REF] Conway | Unprecedented High-Resolution View of Bacterial Operon Architecture Revealed by RNA Sequencing[END_REF]. Immunoprecipitation of doublestranded RNAs using specific antibodies allowed the identification of 200 asRNAs of which 21 were validated as RNase III-degraded asRNAs [START_REF] Lybecker | The double-stranded transcriptome of Escherichia coli[END_REF]. In a fourth study, primary transcripts were isolated by selective tagging allowing the identification of 212 asRNA transcription start sites (asTSSs) [START_REF] Thomason | Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli[END_REF].

More recently, p19 viral protein capture of double-stranded RNAs identified 436 asRNAs [START_REF] Huang | Tombusvirus p19 Captures RNase III-Cleaved Double-Stranded RNAs Formed by Overlapping Sense and Antisense Transcripts in Escherichia coli[END_REF].

Unexpectedly, there is a little overlap between the identified asRNAs from these different studies, which may be due to technical bias, but may also depend on the genetic context and/or the environmental conditions.

While previously published works focused on the identification of asRNAs, we aimed to characterize physiologically relevant asRNAs. Some time ago we performed a transcriptome analysis of an rnc mutant compared to its isogenic wt strain. We used a tailored RNA-seq approach described previously [START_REF] Maes | Landscape of RNA polyadenylation in E. coli[END_REF]. In agreement with other published genomics experiments [START_REF] Lybecker | The double-stranded transcriptome of Escherichia coli[END_REF][START_REF] Thomason | Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli[END_REF][START_REF] Stead | Analysis of Escherichia coli RNase E and RNase III activity in vivo using tiling microarrays[END_REF] several candidate asRNA were detected, which were stabilized in the strain lacking RNase III activity. We were surprised to see that many were asRNA to genes of important regulators which raised the question of whether or not they could have a physiological impact on the expression and function of their target regulator and hence on the downstream regulon. We first confirmed that RNase III modulates the level of 4 of these antisense transcripts and then concentrated on the asRNA to flhD. flhD is the first gene of the flhDC operon encoding the master regulator of swimming motility. We find that AsflhD is involved in the direct repression of the transcription elongation of flhD, which provides an additional regulatory layer to the complex cascade of motility in enterobacteria.

Results

Characterization of asRNAs stabilized upon RNase III inactivation

An RNA-seq analysis in a wt and its rnc105 derivative strain was performed by tagging transcripts according to their 5′-phosphorylation status, allowing to distinguish between 5'-triphosphate fragments (primary transcripts TSS), monophosphate 5'-fragments (processed transcripts, PSS) and internal fragments resulting from the fragmentation (INT) [START_REF] Maes | Landscape of RNA polyadenylation in E. coli[END_REF]. The depth of sequence coverage was not sufficient for a compilation of all asRNAs. Instead we looked manually for antisense reads covering the translation signals of mRNAs that were enriched upon RNase III inactivation. Potential asRNA promoter regions were deduced by examination of the TSS and PSS fractions in the rnc strain. The RNase III processing sites in the wild-type were usually not obvious since they presumably provoked the rapid degradation of the asRNA. We selected 4 asRNAs to the crp, ompR, phoP and flhD transcripts encoding important global regulators for verification by northern blot (Fig. 1). We note that they had all been proposed as antisense transcripts in one or more of the previous genomic studies [START_REF] Dornenburg | Widespread Antisense Transcription in Escherichia coli[END_REF][START_REF] Conway | Unprecedented High-Resolution View of Bacterial Operon Architecture Revealed by RNA Sequencing[END_REF][START_REF] Lybecker | The double-stranded transcriptome of Escherichia coli[END_REF][START_REF] Thomason | Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli[END_REF][START_REF] Huang | Tombusvirus p19 Captures RNase III-Cleaved Double-Stranded RNAs Formed by Overlapping Sense and Antisense Transcripts in Escherichia coli[END_REF].

The crp gene encodes the major regulator of carbon catabolite repression and it was shown previously to be transcriptionally regulated by a transcript initiated from a divergently expressed promoter 3 base-pairs upstream and on the opposite strand [START_REF] Hanamura | Molecular mechanism of negative autoregulation of Escherichia coli crp gene[END_REF][START_REF] Okamoto | Mechanism for the autogenous control of the crp operon: transcriptional inhibition by a divergent RNA transcript[END_REF]. This transcript, now known to express the yhfA gene, was detected in the wt strain (Fig. 1-A). Additional asRNAs, were stabilized in the rnc strain in the 3 fractions. The TSS of one species is located 20 nts upstream of the crp translation start on the opposite strand (shown by an orange dotted arrow on Fig. 1A) and in addition there is extensive asRNA, complementary to the crp coding sequence and 5'-UTR (INT). We performed northern blots using complementary probes hybridizing to positions 13 to 441 of the crp ORF to detect both the crp mRNA and its antisense transcript. An asRNA, named Ascrp, of about 350 nts accumulates only in the mutant (Fig. 1-A).

The ompR gene encodes the response regulator of a two-component system involved in cell wall homeostasis and response to low pH, EnvZ-OmpR [START_REF] Wurtzel | Osmoregulation of gene expression. I. DNA sequence of the ompR gene of the ompB operon of Escherichia coli and characterization of its gene product[END_REF][START_REF] Kenney | EnvZ/OmpR Two-Component Signaling: An Archetype System That Can Function Noncanonically[END_REF][START_REF] Pratt | From acids to osmZ: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli[END_REF][START_REF] Stincone | A systems biology approach sheds new light on Escherichia coli acid resistance[END_REF]. We observed an asTSS 147 nts downstream from the AUG (Fig. 1-B) in wt and enhanced in the rnc mutant. Northern blotting with complementary probes corresponding to the 5'-end of the ompR ORF confirmed the presence of the ompR-envZ transcript and a long asRNA transcript, AsompR, of about 2000 nts, which is likely to also encode the divergently expressed greB gene. In addition, in the mutant, smaller fragments (less than 500 nts) are detected for both ompR and AsompR (Fig. 1-B), likely corresponding to a stable duplex between the sense and asRNA transcripts in the absence of RNase III.

The phoP gene encodes the response regulator of the PhoQ-PhoP two-component system, involved in cell wall homeostasis and in response to low magnesium [START_REF] Zwir | Dissecting the PhoP regulatory network of Escherichia coli and Salmonella enterica[END_REF][START_REF] Groisman | Bacterial Mg2+ homeostasis, transport, and virulence[END_REF]. We observed an asRNA upon RNase III inactivation in the INT fraction about 282 nts downstream from the phoP AUG. Northern blot with complementary probes corresponding to the 5'-end of the phoP ORF confirmed the accumulation of two fragments (AsphoP) about 300-320 nts long in the mutant (Fig. 1-C).

The flhDC genes are co-transcribed and together they encode the master regulator of motility, FlhD 4 C 2 [START_REF] Bertin | The H-NS protein is involved in the biogenesis of flagella in Escherichia coli[END_REF]. We detected an asRNA to flhD mRNA (AsflhD) accumulating in the mutant, initiated 22 nts downstream from the flhD AUG. Northern blot analysis with probes hybridizing to the 5'-UTR of flhD confirmed the accumulation of AsflhD, with a major fragment about 220 nts and one minor fragment about 160 nts upon RNase III inactivation. Together, there was an increase in the amount of the full-length flhD mRNA in the mutant and the stabilization of a flhD fragment of approximate size 220 nts (Fig. 1-D).

All these asRNAs are partially or completely processed by RNase III since they are only visible in the rnc strain. Crp, OmpR, PhoP and FlhD are major regulators of gene expression in E. coli, all involved in the control of large regulons (RegulonDB v 10.5 [START_REF] Santos-Zavaleta | RegulonDB v 10.5: tackling challenges to unify classic and high throughput knowledge of gene regulation in E. coli K-12[END_REF]). We wondered whether these asRNAs and RNase III have a functional regulatory role and thus affect cell physiology. We studied in more details asRNAs to phoP and flhD, two regulators tightly controlled at both the transcriptional and post-transcriptional levels.

Regulation of phoP and AsphoP by RNase III

The RNA-seq profiles suggested that AsphoP may be transcribed from an asTSS located 282 nts downstream from the translation start of phoP mRNA. An asRNA derived from this TSS was confirmed by northern blot, probing for the 5'-region of the phoP ORF (Fig 1-C). In addition, the rnc mutation slightly increases phoP stability and amount but induces very large increases in the stability and level of AsphoP (Fig. 2-AB). Candidate consensus -10 and -35 sequences are located just upstream of AsphoP TSS (Fig. 2-C). To validate this potential promoter, we constructed a P AsphoP -lacZ transcriptional fusion containing 150 nts before and 15 nts after the putative TSS of AsphoP, with the wt sequence (P AsphoP wt ) and also with mutations decreasing the agreement with the consensus in the predicted -35 and -10 boxes (P AsphoP -) (Fig. 2-D). The mutated AsphoP promoter (P AsphoP -) strongly decreased the expression of P AsphoP -lacZ (20-fold), confirming it as the endogenous AsphoP promoter (Fig. 2-E). The activity of the P AsphoP wt -lacZ fusion decreased 2-fold in the mutant implying that RNase III positively regulates AsphoP. In summary, RNase III positively controls the transcription of AsphoP and also participates to the degradation of both phoP and AsphoP transcripts.

Sequence comparison with other bacterial species showed that although the region of the AsphoP promoter is moderately well conserved, there are several A to G substitutions in the -10 box at positions -9 and -12, suggesting that this promoter may be inactive in these genomes (Fig. 2-C). This, in turn, implies that, if AsphoP has any function, it could be limited to E. coli K-12 and have been counter-selected in these other species or more likely, represents a novel, evolving trait.

Physiological expression of AsflhD

Figure 1 shows a corresponding increase in the amounts of flhD and the appearance of a smaller mRNA fragment in the rnc mutant. Intriguingly 40 years ago it was noted that RNase III was involved in the swimming activity of E. coli and rnc mutants were immotile [START_REF] Apirion | Ribonuclease III is involved in motility of Escherichia coli[END_REF]. In this work we have investigated whether RNase III could exert this effect via AsflhD.

The RNA-seq profiles revealed an asTSS 22 nts downstream from the translation start of flhD as reported by Dornenberg et al. [START_REF] Dornenburg | Widespread Antisense Transcription in Escherichia coli[END_REF]. A candidate promoter exists upstream of this asTSS. Sequence alignment of this region in other enterobacteria shows a good conservation of a promoter with an extended -10 5'-TG box [START_REF] Mitchell | Identification and analysis of 'extended -10' promoters in Escherichia coli[END_REF], suggesting that this promoter is conserved and active (Fig. 3-A).

To validate the presence of a functional promoter, a P AsflhD -lacZ transcriptional fusion (P AsflhD wt ) was constructed containing 165 nts before and 15 nts after the putative TSS of AsflhD (Fig 3-B). This fusion showed a relatively low level of -galactosidase activity (Fig. 3-C). Its expression was strongly increased when the -10 motif was improved towards the RpoD consensus (P AsflhD + ) while mutating the -35 to a less consensus sequence (P AsflhD -) decreased expression 2-fold (Fig. 3-C) confirming that we had identified the AsflhD promoter. It should be noted that mutations were designed to be used in the endogenous flhD locus, and chosen to minimally affect the coding sequence of flhD and to avoid introduction of rare codons. The low level of expression made us wonder if AsflhD was expressed using an alternative sigma factor. A heat-shock increased P AsflhD -lacZ expression 2-fold after 15 minutes and 5-fold after 60 minutes and also increased the level of the AsflhD RNA in the rnc strain (Fig. 3-DE). Comparison of the asflhD promoter with the consensus sequences for the two heat-shock sigma factors,  H and  E (rpoH and rpoE) shows better correlation with the  E consensus than with  H (Fig. 3-A) [START_REF] Koo | Dissection of recognition determinants of Escherichia coli sigma32 suggests a composite -10 region with an 'extended -10' motif and a core -10 element[END_REF][START_REF] Rhodius | Conserved and Variable Functions of the σE Stress Response in Related Genomes[END_REF].

We then examined whether the P AsflhD promoter is under the control of RpoE by using a strain deleted for rseA (anti-σ factor inhibitor of RpoE), which leads to strong induction of the RpoE regulon [START_REF] Thompson | SigmaE regulates and is regulated by a small RNA in Escherichia coli[END_REF][START_REF] Ades | The Escherichia coli sigma(E)-dependent extracytoplasmic stress response is controlled by the regulated proteolysis of an anti-sigma factor[END_REF]. Deletion of rseA increased 2-fold the expression of the wt P AsflhD -lacZ fusion and of the improved P AsflhD + -lacZ construct (Fig. 3-FG) comparable with the effect of the heat-shock at 46°C, known to induce the RpoE regulon [START_REF] Rouvière | rpoE, the gene encoding the second heat-shock sigma factor, sigma E, in Escherichia coli[END_REF]. To test for an effect of rpoH, we introduced the P lac promoter in front of the endogenous rpoH gene and compared AsflhD RNA levels in wt and rnc mutant cells after 15 minutes of heat-shock at 45°C, in the presence or absence of IPTG. rpoH mRNA expressed from its own promoter or from the P lac promoter in the presence of IPTG was strongly increased by the heat-shock (Fig. 3-E). AsflhD was only detected in the rnc mutant and there was a correlation between the levels of rpoH and AsflhD transcripts at 30°C and 45°C with and without IPTG (Fig. 3-E). However, RpoH overexpression at 37°C revealed a slight repression of the transcription of AsflhD on the wt P AsflhD -lacZ fusion (Fig. S1-A) even though RNase III-stabilized AsflhD accumulated when RpoH was induced at 45°C. This would seem to rule out a direct role for RpoH in AsflhD transcription. We also tested RpoS overexpression but found it had no effect on the transcription of AsflhD (Fig. S1-B). These experiments indicate that P AsflhD is functional and induced during a heat-shock due primarily to the activity of RpoE, acting directly or indirectly on the promoter of AsflhD.

To confirm the identification of the AsflhD promoter and to allow variation of the AsflhD expression, P AsflhD -and P AsflhD + were introduced at the endogenous flhDC locus and the expression of the AsflhD asRNA was examined by northern blot. The inactivation of the native promoter prevented the detection of AsflhD in the rnc mutant bacteria. Conversely, the mutation overexpressing AsflhD, led to the detection of a faint smear in the wt strain and to the accumulation of a high level of AsflhD in the mutant (Fig. 3-H). In summary, we have identified the AsflhD promoter and shown that the mutations in the promoter of AsflhD can be used as tools to study the function of AsflhD at the genomic locus of flhD.

Characterization of the 3'-end of AsflhD

AsflhD RNA is only detected in the rnc strain, implying that it is very labile when RNase III is active (Fig. 3-E). Circular RT-PCR experiments (cRT-PCR) confirmed that AsflhD is indeed expressed in both the wt and rnc strains from the predicted asTSS promoter but that the 3′-extremities of the different AsflhD transcripts are highly heterogeneous and can extend up to 345 nts in the mutant (Fig. 4-A). Surprisingly, no 220 nts long RNA (Figs. 1-C, 3-EH) was detected in the mutant by cRT-PCR while a 149 nts long fragment was found several times exclusively in the wt strain, which might suggest that it is an intermediate in the degradation of AsflhD. It should be noted that the requirement for a ligation step during the cRT-PCR may lead to a bias towards more accessible single-stranded RNA fragments and could have excluded double-stranded RNAs from this analysis.

The stable AsflhD 220 nts transcript detected in rnc (Fig. 5-A) should correspond to duplex RNA formation between transcripts of the convergent flhD and AsflhD promoters. The equivalent sensetranscript is also detected (Figs. 1-D, 5-B below). RNase III is clearly a major factor in the degradation of AsflhD. We also investigated the role of RNase E, the major endonuclease involved in mRNA turnover in E. coli, and of PNPase an exoribonuclease negatively controlled by RNase III. We found that the low stability of AsflhD is independent of the activity of PNPase but depends on RNase E, since a longer transcript of an approximate size of 300 nts is detected upon RNase E inactivation (Fig. 4-B). Hence, RNase III and RNase E are both involved in the rapid turnover of AsflhD but they act independently.

Independent degradation of flhD and AsflhD transcripts by RNase III

We further investigated the role of RNase III in the degradation of flhD mRNA and AsflhD asRNA.

First, we analyzed the stability of both AsflhD and flhD transcripts upon RNase III inactivation. In the mutant the 220 nts long AsflhD transcript and also a somewhat shorter about 160 nts long transcript were strongly stabilized, while both the amount and the stability of the long flhDC mRNA increased only 2-fold (Fig. 5-AB). In addition, a 220 nts long flhD RNA fragment was highly stabilized in the rnc strain. It is derived from the 5′-UTR, where the probe used in this study to detect flhD mRNA is located (Table S2). It presumably corresponds to a fragment of the flhD mRNA extending from its promoter to the AsflhD promoter located in the beginning of the flhD ORF (Fig. 3-A) and thus is complementary to AsflhD. The interaction of the 5'-UTR of flhD mRNA with AsflhD should generate an RNA duplex (Fig. 4-A) whose degradation depends on cleavage by RNase III.

We examined the interaction between AsflhD and flhD RNAs and their cleavage by RNase III in vitro.

A 308 nts long flhD transcript corresponding to the 5'-UTR and part of the ORF of the flhD mRNA and a 256 nts long AsflhD asRNA were synthesized and labeled at their 5'-extremity. These two RNAs form a duplex when present in equimolar concentrations, which is completely degraded upon addition of RNase III (Fig. S2-AB). Remarkably, under the same condition, RNase III cleaves the individual RNAs independently at 2 sites on AsflhD and 4 sites on flhD (Fig. S2-CD). These cleavage sites are located within regions able to form secondary structure on each molecule (6, 44) (Fig. S2-C). RNase III is thus able to process both AsflhD and flhD RNAs in vitro, at specific sites but is also able to drive the complete degradation of the 5'-UTR of flhD when it is bound to the asRNA AsflhD.

As AsflhD is never detected in the wt strain, this implies that it immediately base-pairs with flhD and both are degraded, so changes in AsflhD expression will directly modulate the level of flhD mRNA.

AsflhD represses the expression of flhD

To investigate the function of AsflhD we determined the effect of AsflhD silencing and overexpression on flhD expression by following flhD mRNA abundance and stability using the endogenous P AsflhD mutations described above. While a slight decrease (35%) of flhD mRNA abundance results from both silencing (P AsflhD -) and overexpression (P AsflhD + ) of AsflhD, the stability of flhD mRNA was not significantly affected in either P AsflhD mutants (Table 1).

Two translational lacZ reporter fusions encompassing the 5'-UTR and the first 34 amino-acids of FlhD (including P AsflhD ) were introduced at the lacZ chromosomal locus. The P flhD -flhD-lacZ fusion allows simultaneous monitoring of the transcriptional and translational regulation of flhD and the P tet -flhD-lacZ, monitors only the post-transcriptional regulation (Fig. 6-A). The mutations in the AsflhD promoter producing silencing and overexpression of AsflhD, were also introduced into both fusions. While loss of AsflhD (P AsflhD -) had no impact on the expression of FlhD, overexpression of AsflhD (P AsflhD + ) resulted in decreased expression of flhD-lacZ expression from both the native and P tet promoters (Fig. 6-BC). Hence, overexpression of AsflhD leads to the reduction of flhD expression irrespective of its promoter which suggests that AsflhD is involved in the direct regulation of flhD mRNA and/or translational levels and not via an effect on the flhD promoter.

Mutual repression of transcriptional elongation by AsflhD and flhD in vitro

To determine whether AsflhD can repress the transcription of flhD, we performed in vitro transcription experiments using a DNA template corresponding to the flhD gene from 76 nts before to 388 nts after the transcription start site of flhD, which allows the transcription of a 388 nts flhD RNA and of a 335 nts AsflhD RNA (Fig. 7-A). We compared the abundance of both transcripts synthesized from this latter DNA fragment to those generated from templates carrying the promoter mutations leading to either silencing (P AsflhD -) or overexpression (P AsflhD + ) of AsflhD. In vitro transcription assays were performed in a single round of elongation in the presence of heparin and with RNA polymerase (RNAP) pre-bound to templates in the absence of RNA, hence observed effects are restricted to the elongation step and should be independent of the initiation of transcription. The results correlate with in vivo data even though the amplitude of the effects is different. Figure 7 shows that expression of AsflhD is strongly impaired (10-fold) on the template carrying the silencing (P AsflhD -) mutation and increased (2.5-fold) from the template carrying the overexpression (P AsflhD + ) mutation of AsflhD (Fig. 7-B orange bars). Also, as in vivo (Fig. 6-B), AsflhD silencing does not affect the level of the flhD RNA, while its overexpression in cis results in a decrease of the transcription of the flhD RNA (40%) (Fig. 7B purple bars). cAMP/CAP is known to activate the transcription of flhD by binding to a sequence located 72 nts upstream from the TSS of flhD [START_REF] Soutourina | Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon[END_REF]. As expected, its addition increased the transcription of flhD, which was still reduced by AsflhD overexpression (Fig. 7-B left). The role of AsflhD in the repression of flhD transcription elongation, was further confirmed by using a template where the P tet promoter replaced the P flhD promoter (Fig. S3-A) producing the same 388 nts flhD RNA but a shorter (260 nts) AsflhD RNA.

Similar results were observed from the templates carrying the P AsflhD silencing and overexpression mutations (Fig. S3-B). We conclude that overexpression of AsflhD in cis leads to the repression of transcription elongation of flhD, which seems to be independent of the transcription level and of the promoter expressing flhD.

We then determined the effect of purified AsflhD or flhD RNA addition on the transcription of both flhD and AsflhD using the same linear DNA templates. Figure 7-C shows that addition of increasing amount of AsflhD led to a linear decrease of flhD while not affecting the accumulation of AsflhD.

The reciprocal assay by adding increasing concentrations of purified flhD RNA decreased linearly the amount of AsflhD synthesized, while the amount of flhD was not affected. We performed the same assay with the shorter template carrying the P tet promoter and observed similar results (Fig. S3-C).

In summary, AsflhD represses the transcription elongation of flhD both in cis and in trans, independently of the promoter and its expression level. Thus, we propose that AsflhD asRNA and flhD mRNA are involved in their mutual transcriptional attenuation in which the interaction of one molecule with the other leads to a reduction in transcription via an alteration of transcription elongation.

AsflhD represses the transcription of flhD in trans in vivo

To confirm in vivo the ability of AsflhD to repress the transcription of flhD in trans, AsflhD was overexpressed from a plasmid, under the control of a P tac promoter inducible by IPTG. The short 242 nts long AsflhD is transcribed from the +1 to the +220 nt relative to the TSS of AsflhD with a rrnBT2

terminator to enable its stabilization. In the rnc mutant, this transcript is further processed with the appearance of the characteristic 160 nts intermediate (Figs. 8-A, 1-D). It is noteworthy that this smaller fragment is slightly longer than in the endogenous AsflhD suggesting that it may correspond to a 3'-fragment of AsflhD. Overexpression of AsflhD upon addition of IPTG decreases the abundance of flhD mRNA both in the wt strain (30%) and in the mutant (20%) (Fig. 8-B), in agreement with our in vitro data (Figs. 7-C, S3-C). Thus, consistent with previous experiments, trans overexpression of AsflhD in vivo reduces the abundance of flhD mRNA but it is less effective than AsflhD expressed from the flhD locus in cis and this effect is mostly independent of RNase III activity.

To further confirm that AsflhD can impact flhD expression in vivo, we measured the expression of the flhD-lacZ reporter fusions when AsflhD is overexpressed from the plasmid. The overexpression induces about a 30-40% decrease in the expression of flhD both from the P flhD -flhD-lacZ and the P tet -flhD-lacZ fusions (Fig. 8-CD, dark grey). However, overexpression of AsflhD from the plasmid in the strain where expression of AsflhD is already upregulated, by the presence of P AsflhD + mutation in cis, has no or little additive effect on the final repression (Fig. 8-CD, dark green). It should be emphasized that these effects are independent of the flhD promoter (native P flhD or P tet ). In summary, we demonstrate that AsflhD RNA is involved in the transcriptional attenuation of flhD both in cis and in trans, and that it does not involve the native P flhD promoter.

AsflhD controls the motility cascade

The flhDC operon encodes the FlhD 4 C 2 transcriptional master regulator of the swimming motility, so we next investigated the effect of AsflhD overexpression on the expression of key factors belonging to the motility cascade. This cascade of gene activation is divided into three classes of genes [START_REF] Fitzgerald | Comprehensive mapping of the Escherichia coli flagellar regulatory network[END_REF]. ) or from the plasmid decreases the swimming speed on plates (50% and 30% respectively) while the combined cis and trans overexpression of AsflhD induces a stronger reduction of the swimming speed (70%) (Fig. 9-D).

These results enforce our hypothesis that the observed strong reduction of the motility cascade effectors (fliA, flgB and fliC genes) in mid-log phase reflects a delayed induction rather than actually inhibiting expression.

In summary, both cis and trans overexpression of AsflhD reduces transcription of flhD, which in turn leads to repression of the whole cascade of motility and a reduction in swimming speed. Of note, cis-expressed AsflhD appears to be more effective than the trans-expressed to control flhD expression (Fig. 8-CD) and bacterial motility (Fig. 9-D). We hypothesize that the overexpression of AsflhD delays the induction of flhD to maintain the timing of the motility program.

Discussion

Regulatory RNA molecules are often part of complex genetic networks in bacteria. They correspond to a heterogeneous class of molecules that differ in gene organization, size and function. Our goal was to detect, identify and investigate the function of some antisense transcripts in E. coli. We compared the transcriptomes of an rnc mutant to that of a wt strain and selected candidate asRNAs. We validated the presence of discrete transcripts, only detected in the rnc mutant, which were complementary to crp, ompR, phoP and flhD genes. We identified the promoters encoding AsphoP and AsflhD, both producing transcripts convergently expressed towards the promoters of their target genes phoP and flhD. We show that RNase III is involved in the decay of both AsphoP and phoP. AsflhD is highly unstable and it is degraded independently by RNase E and RNase III. The promoter of AsflhD is induced by a heat-shock and is partially dependent on RpoE. We reveal that AsflhD is involved in transcriptional attenuation of flhD by being able to repress flhD transcription both in vitro and in vivo when overexpressed in cis or in trans (Figs. 78). Remarkably, in vitro, flhD RNA in trans could also drive the transcriptional attenuation of AsflhD asRNA (Fig. 7), suggesting that the interaction of the two RNAs can perturb the transcription elongation of the other transcript in vivo. The relatively small effect of AsflhD on flhD expression has important consequences for the FlhD 4 C 2 regulon since, the cis overexpression of AsflhD lead to decreased expression of three representative genes of the motility cascade; fliA, flgB and fliC, and reduced the swimming speed (Fig. 9). In conclusion, this work demonstrates that AsflhD is an additional player acting in the already complex regulatory process controlling flhDC. Our view of how AsflhD, by cotranscriptionally fine-tuning the expression of flhD, reinforces the control of the motility cascade, is shown in Figure 10.

Conservation of the promoters of AsflhD and AsphoP

asRNAs are poorly conserved among bacteria ( 50), but if this can be expected for asRNAs from intergenic regions with low sequence constraints, nucleotide changes within the coding region of the target risk to upset the function of the ORF and could be counterselected. AsflhD corresponds almost entirely to the 5'-UTR of flhD but with the promoter located in the ORF, which is fairly well conserved in enterobacteria (Fig. 3-A). The presence of multiple critical amino-acids along the protein, whose modification leads to decreased motility, could explain the conservation of the amino sequence of FlhD among Gram-negative bacteria [START_REF] Campos | Extensive alanine scanning reveals protein-protein and protein-DNA interaction surfaces in the global regulator FlhD from Escherichia coli[END_REF]. Thus, the conservation of the promoter of AsflhD could be the result of direct selection for FlhD activity or for the regulatory function of AsflhD controlling the expression of FlhD in these bacteria. In spite of the close similarity between the AsflhD promoter regions of Salmonella enterica sv Typhimurium and that of E. coli (Fig. 3-A), it would be interesting to examine whether AsflhD is also expressed and controls flhD levels in S. enterica, since it was reported that E. coli flhDC operon is expressed in S. enterica but at significantly lower levels [START_REF] Albanna | Driving the expression of the Salmonella enterica sv Typhimurium flagellum using flhDC from Escherichia coli results in key regulatory and cellular differences[END_REF].

The promoter of AsflhD shows low activity in vivo despite a -10 consensus with an extended -10 5′-TG-3' element and a -35 element which is functional since the P AsflhD -mutation decreased the promoter activity. In the case of AsphoP, the promoter located in the central region of the phoP ORF is not conserved in other enterobacteria. In E. coli, the two G to A changes in the -10 sequence of the promoter of AsphoP, compared to other bacteria make a relatively good consensus -10 (CATAAT) which can account for the high level of expression of AsphoP (Fig. 2-E) compared to AsflhD (Fig. 3-C). The lack of conservation in other bacteria suggests that, on the contrary to AsflhD, any function of AsphoP may be unique to E. coli where it was most likely acquired.

Role of RNase III in the degradation of asRNAs-mRNAs

We demonstrate that RNase III initiates the rapid degradation of AsphoP and AsflhD RNAs, at the same time it is also involved in the flhD degradation. The formation of an intermolecular RNA duplex can trigger the RNase III-mediated degradation of a mRNA as in the case of the puf mRNA upon binding to asPcrL in Rhodobacter sphaeroides or a sRNA upon binding to its target, as in the case of the sRNA RyhB upon binding to the sodB mRNA [START_REF] Afonyushkin | Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB[END_REF]. However, RNase III cleavage may generate a shorter but more stable mRNA and so can have a positive role on gene expression [START_REF] Opdyke | RNase III Participates in GadY-Dependent Cleavage of the gadX-gadW mRNA[END_REF][START_REF] Aiso | Overexpression of an antisense RNA, ArrS, increases the acid resistance of Escherichia coli[END_REF]. In the case of AsphoP the rnc mutation reduces promoter activity (Fig. 2-E) but greatly increases AsphoP stability (Fig. 2-A). In the case of AsflhD, RNase III allows the recycling of the products of transcriptional attenuation between flhD and AsflhD (Fig. 7-A).

Mechanism of regulation by AsflhD

Cis-encoded regulatory elements and asRNAs have the advantage of their close location facilitating their access to their target. Binding of AsflhD to the 5'-UTR of flhD mRNA can have multiple consequences on the interaction of other trans-acting regulators, known to affect flhD expression.

For example, after removal of the 5'-triphosphate, CsrA binds two regions of the flhD mRNA and protects it by competing with RNase E [START_REF] Yakhnin | CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage[END_REF][START_REF] Wei | Positive regulation of motility and flhDC expression by the RNA-binding protein CsrA of Escherichia coli[END_REF]. Closer to the translational start, the binding of the does not contain any canonical terminator motif [START_REF] Stork | Transcription Termination within the Iron Transport-Biosynthesis Operon of Vibrio anguillarum Requires an Antisense RNA[END_REF]. The asRNA RnaG was also shown to stabilize a terminator structure upon binding to the icsA mRNA in Shigella flexneri [START_REF] Giangrossi | A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri[END_REF]. Similarly, the asRNA RNAIII binds to the leader region of the repR mRNA and favors the formation of a terminator structure in Bacillus subtilis [START_REF] Brantl | RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism[END_REF]. The asRNA anti-Q in Enterococcus faecalis is responsible for both transcriptional interference due to RNAPs collisions and attenuation by an uncharacterized mechanism [START_REF] Chatterjee | Convergent transcription confers a bistable switch in Enterococcus faecalis conjugation[END_REF]. Our experiments do not detect the accumulation of a shorter transcript in vitro upon addition of one or the other of the transcripts suggesting that binding of AsflhD to flhD does not stabilize a terminator structure but could rather modify the stability of the elongating RNAP leading to heterogenous 3'-termini as observed for AsflhD in vivo by cRT-PCR (Fig. 4-A).

Physiology of AsflhD

Motility depends on the growth-rate due to the regulation of flhD [START_REF] Sim | Growth rate control of flagellar assembly in Escherichia coli strain RP437[END_REF]. The expression of flhD peaks at the end of the exponential phase then decreases and is stabilized to an intermediate level during the stationary phase in E. coli [START_REF] Pruss | Cell cycle regulation of flagellar genes[END_REF]. The temporal control of the motility cascade is maintained via, among other mechanisms, the expression of anti-σ factor, FlgM, at the same time as FliA, in order to allow the expression of class II genes without inducing class III genes expression prematurely.

When the basal part of the flagellum is assembled, FlgM is exported from the cytoplasm and FliA induces the expression of class III genes.

We show here that the P AsflhD promoter is induced during a heat-shock likely via RpoE. Remarkably, the swimming motility behavior is down-regulated during a heat-shock. This was proposed to be due to both a lowered level of FlhD and the inefficient export of FlgM [START_REF] Maeda | Effect of temperature on motility and chemotaxis of Escherichia coli[END_REF][START_REF] Rudenko | Inefficient Secretion of Anti-sigma Factor FlgM Inhibits Bacterial Motility at High Temperature[END_REF]. Our results show that the up-regulation of AsflhD during the heat-shock reducing flhD expression could also contribute to the decrease in swimming motility. Thus, as well as providing a fine-tuning mechanism to coordinate the expression of flhD, a function of AsflhD could be help to maintain the motility cascade off in conditions where motility would be detrimental and/or too costly.

Outlook

Our results demonstrate that the asRNA AsflhD is involved in a mutual transcriptional attenuation mechanism with its target flhD mRNA. Regulatory RNAs are far from being fully understood in bacteria and new mechanisms of action are likely to be discovered. Development of global approaches able to capture RNA-RNA and RNA-protein interaction (66-67) as well as prokaryotic single cell RNA-seq ( 68) are likely to pave the way for the elucidation of the role of the widely distributed asRNAs which were, until recently mostly considered as pervasive transcriptional noise but for which the study of individual cells and molecules could be critical for the understanding of their function.

Materials and methods

Bacterial strains and culture conditions

Strains and plasmids used in this work are listed in table S1. Constructions and mutations were made by using primers given in table S2 and are described in Supplementary Materials and methods. Strains were grown in LB Miller medium at 37°C, or at 30°C and shifted to 42°C, 45°C or 46°C for the heat-shock experiments. Appropriate antibiotics were added when required. IPTG was used at the indicated concentrations for induction of AsflhD from the pCA24N AsflhD plasmid and arabinose for RpoS from the pBAD18 plasmid.

Northern blotting and RNA-seq analysis

Total RNA was prepared from bacteria grown to the A 600 0.4 using the hot-phenol procedure [START_REF] Braun | Polynucleotide phosphorylase is required for the rapid degradation of the RNase E-processed rpsO mRNA of Escherichia coli devoid of its 3' hairpin[END_REF].

Five g of total RNA were electrophoresed either on 1% agarose, 1xTBE or 6% polyacrylamide gels (19/1), 7M urea, 1xTBE for analysis by northern blotting (70-71) along with RiboRuler High-Range marker (ThermoFisher) or radio-labeled Msp1-digested pBR322 (NEB). Membranes were hybridized with complementary RNA probes. Templates for the synthesis of the RNA probes were obtained by PCR amplification using the pair of "m" and "T7" oligonucleotides (Table S1). Probes were synthesized by T7 RNAP with [-32 P]-UTP yielding uniformly labeled RNAs [START_REF] Hajnsdorf | Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I[END_REF]. Membranes were also probed with M1 or 5S as loading control by using 5'-end labeled primers (Table S2). An RNA-seq analysis was performed to compare the transcriptomes of the wild-type (N3433) and the RNase III deficient strain (IBPC633). Sample preparation for RNA-seq, 5′-RNA tagging and RNA-seq analysis were performed as in [START_REF] Maes | Landscape of RNA polyadenylation in E. coli[END_REF]. Data have been deposited in the ArrayExpress database at EMBL-EBI under accession number E-MTAB-9507 (M. Lejars, L.Kuhn, A. Maes, P. Hammann, E. Hajnsdorf manuscript in preparation).

-galactosidase assays

Cultures were initiated at A 600 0.05 and sampled at A 600 0.4 or in the case of the results presented in figure S2 also at A 600 1.2. Samples (200 µL) were lysed in 800 µL PBS buffer with 10 µL 0.1% SDS and 20 µL chloroform. β-galactosidase activity was assayed as described [START_REF] Miller | Experiments in molecular genetics[END_REF], results are the mean of at least three biological replicates.

Circular RT-PCR

Circular RT-PCR was performed with total RNA extracted from N3433 and IBPC633 treated with 5′polyphosphatase. After circularization with T4 RNA ligase, mflhD2 was used to prime reverse transcription and mflhD6 and masflhD10 to generate PCR products (Table S2), which were cloned [START_REF] Fontaine | The small RNA SraG participates in PNPase homeostasis[END_REF].

RNA band-shift assay and in vitro processing by RNase III

DNA templates carrying a T7 promoter sequence were generated by PCR using the Term and T7 oligonucleotides (Table S2). They allow the transcription of the first 308 nts flhD and of the first 256 nts of AsflhD. RNAs were synthesized by T7 RNAP with [α-32 P]-UTP as a tracer and were gel purified. Transcripts 5′-end-labelling, hybridization, RNase III digestion and sample analysis were described in [START_REF] Fontaine | The small RNA SraG participates in PNPase homeostasis[END_REF][START_REF] Folichon | The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation[END_REF][START_REF] Maikova | Discovery of new type I toxin-antitoxin systems adjacent to CRISPR arrays in Clostridium difficile[END_REF]. Briefly, radioactive AsflhD was incubated with increasing concentrations of flhD mRNA under two conditions referred to as "native" (incubation in TMN buffer (20 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 100 mM sodium acetate for 5 min at 37•C) and "full RNA duplex" (initial denaturation at 90°C for 2 min, then incubation in 1xTE at 37°C for 30 min). The complexes were loaded on native polyacrylamide gels to control for hybridization efficiency or submitted to in vitro processing by RNase III of E. coli. RNase III digestion of free 5'-radiolabeled AsflhD, flhD or complexed AsflhD with flhD was performed at 37°C in TMN buffer containing 1 µg tRNA for 15 min with RNase III (Epicentre). Samples were loaded on denaturing polyacrylamide gels together with an RNA alkaline ladder as in [START_REF] Folichon | The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation[END_REF].

In vitro transcription assay

Single-round in vitro transcription experiments were carried out on linear templates as described in Supplementary materials and methods.

Motility assay

Stationary phase bacterial cultures (MG1655-B, ML241 (P AsflhD + ) carrying the pCA24N control (Ctl) or the pCA24N AsflhD (As) plasmid) were inoculated (2 µL) on soft-agar (0.2 g/L) SOB motility plates (containing 10 -4 M IPTG and 2.4 g/L MgSO 4 ) at 37°C and pictures were taken using a Gel Doc (Biorad) imager at the beginning and the end of the linear swimming motility period (from 5 to 8 hours). Swimming speed was then calculated as a function of time by comparing motility diameters. ) were grown to mid-log phase (A 600 0.4) at 37°C. Sampling was performed at different times after addition of rifampicin (500 μg/mL) (t0) and total RNA was subjected to northern blot analysis. The membranes were probed successively for flhD and M1. The decay-rate of flhD mRNA was calculated as described in the "quantification and statistical analysis" section of the supplementary materials and methods. and its derivatives, pnp, rnc, pnp-rnc, rne ts , rnc-rne ts mutants (respectively N3433-pnp, IBPC633, IBPC633-pnp, N3431, IBPC637), were grown at 37°C until mid-log phase. Where indicated, cells were grown at 30°C and submitted to a heat-shock at 42°C for 15 min, in order to inactivate RNase E in the strain carrying the thermosensitive rne ts allele. Total RNA was analyzed by northern blotting.

Image treatment, quantifications and statistical analysis are given in Supplementary Materials and Methods

Legends

The membrane was probed successively for AsflhD and 5S. ) and their rnc mutant derivatives (ML65 and ML341 respectively)

were grown to mid-log phase (A 600 0.4) at 37°C. Total RNA was extracted and subjected to northern blot analysis. The membrane was probed successively for flhD and for M1. S1) and corresponds to -76 to +388 of the flhD transcript relative to its TSS, with a 40 nts extension carrying the rrnBT2 terminator (fragment length 504 bp).

This fragment carries the native flhD promoter (-10 and -35 sites) and includes the cAMP/CAP site at -72 compared to the flhD TSS, at its upstream extremity.

In vitro transcription assays were performed as described in the Supplementary material and

Finally, we determined

  the effect of RNase III inactivation on flhD mRNA in the mutant overexpressing AsflhD (P AsflhD + ). Northern blot confirms that, as expected, the abundance of flhD mRNA increases in the rnc strains overexpressing or not AsflhD (Fig.6-D). Remarkably, the 220 nts long fragment observed upon RNase III inactivation (Figs.4-B, 5-B) strongly accumulates when AsflhD is overexpressed (Fig.6-D). Hence this suggests that the increase in strength of the AsflhD promoter drives the accumulation of the small fragment corresponding to the 5'-UTR of flhD mRNA, presumably as a duplex with AsflhD and that both are rapidly degraded by RNase III. This short flhD fragment could either be generated by processing of longer flhD mRNA or correspond to premature transcriptional termination of flhD mRNA. In summary, we show that AsflhD is involved in the repression of the expression of flhD at the post-transcriptional level.

The

  flhDC operon encodes the only Class I protein, FlhD 4 C 2 , which is required for expression of class II genes, which in turn control class III genes. We tested the effect of AsflhD on representative Class II and Class III genes. The selected class II genes are fliA that encodes FliA, the sigma factor for class III motility genes, and flgB that encodes FlgB, the main component of the flagella rod. The fliC gene is a class III gene, located upstream from the fliA gene, and it encodes the main component of flagella, FliC. The amounts of fliA, flgB and fliC mRNAs are all very strongly reduced upon overexpression of AsflhD from its endogenous locus in the mid-log phase (Fig.9-ABC). As the reduction is considerably stronger than the effect on flhD itself, this implies that the effect of AsflhD overexpression is amplified compared to that on flhD, similarly to what was previously reported for transcriptional regulators of flhD expression[START_REF] Lehnen | LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli[END_REF][START_REF] Kim | Potential Swimming Motility Variation by AcrR in Escherichia coli[END_REF]. Furthermore, a bioinformatics search (TargetRNA2 (49)) for possible direct trans targets of AsflhD found no candidates amongst genes from the motility cascade. In addition, the abundance of flgB and fliC mRNAs in late-log phase slightly increases relatively to mid-log phase (Fig.S4-AB) therefore this is consistent with the hypothesis that cis-overexpression of AsflhD limits the level but also delays the timing for the induction of the motility cascade.Finally, we analyzed the effect of AsflhD on the motility of bacteria on low-agar plates and observed that overexpression of AsflhD from both the flhD locus (P AsflhD +

  McaS sRNA is required to expose the ribosome binding site and activate translation. On the contrary, binding of the sRNAs OxyS, ArcZ, OmrA and OmrB represses translation[START_REF] Lay | A complex network of small non-coding RNAs regulate motility in Escherichia coli[END_REF][START_REF] Thomason | A small RNA that regulates motility and biofilm formation in response to changes in nutrient availability in Escherichia coli[END_REF]. AsflhD binding can interact at any of these sites along the flhD mRNA to compete with the RNase Emediated degradation pathway and/or the sRNA control of translation. Our results clearly demonstrated that AsflhD represses the transcription of flhD but without excluding that AsflhD may also be involved in the control of the translation of flhD mRNA. However, it is difficult to assess whether AsflhD may have a direct effect on translation or an indirect effect via the competition with the other positive and negative post-transcriptional regulators of translation of flhD.AsflhD and flhD mutually repress their transcription elongation. AsflhD interacting with the 5'-UTR of flhD mRNA, could either provoke the termination of transcription by transcriptional interference or by transcriptional attenuation[START_REF] Naville | Transcription attenuation in bacteria: theme and variations[END_REF], or drive the processing of flhD mRNA (e.g., via RNase III) or inhibit translation. Diverse mechanisms of transcriptional attenuation have been described which are dependent on regulatory RNAs. For example, sRNA binding drives the premature Rhodependent transcription termination of the rpoS mRNA[START_REF] Sedlyarova | sRNA-Mediated Control of Transcription Termination in E. coli[END_REF]. The asRNA RNAβ in Vibrio anguillarum promotes transcription termination on the fatDCBA polycistronic mRNA before RNA polymerase reaches the end of the fatDCBAangRT operon. Remarkably, the region where termination occurs

Figure 1 :

 1 Figure 1: RNase III inactivation stabilizes asRNAs

Figure 2 :

 2 Figure 2: AsphoP and phoP levels are regulated by RNase III

Figure 3 :

 3 Figure 3: Transcriptional regulation of AsflhD

Figure 4 :

 4 Figure 4: Characterization of AsflhD

Figure 5 :

 5 Figure 5: RNase III is involved in the degradation of AsflhD asRNA and flhD mRNA in vivo

Figure 6 :

 6 Figure 6: AsflhD repress the expression of flhD

Figure 7 :

 7 Figure 7: AsflhD is involved in the transcriptional attenuation of flhD

  method (B) with or without addition of 100 nM CAP and 0.2 mM cAMP for 15 min at 37°C before addition of RNA polymerase, (C) with 100 nM CAP and 0.2 mM cAMP and the addition of in vitro purified AsflhD or flhD transcripts to the reaction at the indicated concentrations. Samples were analyzed on sequencing gels. Relative intensity of the indicated bands (flhD in purple and AsflhD in orange) were analyzed. Values are means of 6 (B) or 3 (C) replicates and error bars are standard deviations. Statistical significance was determined by a heteroscedastic two-tailed t test (* for pvalues ≤0.05, ** for p-values ≤0.01 and *** for p-values ≤0.001).

Figure 8 :

 8 Figure 8: AsflhD repress the expression of flhD in trans

Figure 9 :

 9 Figure 9: AsflhD overexpression represses the cascade of motility

Figure 10 :

 10 Figure 10: Schematic representation of the regulatory function of AsflhD

Figure S3 :

 S3 Figure S3: AsflhD repression of the transcription of flhD is independent of the P flhD promoter

Figure S4 :

 S4 Figure S4: AsflhD overexpression repress and delay the induction of the motility cascade

Table 1 :

 1 Effect of AsflhD silencing and overexpression on flhD expression and stability

	Promoter	Relative abundance	flhD Half-
				flhD at t0 (%)	life
					(min)
	P AsflhD	100	0.57 ± 0.15
	P AsflhD	-	65	0.54 ± 0.19
	P AsflhD	+	64	0.45 ± 0.13
	MG1655-B (P AsflhD ), ML73 (P AsflhD	-) and ML241 (P AsflhD	+

Acknowledgements

We thank C. Beloin and J-M Ghigo for providing strains and A. Kolb for the kind gifts of purified RNAP core, σ 70 and CAP. This work was supported by the Centre National de la Recherche Scientifique (UMR8261), Université de Paris, Agence Nationale de la Recherche (asSUPYCO, ANR-12-BSV6-0007-03 to E.H.), (RIBECO ANR-18-CE43-0010 to E. H.) and the "Initiative d'Excellence" program from the French State (Grant "DYNAMO," ANR-11-LABX-0011).

Abbreviations

Antisense RNA (asRNA); RNA-binding protein (RBP); untranslated region (UTR); small RNA (sRNA): open reading frame (ORF); nucleotide (nt); circular RT-PCR (cRT-PCR); RNA polymerase (RNAP)

Supplementary legends

Figure S1: The transcription of AsflhD is not activated by RpoH and RpoS

(A) Expression of AsflhD-lacZ fusion in the wt strain (MG2114 P AsflhD ) and its P lac -rpoH derived strain (ML310) in the presence of 10 -3 M IPTG at 37°C was measured at OD 600 1.