Circadian rhythms in the critical human pathogen Acinetobacter baumannii

Circadian rhythms in the critical human pathogen Acinetobacter baumannii | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Circadian rhythms in the critical human pathogen Acinetobacter baumannii María Alejandra Mussi, Valentín Permingeat, Bárbara Perez Mora, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5277866/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Acinetobacter baumannii is recognized as the paradigm of multidrug resistant superbug, topping the WHO priority list of critical human pathogens. Interestingly, it senses and responds to blue light, which modulates global aspects of its physiology including the pathogenicity. We hypothesized that light could serve as a signal to synchronize the bacterial physiology to the host’s behavior, or to the environment. At environmental temperatures, light regulation is mainly governed by the BLUF-type photoreceptor BlsA. In this work, we identified the existence of daily rhythms in blsA expression displaying a robust response to light, as well as endogenous circadian rhythms in A. baumannii . In fact, we show that blsA gene expression can be synchronized to 24-hour blue light-dark cycles, which immediately resynchronizes after a phase shift due to a longer night. Upon release to constant darkness, bacterial populations present free-running oscillations with a period close to 24 hours. Furthermore, our data indicate that BlsA is involved in synchronization to the zeitgeber during light-dark cycles. Importantly, β-lactamase activity varied along the day in cultures under light-dark period, establishing a new paradigm. Our work contributes to the developing field of circadian clocks in bacterial human pathogens, which could impact the microorganisms’ lifestyle and pathogenicity. Biological sciences/Microbiology/Bacteria/Bacterial physiology Biological sciences/Microbiology/Pathogens Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Circadian rhythms, observed across all domains of life, enable organisms to anticipate and prepare for daily changes in environmental conditions resulting from the periodic movement of the Earth. Among bacteria, circadian rhythms have only been extensively studied in cyanobacteria, which are photosynthetic microorganisms. In fact, genes responsible for the core clock of cyanobacteria have been identified, including kaiA , kaiB , and kaiC , along with other crucial components like the response regulator RpaA and two regulatory histidine kinases, CikA and SasA 1 . Recently, it has been shown that non-photosynthetic soil microorganisms, such as Bacillus subtilis , exhibit a light-dependent circadian system that behaves in a complex fashion, similar to the circadian systems of multicellular eukaryotes 2,3 . Moreover, it was shown that a clinical isolate of the gut bacterium Klebsiella aerogenes , exhibited melatonin and temperature-dependent rhythms in the expression of the motility gene motA 4,5 . Although the relevance of these circadian rhythms in pathogenesis has not been explored, these findings suggest that the circadian clock of K. aerogenes may entrain to host cues in vivo . Although light-dependent circadian rhythms have not been reported in bacterial pathogens, it is well established that important pathogens such as Acinetobacter baumannii , Pseudomonas aeruginosa , Brucella abortus , and Staphylococcus aureus sense and respond to light, showing a global photic modulation of bacterial physiology and, most interestingly, of determinants of pathogenicity 6–11 . A large body of evidence demonstrates that light signal transduction and physiological responses are mainly governed by the blue light using FAD (BLUF)-type photoreceptor BlsA at environmental temperatures in A. baumannii 6,7,12 . In fact, blue light modulates iron uptake, tolerance and susceptibility to antibiotics, desiccation tolerance, competition with other microorganisms, antioxidant enzyme levels, metabolism, biofilm formation, and quorum sensing 6,7,13–18 . BlsA is a global regulator able to bind and antagonize the functioning of different transcriptional regulators such as Fur, the iron metabolism repressor; and AcoN, the acetoin catabolism repressor, in a light-dependent manner 15,16 . We have also shown that A. baumannii responds to light modulating virulence in a human keratinocyte epithelial infection model 7 as well as quorum sensing 17 at 37ºC, through a BlsA-independent mechanism 7,17 . Moreover, the two-component system BfmRS has been shown to transduce light signaling as it is involved in photoregulation of motility and desiccation tolerance in A. baumannii strain V15, while evidence strongly indicates that BfmR phosphorylation levels are also modulated by light 18 . Finally, BfmRS antagonizes blsA transcription 18 , and has been shown to physically interact with BlsA 19 . In this work, we studied the existence of circadian rhythms in the critical pathogen A. baumannii , which presents an alarming propensity to develop multi-drug resistance 17–20 leading to serious outbreaks in the hospital setting 20,21 . Infections associated with multi-drug resistant (MDR) A. baumannii are linked to higher rates of morbidity and mortality 18, 20 . Moreover, there are increasing reports of community-acquired A. baumannii infections across the globe, suggesting the existence of extra-hospital reservoirs. Accordingly, carbapenem-resistant Acinetobacter recently topped the WHO priority list of bacteria that require research and development of novel therapeutic strategies 22 . Our results show rhythmic oscillations in blsA expression presenting a robust response to blue light configuring daily rhythms, as well as endogenous circadian rhythms under constant conditions. In fact, we show that blsA gene expression can be synchronized to 24-hour blue light-dark cycles, which resynchronize immediately after a phase shift due to a longer night. Upon release to constant darkness and temperature conditions, bacterial populations present free running (FR) oscillations with a period close to 24 hours showing a phase shift from light-dark conditions, possibly indicative of circadian masking. Our data also indicate that the photoreceptor BlsA is involved in synchronization to the zeitgeber during light-dark cycles but does not influence the endogenous rhythm. Importantly, we detect differences in β-lactamase activity along the day in the light-dark period, establishing a new paradigm. The existence of such rhythms could impact virulence, antibiotic susceptibility or persistence, opening new paradigms for treatment of infections produced by these pathogens. Results Rhythmic oscillations in blsA expression. We have shown that blue light exerts a global modulation of A. baumannii ’s physiology at moderate temperatures such as 23 ºC through the blsA photoreceptor 6,7,12,14–18,23 . To study the existence of rhythms in blsA expression in light-entrained and dark-released cultures, A. baumannii V15 strain cells were inoculated in LB broth, placed in 24 well-plates and incubated under a 12 h blue light (bL)- 12 h dark (D) 12L:12D photoperiod for 5 days and then released to constant darkness at 23ºC. Samples corresponding to individual wells were recovered at 4 hour-intervals for 4 days corresponding to the 4th and 5th days of bLD entrainment as well as the 1st and 2nd days in constant darkness (DD), and blsA RNA levels were quantified by retrotranscription (RT) followed by quantitative PCR (qPCR), RT-qPCR, using blsA specific primers 14 . Our data show oscillations in blsA expression along different times of the day both in entrained as well as dark-released cultures (Fig. 1 ). A JTK_Cycle analysis implemented in the R package MetaCycle further indicated rhythmic components in blsA oscillations 24 (Fig. 1 ). Overall, our results show the existence of rhythmic oscillations in expression of the blsA gene determined by RT-qPCR. Daily and free-running rhythms of blsA promoter activity in A. baumannii. To validate the above results and characterize the rhythms more accurately, we next evaluated blsA promoter activity using a luciferase reporter transcriptional fusion. For this, A. baumannii V15 pLPV1Z- pblsA::luc clone 1 cultures were grown stagnantly in LB broth in 12L:12D cycles for 4 days and then released to constant darkness for 2.5 more days, at 23ºC. This strain expresses a luciferase reporter under the control of the blsA promoter from the pLPV1Z plasmid 25 . Results shown in Fig. 2 A represent the total of wells (light blue shadow) and average (blue line) from one representative experiment of a total of 3 biological replicates. We first analyzed whether blue light could act as a zeitgeber in A. baumannii . The pblsA::luc reporter activity showed that blsA expression responds to LD cycles establishing a robust diurnal rhythm with a clear phase relationship with the zeitgeber, in which expression increases during the dark phase and decreases during the light phase (Fig. 2 A). The higher or lower maximal values of gene expression tend to anticipate zeitgeber transitions, suggesting that the bacteria are not only passively responding to external stimuli (Fig. 2 A). Interestingly, a free-running (FR) rhythm in blsA promoter activity was maintained when the cultures were released to DD (Fig. 2 A), showing a calculated period of 26 ± 2.4 hours (Fig. 2 B), indicating the existence of a genuine circadian component. Under LD conditions, the bioluminescence peaks occurred at zeitgeber time (ZT) 22.9 ± 0.8 h, which shifted to circadian time (CT) 14.6 ± 1.5 h under DD (Fig. 2 C). The fact that the circadian phase was not maintained when cultures were transferred to DD (Fig. 2 C), suggests that canonical light-entrainment is not occurring but rather a zeitgeber masking effect is taking place. In other words, the blue light zeitgeber is able to synchronize blsA expression rhythms with a robust and clear phase relationship, which is lost under constant conditions although the bacterial populations still remain rhythmic, indicating the presence of an endogenous circadian oscillator. Figure 2 D shows luminescence data retrieved from individual wells representative of the three independent experiments performed. Supplementary Fig. 1 shows similar experiments as above but performed using strains A. baumannii V15 pLPV1Z- pblsA::luc clones 2–4, recovered from independent transformation events. The data for these clones is consistent with that shown for clone 1. A. baumannii instantaneously resynchronizes to changes in the blue light zeitgeber. To further characterize the rhythm response to blue light in A. baumannii , we lengthened the third LD night by 6 hours so that the bacteria faced 18 hours of darkness, followed by reestablishment of the 12bL:12D photoperiod for additional 3 days and a final release to darkness for 2 days. Figure 3 shows a robust adaptation of the A. baumannii ’s daily rhythm to the zeitgeber, presenting an instantaneous resynchronization to the zeitgeber’s new phase. It is interesting to note that the amplitude of the peaks remained conserved respect to the previous LD days before night lengthening, indicating that blsA expression is not affected by culture aging or nutrient deprivation. From this, it follows that the reduction in amplitude observed when cultures are released to DD in Fig. 2 A or D is a characteristic of the endogenous rhythms and not an artifact resulting from those conditions. Finally, when cultures were released to DD after resynchronization, endogenous circadian rhythms were observed, while the phase was not conserved with respect to the new LD phase, indicating masking and consistency with previous results shown in Fig. 2 and Supplementary Fig. 1. Overall, there is a strong response to the blue light zeitgeber in blsA expression, where masking appears as a predominant component leading to instantaneous resynchronization, again showing endogenous circadian rhythms under constant illumination conditions. Rhythms are maintained under constant dark conditions in A. baumannii. To further explore endogenous circadian rhythms in A. baumannii , we next decided to study the blsA promoter activity under constant dark conditions measuring emitted bioluminescence at discrete time points for at least 3 continuous days spanning a total of 6 days in at least 4 independent experiments. Representative experiments are shown in Fig. 4 , in which the presence of rhythmic oscillations is observed in blsA expression with a calculated period of 23.45 h. Thus, endogenous circadian rhythms are observed under constant dark conditions. Supplementary Fig. 2 shows results for the other independent experiments. BlsA is involved in synchronization to blue light in A. baumannii. We further evaluated the behavior of another A. baumannii strain, ATCC 17978, under similar conditions as those described above. Our results show that 17978 harboring plasmid pLPV1Z- pblsA::luc presented a robust response to the light zeitgeber during the LD photoperiod similarly to the V15 strain, with a nocturnal increase in expression (Fig. 5 A). Moreover, the presence of an endogenous rhythm was observed in DD, which exhibited characteristics of canonical light entrainment (64% of synchronized and rhythmic wells were also entrained; Fig. 5 A). On the other hand, the ΔblsA mutant strain induced a significant decrease in circadian amplitude under LD conditions, while the endogenous rhythm was maintained in DD (Fig. 5 A-B). Most interestingly, synchronization to the light zeitgeber as well as entrainment were both compromised in ΔblsA mutant populations (approximately 30% of wells; Chi-square test, p < 0.005), consistent with synchronization being required for entrainment. The acrophase dispersion to rhythmic populations was higher in the LD for 17978 ΔblsA strain (Watson-Wheeler test for homogeneity of angles, p = 0.0312, * p < 0,05) (Fig. 5 C). We did not observe any significant differences in the circadian period in ΔblsA mutant (24.5 ± 1.1 h, n = 12 mutant rhythmic, n total = 36, 33% rhythmic vs. 24.8 ± 1.4 h, n = 14 control rhythmic, n total = 23, 61% rhythmic) (Fig. 5 D). Thus, the overall results indicate that BlsA is involved in synchronization to the zeitgeber and entrainment, but does not affect the endogenous rhythm component. β-lactamase activity fluctuates along the day. A. baumannii is the paradigm of bacterial multidrug resistance, as circulating strains are invariably resistant to last generation antibiotics including the β-lactams carbapenems, which seriously complicate therapeutics. As we previously showed the existence of daily rhythms in this microorganism, we decided to evaluate next whether β-lactamase activity also oscillates along the day. For this purpose, we used the A. baumannii multidrug resistant strain Ab825, since both V15 and ATCC 17978 are sensitive to multiple drugs. Ab825 cells were grown under 12L:12D photocycle for 4 days, and β-lactamase activity levels were determined at two different times along the day of the 3rd and 4th days (LD3 and LD4): 7 am and 7 pm, which reflect the end of the dark and light phases, respectively. It should be noted that we performed β-lactamase activity determination to acquire an instantaneous measurement at different hours of the day, in a growth-independent manner. Our results show oscillations in β-lactamase activity levels along the day both in L3 and L4 (mean ratio of the ratio/OD different for at least one measurement time, p = 0.0045), which were higher at the end of the light phase respect to the end of the dark one (post-hoc multiple comparisons significant at 10%, Fig. 6 ). These results indicate that oscillations are observed not only at the gene expression level but also in cellular processes as important in A. baumannii lifestyle as is antibiotic-inactivating activity. These findings establish a new paradigm. Discussion It has been recently ascertained that many non-phototrophic pathogens are capable of sensing and responding to light via specialized molecular systems such as photoreceptors. In fact, we have extensively shown that the critical human pathogen A. baumannii presents a global response to light, including modulation of pathogenicity and virulence. This prompted us to determine whether this response to light is actually part of a more specialized mechanism such as a light-dependent circadian pacemaker. In this work, we provide evidence indicating that blsA expression shows a robust response to LD cycles leading to daily rhythms, while endogenous free-running rhythms were observed under constant darkness. Daily rhythms are very important as they show variations in bacterial physiology due to changing environmental conditions throughout the day, while the existence of endogenous rhythms indicate that this pathogen has evolved mechanisms to anticipate periodic variations in these conditions. Interestingly, our data show no phase conservation between LD and DD in V15 strain, suggesting that canonical light entrainment of blsA expression is not taking place in this case, but rather a masking mechanism induced by the zeitgeber. Masking is an important evolutionary mechanism that allows an organism to respond to changes in exogenous stimuli (e.g., light-dark cycle, social cues, temperature, food, drugs), thereby enabling the organism to act immediately and appropriately 26 . In addition, masking can complement and integrate with entrainment if the cues align with arousal and the circadian system 26 . The circadian response to light depends on the timing of light exposure, as well as its intensity, duration, wavelength, and prior light exposure history. For example, the threshold for human entrainment requires relatively bright light for a long duration; on the contrary, the circadian system of mice is exquisitely sensitive to light 27 : a 12L:12D photocycle using light intensities as low as 0.01–0.1 lux will photoentrain murine rhythms. While it is possible that the minimum light timing information necessary for entrainment has not been reached in our experiments, this possibility would be unlikely in our setup given the robust response observed in the LD cycle. On the other side, short-wavelength (‘blue’) light is significantly more effective for photoentrainment compared to longer wavelengths of light 27 . In fact, blue light sensing is an integral part of circadian rhythms in all the experimental models examined so far. Moreover, it should be noted that we are using very low light intensity (8 µE m − 2 s − 1 ), more than 4 times lower than that reported in photoentrained circadian rhythms evidenced in B. subtilis 2 . Thus, a priori , we would not expect the blue light intensity used in our setup to be too high to lead to masking effects prevailing over entrainment. Also interesting is that in DD the whole bacterial population behaves similarly, i.e. blsA expression increases and decreases following the same pattern in different wells, indicating that they are synchronized to some external clue, which our experiments suggest is environmental light. Indeed, as non-photosynthetic bacteria constitute poorly characterized organisms in terms of circadian rhythmicity, we can conclude that synchronization by light is a conserved mechanism, rather than phase conservation between LD and DD. In fact, bacteria introduce a new dimension in the understanding of circadian rhythms. It would thus be not surprising that non-canonical features are discovered regarding circadian rhythms in these microorganisms. It is also likely that different strains present differential characteristics regarding synchronization and entrainment by light, as well as endogenous circadian components; most probably because of differential lifestyle, antibiotic susceptibility profile, pathogenicity, etc. Such is the case of strain ATCC 17978, which, in contrast to V15 strain, shows not only synchronization but also entrainment of the endogenous rhythm. Moreover, it is possible that the entry route is also part of the clock input pathway; in other words, BlsA could serve both as the photoreceptor sensing blue light and entraining the clock. In fact, our data indicate that the photoreceptor BlsA is involved in synchronization to the zeitgeber during light-dark cycles as well as in entrainment. In this work, we characterized blsA as it is the photoreceptor governing photoregulation at environmental temperatures in A. baumannii . Further work studying other genes and phenotypic responses will lead to gain full understanding into rhythms in this critical pathogen. A. baumannii ’s rhythm was detected in conditions compatible with biofilm formation, as occurred with B. subtilis 2 , which is considered a bacterial social behavior. In this context, another interesting aspect to further explore is the communication among these “independent individuals” that constitute the bacterial population. Several questions arise, such as whether the bacteria interact through quorum sensing, or how is the circadian mechanism transmitted to new generations in dividing short-lived bacteria, considering that the circadian clock is thought to be dependent on state variables, i.e., substances that reflect time, whose concentrations might be disrupted by the cell division process. In this sense, in cyanobacteria a memory effect has been identified, which spans over the cell cycle interval (i.e., the physiological state of a bacterium is sustained for several cell divisions) 25 . In fact, acyl-homoserin lactones, the molecules signaling quorum sensing in Gram-negative bacteria, shares with melatonin the presence of specific motifs with associated functional groups. Since melatonin has been shown to couple circadian rhythms (e.g., Pilorz et al. , 2020 28 ), we suggest the intriguing hypothesis that quorum sensing might serve a similar function in bacteria. A. baumannii is a recent human pathogen, i.e., it is known as an old friend but a new human enemy whose pathogenic character originated from selection due to extensive antibiotic use during the last decades 29 . Therefore, the possibility that it evolved a circadian rhythm to synchronize to the human host is not very straightforward. Yet, A. baumannii is a dual microorganism capable of environmental as well as pathogenic lifestyles. Interestingly, and despite A. baumannii is mainly described as a non-internalizing pathogen, the ability to invade the host’s cells is increasingly recognized in modern strains 30 . This work contributes to establishing that bacterial pathogens are subjected to circadian regulation, which defines a new paradigm, and is the first reporting rhythmicity in Acinetobacter , which opens a new full area of research that will likely lead to reinterpretation of previous data on the pathogen’s behavior. We expect this work will inspire future efforts to investigate whether bacterial pathogens can synchronize their behavior to the host’s circadian rhythm and its immune response, to optimize infection or its persistence in the environment. This would not be surprising given that it has been recently shown that internal timekeeping mechanisms in the malaria parasite synchronize with the host’s circadian rhythm. An example of this is the synchronization of the rupture of red blood cells with the completion of the parasite's asexual cell cycle 31 . Most interestingly, the significance is that bacterial circadian rhythms could potentially impact bacterial persistence in the environment, virulence or antibiotic susceptibility, as we have shown that the activity of β-lactamases fluctuate along the day. Changes on bacterial antibiotic susceptibility, infection outcomes or persistence in the environment influenced by the time of the day could introduce modification of treatment schedules to optimize medical interventions and prevention of critical infections, offering new opportunities for the development of targeted therapeutic strategies to combat infectious diseases and constitute a change in paradigm. Our work contributes to the developing field of circadian clocks in bacterial human pathogens, indicating the existence of daily as well as endogenous circadian rhythms in a critical pathogen, which could impact the microorganisms’ lifestyle and its pathogenicity. Methods Strain and constructions. The promoter region of the blsA gene was amplified using primers blsA _ Eco RI_FW (5′- GAATTCagtattacaaattgaacgtgt − 3′) and blsA _ Bam HI_REV (5′- GGATCCaagacttccgtgaaatataaa − 3′). High fidelity polymerase chain reaction products were digested with Eco RI and Bam HI enzymes (Promega) and cloned into the corresponding sites of pLPV1Z harboring the promoterless luxABCDE genes 25 . The correct construction was verified by sequencing the cloned fragment and pLPV1Z-P blsA -luc was subsequently incorporated into A. baumannii V15 strain by transformation. Light settings. Samples were exposed to blue light emitted by nine-LED (light-emitting diode) arrays with an intensity of 6 to 10 µmol photons/m 2 /s and peak emission centered at 462 nm 6 . Light intensity was measured using a radiometer/photometer (Flame-T, OceanOptics). Temperature was set at 23ºC and fluctuations in the incubator were less than 0.5°C. Zeitgeber (i.e., “time giver” or entraining agent) time 0 or ZT0 (9:00 am) indicates the time at which lights were turned on. Circadian Time (CT) refers to a specific time in the free running conditions (constant darkness, DD, and constant temperature of 23ºC). Photo and thermal conditions were controlled with an I-291PF incubator (INGELAB, Argentina) and temperature was monitored using DS1921H-F5 iButton Thermochrons (Maxim Integrated, USA). Luminescence assays. For all assays, A. baumannii V15 or ATCC 17978 cells harboring plasmid pLPV1Z-P blsA -luc were cultured in white 96-well plates (Greiner) under stagnant conditions in LB broth (250 µl for well) at 23°C from an initial OD 660 of 0,05. Plates were sealed with a transparent optical film (ThermalSeal RT2RRTM, EXCEL Scientific) to avoid evaporation and contamination, and the seal over each well was perforated twice to avoid condensation and allow oxygen exchange. Cultures were exposed to 12 h blue light (bL) and 12 h dark (D) photoperiod (12L:12D) for 4 days and then released to constant darkness, at a constant temperature of 23°C. We measured bioluminescence using a Berthold Centro LB 960 microplate luminometer (Berthold Technologies) stationed inside an incubator (INGELAB) to allow tight control of the blue light and temperature in each experiment. Microwin 2000 software version 4.43 (Mikrotek-441 Laborsysteme) was programmed to leave the plate outside the luminometer after each recording to expose A. baumannii V15 to the environmental cues. The luminescence of each well was integrated for 10 s every 30 min. Temperature fluctuations in the incubator due to lights being on or off were less than 0.5°C. For phase-shift assays, A. baumannii V15 was entrained for 3 days under a bLD cycle and then were subjected to a phase shift caused by a 6-h night extension. After 3 more days, A. baumannii V15 was released into free running (FR) conditions for 2 days. Data acquisition and analysis. Luminescence was sampled at 30 min intervals. Background noise was extracted from the raw data obtained from the luminometer. In all cases, the first 24 to 36 hours of recording were removed due to accumulation of the luciferase enzyme. All raw data was analyzed using the CircaLuc v0.7 program ( https://ispiousas.shinyapps.io/circaluc/ ). The raw data was detrended, smoothed and normalized to the initial maximum value of each sample and plotted using the same program. The data is shown as mean ± SD of luminescence. Subsequently, the circadian period was calculated from the data using the Lomb-Scargle (LS) periodogram within the lomb R package (DOI: 10.1076/brhm.30.2.149.1424 ). In the case of FR rhythms, any signal resulting from the analysis with a period range between 18 h and 33 h, and an R2 adjustment ≥ 0.5 was considered “Circadian”. Final figures were generated using Biorender ( https://app.biorender.com/ ). Background signals lower than 10 folds of magnitude resulting from the bacteria transformed with the empty plasmid or LB broth alone were discarded from the analysis. qRT-PCR bacteria. A. baumannii V15 cells were cultured in 24-well microplates under stagnant conditions in LB broth at 23 ºC from an initial OD 660 of 0,05. The bacteria were incubated for 5 days under 12L/12D photoperiod and then released to constant darkness. 2 ml samples were retrieved every 4 hours from the 4th day of LD entrainment until and including the 2nd day released to darkness. The samples were centrifuged and the pellets were saved at -80ºC until further use. RNA was extracted following procedures described in Muller et al. , 2017 14 . qRT-PCR data analyses. blsA expression data from the last day under LD entrainment, the first day released to darkness and the 2 following points was analyzed using JTK_CYCLE 24 and Lomb-Scargle 32 methods, implemented in the R package MetaCycle 33 . p -values from both procedures were integrated using Fisher’s method, while estimates of period and phase parameters were combined using the arithmetic and circular mean, respectively. RNA extraction. RNA was extracted following procedures described in Mûller et al. , 2017 14 . Instantaneous β-lactamase activity determination along the day in LD cultures. A. baumannii Ab825 cells were grown overnight in LB at 37 ºC in the dark, and then inoculated in fresh new LB media at a DO 600 = 0.05. The bacteria were then grown under 12L:12D photoperiod for 4 days at 23ºC. At 7 am and 7 pm of the 3rd and 4th days (LD3 and LD4), samples were retrieved and processed using a nitrocephin-based colorimetric method for β-lactamase activity detection following the manufacturer recommendations (Amplite Colorimetric Beta-Lactamase Activity, AAT Bioquest). The reactions were incubated at room temperature, with the plate protected from light, and after 60 minutes absorbances at 490, 380, and 600 nm were determined using in a microplate reader (Bio Tek Instruments EPOCH2T). A 490/380 ratio was calculated using these values and normalized to the OD 600 corresponding to the time point for each sample. Four replicates of the experiment were carried out, one of them only measured on the third day. Statistical analysis. For the analysis of antibiotic susceptibility, the effect of measurement time on the mean of ratio/OD was analyzed using a mixed effects model with replicates included as a random effect. Residuals of the fitted model confirmed that necessary assumptions were met. Post-hoc pairwise mean comparisons were conducted using the Kenward-Roger correction. Declarations Competing interests The authors declare no competing interests. Author Contributions Conceptualization: V.P., B.E.P.M., M.L.M., D.A.G. and M.A.M. Formal analysis: V.P., B.E.P.M., M.L.M., N.A., M.L.L., J.I.F., M.B.A., D.A.G. and M.A.M. Funding acquisition: M.A.M and D.A.G. Investigation: V.P., B.E.P.M., M.L.M., M.L.L., J.I.F., M.B.A., G.D.V., M.F., D.A.G. and M.A.M. Methodology: V.P., B.E.P.M., M.L.M., N.A., D.A.G. and M.A.M. Project administration: M.A.M. Visualization: V.P., M.L.M., J.I.F., M.B.A. and M.A.M. (Writing—original draft: M.A.M and D.A.G. Writing—review & editing: V.P., B.E.P.M., M.L.M., J.I.F., M.B.A., G.D.V., M.F., D.A.G. and M.A.M. Acknowledgements This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica to MAM (PICT 2019 − 01484) and DG (PICT 2021 − 1051). MAM, BPM and DG are career investigators of CONICET, while VP and NA are fellows from the same institution. We thank Dr. Adrián E. Granada (Universitat Medizin, Berlin, Germany) for his kind assistance using pyBoat and data analyses. References Swan, J. A., Golden, S. S., LiWang, A. & Partch, C. L. Structure, function, and mechanism of the core circadian clock in cyanobacteria. The Journal of biological chemistry 293 , 5026-5034, doi:10.1074/jbc.TM117.001433 (2018). Eelderink-Chen, Z. et al. A circadian clock in a nonphotosynthetic prokaryote. Sci Adv 7 , doi:10.1126/sciadv.abe2086 (2021). Sartor, F. et al. 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Genome biology 8 , R19, doi:10.1186/gb-2007-8-2-r19 (2007). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryFigure1.png Supplementaryfigure2.tif Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5277866","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":368661935,"identity":"6e510263-1dd3-4af9-8bcd-dbcd63e3c59f","order_by":0,"name":"María Alejandra Mussi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIie2SMUvDQBTH3y3J8jDrTfETCAmFTKGf5R6Fy1JcXBwK3hSX6NxB6rfo6slBu1TnjpGCk0O6FangacUsOa1bkfsNBw/ej/97jwPweA4VcQ4QKgT9UQT7KQsA1K3C1K8KK3fKd/2jcjIuVs3TpB9jeHWvYZSfHoVmXsNrTip8qLuUbDnMOE0HPcRHoWEmzwKUpNi1fbBIHEoANNV0x4eJeVOGSo6pYpXpAcjOwbKlHYxu9EVlFc0+lWi9U6JnhyISTkoLbBW0629MDNyRsnjJuJgN0uprFypRpmNSMg64I2VerNabUf/YXsw09mJ0e2nqptnmGEXdKQ6o3PcbtGz/2O/xeDz/mXdW8mGm290EkQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4168-3624","institution":"Centro de Estudios Fotosintéticos y Bioquímicos - CONICET","correspondingAuthor":true,"prefix":"","firstName":"María","middleName":"Alejandra","lastName":"Mussi","suffix":""},{"id":368661936,"identity":"edc49772-ecb3-4428-91e1-54b7ce31a071","order_by":1,"name":"Valentín Permingeat","email":"","orcid":"","institution":"Centro de Estudios Fotosintéticos y Bioquímicos - CONICET","correspondingAuthor":false,"prefix":"","firstName":"Valentín","middleName":"","lastName":"Permingeat","suffix":""},{"id":368661937,"identity":"ecea38c7-7560-4964-98ac-1a85454579cb","order_by":2,"name":"Bárbara Perez Mora","email":"","orcid":"","institution":"Centro de Estudios Fotosintéticos y Bioquímicos - CONICET","correspondingAuthor":false,"prefix":"","firstName":"Bárbara","middleName":"Perez","lastName":"Mora","suffix":""},{"id":368661938,"identity":"ff1a2850-d13c-4846-8605-bff773181260","order_by":3,"name":"María Migliori","email":"","orcid":"https://orcid.org/0009-0004-3588-8430","institution":"","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Migliori","suffix":""},{"id":368661939,"identity":"0a37ef3a-8bc8-4696-9d0a-91a70d235e4c","order_by":4,"name":"Natalia Arana","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Arana","suffix":""},{"id":368661940,"identity":"9ef72e94-340f-4e77-aaaf-0801b4f4c22e","order_by":5,"name":"Julia Fernández","email":"","orcid":"https://orcid.org/0000-0001-6600-1442","institution":"Facultad de Ciencias Bioquímicas y Farmacéuticas","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Fernández","suffix":""},{"id":368661941,"identity":"ac161c78-f00a-4efd-998f-74df24d4e1d3","order_by":6,"name":"María Belén Allasia","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Belén","lastName":"Allasia","suffix":""},{"id":368661942,"identity":"2c4b1ade-9448-4c51-bd20-854b9a3354a5","order_by":7,"name":"Melisa Lamberti","email":"","orcid":"","institution":"Univerisad Nacional de Quilmes","correspondingAuthor":false,"prefix":"","firstName":"Melisa","middleName":"","lastName":"Lamberti","suffix":""},{"id":368661943,"identity":"2aad4652-90d5-4de6-91b9-659ff342960e","order_by":8,"name":"Gisela Di Venanzio","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Gisela","middleName":"Di","lastName":"Venanzio","suffix":""},{"id":368661944,"identity":"b166e8d4-cd8f-4454-90a0-4fc1d83be8c3","order_by":9,"name":"Mario Feldman","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"prefix":"","firstName":"Mario","middleName":"","lastName":"Feldman","suffix":""},{"id":368661945,"identity":"bbe9e4c0-8367-4d7c-99ca-d3da59ca741a","order_by":10,"name":"Diego Golombek","email":"","orcid":"","institution":"Universidad de San Andrés","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"Golombek","suffix":""}],"badges":[],"createdAt":"2024-10-16 18:16:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5277866/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5277866/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-08732-2","type":"published","date":"2025-09-30T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68143195,"identity":"d1c703c0-a69d-4df3-a652-060034bc6ef4","added_by":"auto","created_at":"2024-11-04 05:25:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":148826,"visible":true,"origin":"","legend":"\u003cp\u003eRhythmic oscillations in \u003cem\u003eblsA\u003c/em\u003e expression. \u003cem\u003eblsA\u003c/em\u003e expression levels determined by RT-qPCR in \u003cem\u003eA. baumannii\u003c/em\u003e V15 wild-type cultured under blue light (L) and in the dark (D) in 12L:12D photoperiod cycles for 5 days, and then released to constant darkness at 23ºC. The data shown are mean ± SE of normalized relative quantities (NRQs) \u003csup\u003e34\u003c/sup\u003e calculated from transcript levels measured in samples grown in LB broth, in at least two biological replicates. Gray and white backgrounds correspond to dark and blue light incubations, respectively.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5277866/v1/375eaa6e3ed16f387a814409.jpg"},{"id":68143192,"identity":"4c9ec2e9-c748-45a9-85d3-43e0ae70fd3d","added_by":"auto","created_at":"2024-11-04 05:25:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1866971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eblsA\u003c/em\u003e–driven luminescence is rhythmic under entrained and FR conditions. (A) Average reporter activity of \u003cem\u003eA. baumannii\u003c/em\u003e V15 pLPV1Z-\u003cem\u003epblsA::luc\u003c/em\u003e clone 1 under blue light/dark (bLD) and FR conditions. This strain was incubated in bLD cycles for 4 days and then released to DD. Black/blue bars indicate dark/light periods. Luminescence signals are shown as mean ± SEM. (B) Average endogenous period of rhythmic populations of \u003cem\u003eA. baumannii\u003c/em\u003e V15 pLPV1Z-\u003cem\u003epblsA::luc\u003c/em\u003eclone 1 (26 ± 2.4 h, n=123). (C) Rayleigh plots showing the phase of the bioluminescent peak under cyclic conditions (bLD, black dots) and the first bioluminescent peak on the first day of release to FR (DD, white dots) for the rhythmic population (bLD: 22.9 ± 0.8 h, n=123; R=0.98 and DD: 14.6 ± 1.5 h, n=123; R=0.92). Lines represent the average peak phase of \u003cem\u003epblsA::luc\u003c/em\u003e expression (mean vectors for the circular distributions) of each group. The length of the vector represents the strength of the phase clustering while the angle of the vector represents the mean phase. Individual data points are plotted outside the circle. The central circle represents the threshold for \u003cem\u003ep \u003c/em\u003e= 0.05. (D) Representative single traces of luciferase activity rhythms from single wells. Bacteria were grown at a constant temperature of 23°C. (B) and (C) analysis included three biological replicates. Number of wells included: 123. One representative biological replicate is shown in (A).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5277866/v1/43802db48f9f5b6c7c013341.png"},{"id":68143191,"identity":"2bd5983e-c4da-421b-bced-38204f4d73dd","added_by":"auto","created_at":"2024-11-04 05:25:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":860642,"visible":true,"origin":"","legend":"\u003cp\u003eLuminescence rhythms respond to a phase shift in the photic periodic conditions. (A) Average population luminescence rhythms after a 6-h phase shift (n = 44) in \u003cem\u003eA. baumannii\u003c/em\u003e V15 pLPV1Z-\u003cem\u003epblsA::luc\u003c/em\u003eclone 1. The arrow indicates the time of the phase shift. Luminescence signals are shown as mean ± SEM. (B) Rayleigh plots of bLD 1 (black dots: 23.2 ± 0.2 h, n = 44; R = 0.99), bLD 2 (blue dots: 22 ± 0.1 h, n = 44; R = 0.99), and DD (white dots: 13.8 ± 0.8 h, n = 44; R = 0.79). Rayleigh test, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Bacteria were grown at a constant temperature of 23°C.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5277866/v1/f29dec6324b14cc6bd0506c6.png"},{"id":68143194,"identity":"f9643936-1d76-431a-a713-3455ffb67385","added_by":"auto","created_at":"2024-11-04 05:25:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":357602,"visible":true,"origin":"","legend":"\u003cp\u003eEndogenous \u003cem\u003eA. baumannii\u003c/em\u003e’s rhythms. \u003cem\u003eA. baumannii\u003c/em\u003e V15 pLPV1Z-\u003cem\u003epblsA::luc\u003c/em\u003eclone 1 was grown stagnantly at 23ºC for 4-6 days under constant dark conditions. Discrete bioluminescent measurements were analyzed using Cosinor. Shown is a representative result from 4 independent experiments.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5277866/v1/32dc3ad77104007c1f3c859a.png"},{"id":68143510,"identity":"a6b27972-565e-41cf-9546-0f445b6e8383","added_by":"auto","created_at":"2024-11-04 05:33:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2083828,"visible":true,"origin":"","legend":"\u003cp\u003eBlue light synchronization of bioluminescent rhythms requires the photoreceptor BlsA.\u003cstrong\u003e \u003c/strong\u003e(A) Reporter activity of \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 strain (n = 23) and isogenic \u003cem\u003eΔblsA\u003c/em\u003e mutant (n = 36) containing pLPV1Z-\u003cem\u003epblsA::luc\u003c/em\u003e under blue light/dark (bLD) and FR conditions. The strains were incubated in bLD cycles for 4 days and then released to DD. Black/blue bars indicate dark/light periods. Luminescence signals are shown as mean ± SEM in red line and all the individual wells are represented in the grey lines. (B) Average amplitude of the luminescence rhythm of rhythmic populations of \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 strain and \u003cem\u003eΔblsA\u003c/em\u003emutant. Two-way ANOVA followed by Sidak's multiple comparisons test. (C) Rayleigh plots showing the acrophases of the bioluminescent signal in bLD, (black dots) and the first bioluminescent peak on the first day of release to FR (DD, white dots); n = 14 for control and n = 12 for mutant. The remaining populations were arrhythmic under FR conditions. Lines represent the average peak phase of \u003cem\u003epblsA::luc\u003c/em\u003eexpression (mean vectors for the circular distributions) of each group. The length of the vector represents the strength of the phase clustering while the angle of the vector represents the mean phase. Individual data points are plotted outside the circle. The central circle represents the threshold for \u003cem\u003ep\u003c/em\u003e= 0.05. (D) Average endogenous period of rhythmic populations of \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 strain (24.8 ± 1.4 h, n=14) and \u003cem\u003eΔblsA\u003c/em\u003e mutant (24.5 ± 1.1 h, n=12). Bacteria were grown at a constant temperature of 23°C.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5277866/v1/adaeb0ccae36852f6789cfa4.png"},{"id":68143509,"identity":"11dd5acd-7e21-4b42-9d01-911406c9d7cc","added_by":"auto","created_at":"2024-11-04 05:33:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":55089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-lactamase activity fluctuates along the day. \u003c/strong\u003e\u003cem\u003eA. baumannii\u003c/em\u003e Ab825 cells were grown overnight in LB at 37 ºC in the dark, and then inoculated in fresh new LB media at a DO\u003csub\u003e600\u003c/sub\u003e= 0.05. The bacteria were then grown under 12L:12D photoperiod for 4 days at 23ºC. At 7 am and 7 pm of the 3rd and 4th days (LD3 and LD4), samples were retrieved and processed using a nitrocephin-based colorimetric method for β-lactamase activity detection following the manufacturer recommendations (Amplite Colorimetric Beta-Lactamase Activity, AAT Bioquest). The reactions were incubated at room temperature, with the plate protected from light, and after 60 minutes absorbances at 490, 380, and 600 nm were determined using in a microplate reader (Bio Tek Instruments EPOCH2T). 490/380 ratios were calculated using these values and normalized to the OD\u003csub\u003e600\u003c/sub\u003e corresponding to the time point for each sample. Colored lines in each time point show mean values±standard deviation.\u0026nbsp; Segments in black indicate p-values of significant post-hoc comparisons based on a mixed model with random replicate effect.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5277866/v1/218459455f4696e2d4a50a7a.png"},{"id":92567946,"identity":"3eb65856-6446-4539-9654-2d0f71074d13","added_by":"auto","created_at":"2025-10-01 07:07:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4770808,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5277866/v1/51761fb1-4edb-483f-873b-bc0a5a716047.pdf"},{"id":68143508,"identity":"b5d3a7e4-3b6e-4ad9-93c8-18e77a657050","added_by":"auto","created_at":"2024-11-04 05:33:55","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1844642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5277866/v1/82def31588af1f1dd47015fa.png"},{"id":68143189,"identity":"34f5c2c7-383a-44ad-8c4d-dd6650d1f319","added_by":"auto","created_at":"2024-11-04 05:25:55","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1424406,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5277866/v1/0df8573b94bc0b18cf670774.tif"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Circadian rhythms in the critical human pathogen Acinetobacter baumannii","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCircadian rhythms, observed across all domains of life, enable organisms to anticipate and prepare for daily changes in environmental conditions resulting from the periodic movement of the Earth. Among bacteria, circadian rhythms have only been extensively studied in cyanobacteria, which are photosynthetic microorganisms. In fact, genes responsible for the core clock of cyanobacteria have been identified, including \u003cem\u003ekaiA\u003c/em\u003e, \u003cem\u003ekaiB\u003c/em\u003e, and \u003cem\u003ekaiC\u003c/em\u003e, along with other crucial components like the response regulator RpaA and two regulatory histidine kinases, CikA and SasA \u003csup\u003e1\u003c/sup\u003e. Recently, it has been shown that non-photosynthetic soil microorganisms, such as \u003cem\u003eBacillus subtilis\u003c/em\u003e, exhibit a light-dependent circadian system that behaves in a complex fashion, similar to the circadian systems of multicellular eukaryotes \u003csup\u003e2,3\u003c/sup\u003e. Moreover, it was shown that a clinical isolate of the gut bacterium \u003cem\u003eKlebsiella aerogenes\u003c/em\u003e, exhibited melatonin and temperature-dependent rhythms in the expression of the motility gene \u003cem\u003emotA\u003c/em\u003e \u003csup\u003e4,5\u003c/sup\u003e. Although the relevance of these circadian rhythms in pathogenesis has not been explored, these findings suggest that the circadian clock of \u003cem\u003eK. aerogenes\u003c/em\u003e may entrain to host cues \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAlthough light-dependent circadian rhythms have not been reported in bacterial pathogens, it is well established that important pathogens such as \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eBrucella abortus\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e sense and respond to light, showing a global photic modulation of bacterial physiology and, most interestingly, of determinants of pathogenicity \u003csup\u003e6\u0026ndash;11\u003c/sup\u003e. A large body of evidence demonstrates that light signal transduction and physiological responses are mainly governed by the blue light using FAD (BLUF)-type photoreceptor BlsA at environmental temperatures in \u003cem\u003eA. baumannii\u003c/em\u003e \u003csup\u003e6,7,12\u003c/sup\u003e. In fact, blue light modulates iron uptake, tolerance and susceptibility to antibiotics, desiccation tolerance, competition with other microorganisms, antioxidant enzyme levels, metabolism, biofilm formation, and quorum sensing \u003csup\u003e6,7,13\u0026ndash;18\u003c/sup\u003e. BlsA is a global regulator able to bind and antagonize the functioning of different transcriptional regulators such as Fur, the iron metabolism repressor; and AcoN, the acetoin catabolism repressor, in a light-dependent manner \u003csup\u003e15,16\u003c/sup\u003e. We have also shown that \u003cem\u003eA. baumannii\u003c/em\u003e responds to light modulating virulence in a human keratinocyte epithelial infection model \u003csup\u003e7\u003c/sup\u003e as well as quorum sensing \u003csup\u003e17\u003c/sup\u003e at 37\u0026ordm;C, through a BlsA-independent mechanism \u003csup\u003e7,17\u003c/sup\u003e. Moreover, the two-component system BfmRS has been shown to transduce light signaling as it is involved in photoregulation of motility and desiccation tolerance in \u003cem\u003eA. baumannii\u003c/em\u003e strain V15, while evidence strongly indicates that BfmR phosphorylation levels are also modulated by light \u003csup\u003e18\u003c/sup\u003e. Finally, BfmRS antagonizes \u003cem\u003eblsA\u003c/em\u003e transcription \u003csup\u003e18\u003c/sup\u003e, and has been shown to physically interact with BlsA \u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this work, we studied the existence of circadian rhythms in the critical pathogen \u003cem\u003eA. baumannii\u003c/em\u003e, which presents an alarming propensity to develop multi-drug resistance \u003csup\u003e17\u0026ndash;20\u003c/sup\u003e leading to serious outbreaks in the hospital setting \u003csup\u003e20,21\u003c/sup\u003e. Infections associated with multi-drug resistant (MDR) \u003cem\u003eA. baumannii\u003c/em\u003e are linked to higher rates of morbidity and mortality \u003csup\u003e18, 20\u003c/sup\u003e. Moreover, there are increasing reports of community-acquired \u003cem\u003eA. baumannii\u003c/em\u003e infections across the globe, suggesting the existence of extra-hospital reservoirs. Accordingly, carbapenem-resistant \u003cem\u003eAcinetobacter\u003c/em\u003e recently topped the WHO priority list of bacteria that require research and development of novel therapeutic strategies \u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur results show rhythmic oscillations in \u003cem\u003eblsA\u003c/em\u003e expression presenting a robust response to blue light configuring daily rhythms, as well as endogenous circadian rhythms under constant conditions. In fact, we show that \u003cem\u003eblsA\u003c/em\u003e gene expression can be synchronized to 24-hour blue light-dark cycles, which resynchronize immediately after a phase shift due to a longer night. Upon release to constant darkness and temperature conditions, bacterial populations present free running (FR) oscillations with a period close to 24 hours showing a phase shift from light-dark conditions, possibly indicative of circadian masking. Our data also indicate that the photoreceptor BlsA is involved in synchronization to the zeitgeber during light-dark cycles but does not influence the endogenous rhythm. Importantly, we detect differences in β-lactamase activity along the day in the light-dark period, establishing a new paradigm.\u003c/p\u003e \u003cp\u003eThe existence of such rhythms could impact virulence, antibiotic susceptibility or persistence, opening new paradigms for treatment of infections produced by these pathogens.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eRhythmic oscillations in blsA expression.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe have shown that blue light exerts a global modulation of \u003cem\u003eA. baumannii\u003c/em\u003e\u0026rsquo;s physiology at moderate temperatures such as 23 \u0026ordm;C through the \u003cem\u003eblsA\u003c/em\u003e photoreceptor \u003csup\u003e6,7,12,14\u0026ndash;18,23\u003c/sup\u003e. To study the existence of rhythms in \u003cem\u003eblsA\u003c/em\u003e expression in light-entrained and dark-released cultures, \u003cem\u003eA. baumannii\u003c/em\u003e V15 strain cells were inoculated in LB broth, placed in 24 well-plates and incubated under a 12 h blue light (bL)- 12 h dark (D) 12L:12D photoperiod for 5 days and then released to constant darkness at 23\u0026ordm;C. Samples corresponding to individual wells were recovered at 4 hour-intervals for 4 days corresponding to the 4th and 5th days of bLD entrainment as well as the 1st and 2nd days in constant darkness (DD), and \u003cem\u003eblsA\u003c/em\u003e RNA levels were quantified by retrotranscription (RT) followed by quantitative PCR (qPCR), RT-qPCR, using \u003cem\u003eblsA\u003c/em\u003e specific primers \u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur data show oscillations in \u003cem\u003eblsA\u003c/em\u003e expression along different times of the day both in entrained as well as dark-released cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A JTK_Cycle analysis implemented in the R package MetaCycle further indicated rhythmic components in \u003cem\u003eblsA\u003c/em\u003e oscillations \u003csup\u003e24\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Overall, our results show the existence of rhythmic oscillations in expression of the \u003cem\u003eblsA\u003c/em\u003e gene determined by RT-qPCR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDaily and free-running rhythms of blsA promoter activity in A. baumannii.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo validate the above results and characterize the rhythms more accurately, we next evaluated \u003cem\u003eblsA\u003c/em\u003e promoter activity using a luciferase reporter transcriptional fusion. For this, \u003cem\u003eA. baumannii\u003c/em\u003e V15 pLPV1Z-\u003cem\u003epblsA::luc\u003c/em\u003e clone 1 cultures were grown stagnantly in LB broth in 12L:12D cycles for 4 days and then released to constant darkness for 2.5 more days, at 23\u0026ordm;C. This strain expresses a luciferase reporter under the control of the \u003cem\u003eblsA\u003c/em\u003e promoter from the pLPV1Z plasmid \u003csup\u003e25\u003c/sup\u003e. Results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA represent the total of wells (light blue shadow) and average (blue line) from one representative experiment of a total of 3 biological replicates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe first analyzed whether blue light could act as a zeitgeber in \u003cem\u003eA. baumannii\u003c/em\u003e. The \u003cem\u003epblsA::luc\u003c/em\u003e reporter activity showed that \u003cem\u003eblsA\u003c/em\u003e expression responds to LD cycles establishing a robust diurnal rhythm with a clear phase relationship with the zeitgeber, in which expression increases during the dark phase and decreases during the light phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The higher or lower maximal values of gene expression tend to anticipate zeitgeber transitions, suggesting that the bacteria are not only passively responding to external stimuli (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Interestingly, a free-running (FR) rhythm in \u003cem\u003eblsA\u003c/em\u003e promoter activity was maintained when the cultures were released to DD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), showing a calculated period of 26\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating the existence of a genuine circadian component.\u003c/p\u003e \u003cp\u003eUnder LD conditions, the bioluminescence peaks occurred at zeitgeber time (ZT) 22.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 h, which shifted to circadian time (CT) 14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 h under DD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The fact that the circadian phase was not maintained when cultures were transferred to DD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggests that canonical light-entrainment is not occurring but rather a zeitgeber masking effect is taking place. In other words, the blue light zeitgeber is able to synchronize \u003cem\u003eblsA\u003c/em\u003e expression rhythms with a robust and clear phase relationship, which is lost under constant conditions although the bacterial populations still remain rhythmic, indicating the presence of an endogenous circadian oscillator.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD shows luminescence data retrieved from individual wells representative of the three independent experiments performed. Supplementary Fig.\u0026nbsp;1 shows similar experiments as above but performed using strains \u003cem\u003eA. baumannii\u003c/em\u003e V15 pLPV1Z-\u003cem\u003epblsA::luc\u003c/em\u003e clones 2\u0026ndash;4, recovered from independent transformation events. The data for these clones is consistent with that shown for clone 1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eA. baumannii instantaneously resynchronizes to changes in the blue light zeitgeber.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further characterize the rhythm response to blue light in \u003cem\u003eA. baumannii\u003c/em\u003e, we lengthened the third LD night by 6 hours so that the bacteria faced 18 hours of darkness, followed by reestablishment of the 12bL:12D photoperiod for additional 3 days and a final release to darkness for 2 days. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows a robust adaptation of the \u003cem\u003eA. baumannii\u003c/em\u003e\u0026rsquo;s daily rhythm to the zeitgeber, presenting an instantaneous resynchronization to the zeitgeber\u0026rsquo;s new phase. It is interesting to note that the amplitude of the peaks remained conserved respect to the previous LD days before night lengthening, indicating that \u003cem\u003eblsA\u003c/em\u003e expression is not affected by culture aging or nutrient deprivation. From this, it follows that the reduction in amplitude observed when cultures are released to DD in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA or D is a characteristic of the endogenous rhythms and not an artifact resulting from those conditions. Finally, when cultures were released to DD after resynchronization, endogenous circadian rhythms were observed, while the phase was not conserved with respect to the new LD phase, indicating masking and consistency with previous results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Fig.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, there is a strong response to the blue light zeitgeber in \u003cem\u003eblsA\u003c/em\u003e expression, where masking appears as a predominant component leading to instantaneous resynchronization, again showing endogenous circadian rhythms under constant illumination conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRhythms are maintained under constant dark conditions in A. baumannii.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further explore endogenous circadian rhythms in \u003cem\u003eA. baumannii\u003c/em\u003e, we next decided to study the \u003cem\u003eblsA\u003c/em\u003e promoter activity under constant dark conditions measuring emitted bioluminescence at discrete time points for at least 3 continuous days spanning a total of 6 days in at least 4 independent experiments. Representative experiments are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, in which the presence of rhythmic oscillations is observed in \u003cem\u003eblsA\u003c/em\u003e expression with a calculated period of 23.45 h. Thus, endogenous circadian rhythms are observed under constant dark conditions. Supplementary Fig.\u0026nbsp;2 shows results for the other independent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBlsA is involved in synchronization to blue light in A. baumannii.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe further evaluated the behavior of another \u003cem\u003eA. baumannii\u003c/em\u003e strain, ATCC 17978, under similar conditions as those described above. Our results show that 17978 harboring plasmid pLPV1Z-\u003cem\u003epblsA::luc\u003c/em\u003e presented a robust response to the light zeitgeber during the LD photoperiod similarly to the V15 strain, with a nocturnal increase in expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Moreover, the presence of an endogenous rhythm was observed in DD, which exhibited characteristics of canonical light entrainment (64% of synchronized and rhythmic wells were also entrained; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). On the other hand, the \u003cem\u003eΔblsA\u003c/em\u003e mutant strain induced a significant decrease in circadian amplitude under LD conditions, while the endogenous rhythm was maintained in DD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Most interestingly, synchronization to the light zeitgeber as well as entrainment were both compromised in \u003cem\u003eΔblsA\u003c/em\u003e mutant populations (approximately 30% of wells; Chi-square test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005), consistent with synchronization being required for entrainment. The acrophase dispersion to rhythmic populations was higher in the LD for 17978 \u003cem\u003eΔblsA\u003c/em\u003e strain (Watson-Wheeler test for homogeneity of angles, p\u0026thinsp;=\u0026thinsp;0.0312, *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). We did not observe any significant differences in the circadian period in \u003cem\u003eΔblsA\u003c/em\u003e mutant (24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 h, n\u0026thinsp;=\u0026thinsp;12 mutant rhythmic, n total\u0026thinsp;=\u0026thinsp;36, 33% rhythmic vs. 24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 h, n\u0026thinsp;=\u0026thinsp;14 control rhythmic, n total\u0026thinsp;=\u0026thinsp;23, 61% rhythmic) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Thus, the overall results indicate that BlsA is involved in synchronization to the \u003cem\u003ezeitgeber\u003c/em\u003e and entrainment, but does not affect the endogenous rhythm component.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eβ-lactamase activity fluctuates along the day.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. baumannii\u003c/em\u003e is the paradigm of bacterial multidrug resistance, as circulating strains are invariably resistant to last generation antibiotics including the β-lactams carbapenems, which seriously complicate therapeutics. As we previously showed the existence of daily rhythms in this microorganism, we decided to evaluate next whether β-lactamase activity also oscillates along the day. For this purpose, we used the \u003cem\u003eA. baumannii\u003c/em\u003e multidrug resistant strain Ab825, since both V15 and ATCC 17978 are sensitive to multiple drugs. Ab825 cells were grown under 12L:12D photocycle for 4 days, and β-lactamase activity levels were determined at two different times along the day of the 3rd and 4th days (LD3 and LD4): 7 am and 7 pm, which reflect the end of the dark and light phases, respectively. It should be noted that we performed β-lactamase activity determination to acquire an instantaneous measurement at different hours of the day, in a growth-independent manner. Our results show oscillations in β-lactamase activity levels along the day both in L3 and L4 (mean ratio of the ratio/OD different for at least one measurement time, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0045), which were higher at the end of the light phase respect to the end of the dark one (post-hoc multiple comparisons significant at 10%, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results indicate that oscillations are observed not only at the gene expression level but also in cellular processes as important in \u003cem\u003eA. baumannii\u003c/em\u003e lifestyle as is antibiotic-inactivating activity. These findings establish a new paradigm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt has been recently ascertained that many non-phototrophic pathogens are capable of sensing and responding to light via specialized molecular systems such as photoreceptors. In fact, we have extensively shown that the critical human pathogen \u003cem\u003eA. baumannii\u003c/em\u003e presents a global response to light, including modulation of pathogenicity and virulence. This prompted us to determine whether this response to light is actually part of a more specialized mechanism such as a light-dependent circadian pacemaker. In this work, we provide evidence indicating that \u003cem\u003eblsA\u003c/em\u003e expression shows a robust response to LD cycles leading to daily rhythms, while endogenous free-running rhythms were observed under constant darkness.\u003c/p\u003e \u003cp\u003eDaily rhythms are very important as they show variations in bacterial physiology due to changing environmental conditions throughout the day, while the existence of endogenous rhythms indicate that this pathogen has evolved mechanisms to anticipate periodic variations in these conditions.\u003c/p\u003e \u003cp\u003eInterestingly, our data show no phase conservation between LD and DD in V15 strain, suggesting that canonical light entrainment of \u003cem\u003eblsA\u003c/em\u003e expression is not taking place in this case, but rather a masking mechanism induced by the zeitgeber. Masking is an important evolutionary mechanism that allows an organism to respond to changes in exogenous stimuli (e.g., light-dark cycle, social cues, temperature, food, drugs), thereby enabling the organism to act immediately and appropriately \u003csup\u003e26\u003c/sup\u003e. In addition, masking can complement and integrate with entrainment if the cues align with arousal and the circadian system \u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe circadian response to light depends on the timing of light exposure, as well as its intensity, duration, wavelength, and prior light exposure history. For example, the threshold for human entrainment requires relatively bright light for a long duration; on the contrary, the circadian system of mice is exquisitely sensitive to light \u003csup\u003e27\u003c/sup\u003e: a 12L:12D photocycle using light intensities as low as 0.01\u0026ndash;0.1 lux will photoentrain murine rhythms. While it is possible that the minimum light timing information necessary for entrainment has not been reached in our experiments, this possibility would be unlikely in our setup given the robust response observed in the LD cycle. On the other side, short-wavelength (\u0026lsquo;blue\u0026rsquo;) light is significantly more effective for photoentrainment compared to longer wavelengths of light \u003csup\u003e27\u003c/sup\u003e. In fact, blue light sensing is an integral part of circadian rhythms in all the experimental models examined so far. Moreover, it should be noted that we are using very low light intensity (8 \u0026micro;E m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), more than 4 times lower than that reported in photoentrained circadian rhythms evidenced in \u003cem\u003eB. subtilis\u003c/em\u003e \u003csup\u003e2\u003c/sup\u003e. Thus, \u003cem\u003ea priori\u003c/em\u003e, we would not expect the blue light intensity used in our setup to be too high to lead to masking effects prevailing over entrainment. Also interesting is that in DD the whole bacterial population behaves similarly, i.e. \u003cem\u003eblsA\u003c/em\u003e expression increases and decreases following the same pattern in different wells, indicating that they are synchronized to some external clue, which our experiments suggest is environmental light. Indeed, as non-photosynthetic bacteria constitute poorly characterized organisms in terms of circadian rhythmicity, we can conclude that synchronization by light is a conserved mechanism, rather than phase conservation between LD and DD. In fact, bacteria introduce a new dimension in the understanding of circadian rhythms. It would thus be not surprising that non-canonical features are discovered regarding circadian rhythms in these microorganisms. It is also likely that different strains present differential characteristics regarding synchronization and entrainment by light, as well as endogenous circadian components; most probably because of differential lifestyle, antibiotic susceptibility profile, pathogenicity, etc. Such is the case of strain ATCC 17978, which, in contrast to V15 strain, shows not only synchronization but also entrainment of the endogenous rhythm.\u003c/p\u003e \u003cp\u003eMoreover, it is possible that the entry route is also part of the clock input pathway; in other words, BlsA could serve both as the photoreceptor sensing blue light and entraining the clock. In fact, our data indicate that the photoreceptor BlsA is involved in synchronization to the zeitgeber during light-dark cycles as well as in entrainment. In this work, we characterized \u003cem\u003eblsA\u003c/em\u003e as it is the photoreceptor governing photoregulation at environmental temperatures in \u003cem\u003eA. baumannii\u003c/em\u003e. Further work studying other genes and phenotypic responses will lead to gain full understanding into rhythms in this critical pathogen.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. baumannii\u003c/em\u003e\u0026rsquo;s rhythm was detected in conditions compatible with biofilm formation, as occurred with \u003cem\u003eB. subtilis\u003c/em\u003e \u003csup\u003e2\u003c/sup\u003e, which is considered a bacterial social behavior. In this context, another interesting aspect to further explore is the communication among these \u0026ldquo;independent individuals\u0026rdquo; that constitute the bacterial population. Several questions arise, such as whether the bacteria interact through quorum sensing, or how is the circadian mechanism transmitted to new generations in dividing short-lived bacteria, considering that the circadian clock is thought to be dependent on state variables, i.e., substances that reflect time, whose concentrations might be disrupted by the cell division process. In this sense, in cyanobacteria a memory effect has been identified, which spans over the cell cycle interval (i.e., the physiological state of a bacterium is sustained for several cell divisions) \u003csup\u003e25\u003c/sup\u003e. In fact, acyl-homoserin lactones, the molecules signaling \u003cem\u003equorum\u003c/em\u003e sensing in Gram-negative bacteria, shares with melatonin the presence of specific motifs with associated functional groups. Since melatonin has been shown to couple circadian rhythms (e.g., Pilorz \u003cem\u003eet al.\u003c/em\u003e, 2020 \u003csup\u003e28\u003c/sup\u003e), we suggest the intriguing hypothesis that \u003cem\u003equorum\u003c/em\u003e sensing might serve a similar function in bacteria.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. baumannii\u003c/em\u003e is a recent human pathogen, i.e., it is known as an old friend but a new human enemy whose pathogenic character originated from selection due to extensive antibiotic use during the last decades \u003csup\u003e29\u003c/sup\u003e. Therefore, the possibility that it evolved a circadian rhythm to synchronize to the human host is not very straightforward. Yet, \u003cem\u003eA. baumannii\u003c/em\u003e is a dual microorganism capable of environmental as well as pathogenic lifestyles. Interestingly, and despite \u003cem\u003eA. baumannii\u003c/em\u003e is mainly described as a non-internalizing pathogen, the ability to invade the host\u0026rsquo;s cells is increasingly recognized in modern strains \u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis work contributes to establishing that bacterial pathogens are subjected to circadian regulation, which defines a new paradigm, and is the first reporting rhythmicity in \u003cem\u003eAcinetobacter\u003c/em\u003e, which opens a new full area of research that will likely lead to reinterpretation of previous data on the pathogen\u0026rsquo;s behavior. We expect this work will inspire future efforts to investigate whether bacterial pathogens can synchronize their behavior to the host\u0026rsquo;s circadian rhythm and its immune response, to optimize infection or its persistence in the environment. This would not be surprising given that it has been recently shown that internal timekeeping mechanisms in the malaria parasite synchronize with the host\u0026rsquo;s circadian rhythm. An example of this is the synchronization of the rupture of red blood cells with the completion of the parasite's asexual cell cycle \u003csup\u003e31\u003c/sup\u003e. Most interestingly, the significance is that bacterial circadian rhythms could potentially impact bacterial persistence in the environment, virulence or antibiotic susceptibility, as we have shown that the activity of β-lactamases fluctuate along the day. Changes on bacterial antibiotic susceptibility, infection outcomes or persistence in the environment influenced by the time of the day could introduce modification of treatment schedules to optimize medical interventions and prevention of critical infections, offering new opportunities for the development of targeted therapeutic strategies to combat infectious diseases and constitute a change in paradigm.\u003c/p\u003e \u003cp\u003eOur work contributes to the developing field of circadian clocks in bacterial human pathogens, indicating the existence of daily as well as endogenous circadian rhythms in a critical pathogen, which could impact the microorganisms\u0026rsquo; lifestyle and its pathogenicity.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eStrain and constructions.\u003c/b\u003e The promoter region of the \u003cem\u003eblsA\u003c/em\u003e gene was amplified using primers \u003cem\u003eblsA\u003c/em\u003e_\u003cem\u003eEco\u003c/em\u003eRI_FW (5\u0026prime;- GAATTCagtattacaaattgaacgtgt \u0026minus;\u0026thinsp;3\u0026prime;) and \u003cem\u003eblsA\u003c/em\u003e_\u003cem\u003eBam\u003c/em\u003eHI_REV (5\u0026prime;- GGATCCaagacttccgtgaaatataaa \u0026minus;\u0026thinsp;3\u0026prime;). High fidelity polymerase chain reaction products were digested with \u003cem\u003eEco\u003c/em\u003eRI and \u003cem\u003eBam\u003c/em\u003eHI enzymes (Promega) and cloned into the corresponding sites of pLPV1Z harboring the promoterless \u003cem\u003eluxABCDE\u003c/em\u003e genes \u003csup\u003e25\u003c/sup\u003e. The correct construction was verified by sequencing the cloned fragment and pLPV1Z-P\u003cem\u003eblsA\u003c/em\u003e-luc was subsequently incorporated into \u003cem\u003eA. baumannii\u003c/em\u003e V15 strain by transformation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLight settings.\u003c/b\u003e Samples were exposed to blue light emitted by nine-LED (light-emitting diode) arrays with an intensity of 6 to 10 \u0026micro;mol photons/m\u003csup\u003e2\u003c/sup\u003e/s and peak emission centered at 462 nm \u003csup\u003e6\u003c/sup\u003e. Light intensity was measured using a radiometer/photometer (Flame-T, OceanOptics). Temperature was set at 23\u0026ordm;C and fluctuations in the incubator were less than 0.5\u0026deg;C.\u003c/p\u003e \u003cp\u003eZeitgeber (i.e., \u0026ldquo;time giver\u0026rdquo; or entraining agent) time 0 or ZT0 (9:00 am) indicates the time at which lights were turned on. Circadian Time (CT) refers to a specific time in the free running conditions (constant darkness, DD, and constant temperature of 23\u0026ordm;C). Photo and thermal conditions were controlled with an I-291PF incubator (INGELAB, Argentina) and temperature was monitored using DS1921H-F5 iButton Thermochrons (Maxim Integrated, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLuminescence assays.\u003c/b\u003e For all assays, \u003cem\u003eA. baumannii\u003c/em\u003e V15 or ATCC 17978 cells harboring plasmid pLPV1Z-P\u003cem\u003eblsA\u003c/em\u003e-luc were cultured in white 96-well plates (Greiner) under stagnant conditions in LB broth (250 \u0026micro;l for well) at 23\u0026deg;C from an initial OD\u003csub\u003e660\u003c/sub\u003e of 0,05. Plates were sealed with a transparent optical film (ThermalSeal RT2RRTM, EXCEL Scientific) to avoid evaporation and contamination, and the seal over each well was perforated twice to avoid condensation and allow oxygen exchange. Cultures were exposed to 12 h blue light (bL) and 12 h dark (D) photoperiod (12L:12D) for 4 days and then released to constant darkness, at a constant temperature of 23\u0026deg;C. We measured bioluminescence using a Berthold Centro LB 960 microplate luminometer (Berthold Technologies) stationed inside an incubator (INGELAB) to allow tight control of the blue light and temperature in each experiment. Microwin 2000 software version 4.43 (Mikrotek-441 Laborsysteme) was programmed to leave the plate outside the luminometer after each recording to expose \u003cem\u003eA. baumannii\u003c/em\u003e V15 to the environmental cues. The luminescence of each well was integrated for 10 s every 30 min. Temperature fluctuations in the incubator due to lights being on or off were less than 0.5\u0026deg;C.\u003c/p\u003e \u003cp\u003eFor phase-shift assays, \u003cem\u003eA. baumannii\u003c/em\u003e V15 was entrained for 3 days under a bLD cycle and then were subjected to a phase shift caused by a 6-h night extension. After 3 more days, \u003cem\u003eA. baumannii\u003c/em\u003e V15 was released into free running (FR) conditions for 2 days.\u003c/p\u003e \u003cp\u003e \u003cb\u003eData acquisition and analysis.\u003c/b\u003e Luminescence was sampled at 30 min intervals. Background noise was extracted from the raw data obtained from the luminometer. In all cases, the first 24 to 36 hours of recording were removed due to accumulation of the luciferase enzyme. All raw data was analyzed using the CircaLuc v0.7 program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ispiousas.shinyapps.io/circaluc/\u003c/span\u003e\u003cspan address=\"https://ispiousas.shinyapps.io/circaluc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The raw data was detrended, smoothed and normalized to the initial maximum value of each sample and plotted using the same program. The data is shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of luminescence. Subsequently, the circadian period was calculated from the data using the Lomb-Scargle (LS) periodogram within the lomb R package (DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1076/brhm.30.2.149.1424\u003c/span\u003e\u003cspan address=\"10.1076/brhm.30.2.149.1424\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). In the case of FR rhythms, any signal resulting from the analysis with a period range between 18 h and 33 h, and an R2 adjustment\u0026thinsp;\u0026ge;\u0026thinsp;0.5 was considered \u0026ldquo;Circadian\u0026rdquo;. Final figures were generated using Biorender (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://app.biorender.com/\u003c/span\u003e\u003cspan address=\"https://app.biorender.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Background signals lower than 10 folds of magnitude resulting from the bacteria transformed with the empty plasmid or LB broth alone were discarded from the analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eqRT-PCR bacteria.\u003c/b\u003e \u003cem\u003eA. baumannii\u003c/em\u003e V15 cells were cultured in 24-well microplates under stagnant conditions in LB broth at 23 \u0026ordm;C from an initial OD\u003csub\u003e660\u003c/sub\u003e of 0,05. The bacteria were incubated for 5 days under 12L/12D photoperiod and then released to constant darkness. 2 ml samples were retrieved every 4 hours from the 4th day of LD entrainment until and including the 2nd day released to darkness. The samples were centrifuged and the pellets were saved at -80\u0026ordm;C until further use. RNA was extracted following procedures described in Muller \u003cem\u003eet al.\u003c/em\u003e, 2017 \u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eqRT-PCR data analyses.\u003c/b\u003e \u003cem\u003eblsA\u003c/em\u003e expression data from the last day under LD entrainment, the first day released to darkness and the 2 following points was analyzed using JTK_CYCLE \u003csup\u003e24\u003c/sup\u003e and Lomb-Scargle \u003csup\u003e32\u003c/sup\u003e methods, implemented in the R package MetaCycle \u003csup\u003e33\u003c/sup\u003e. \u003cem\u003ep\u003c/em\u003e-values from both procedures were integrated using Fisher\u0026rsquo;s method, while estimates of period and phase parameters were combined using the arithmetic and circular mean, respectively.\u003c/p\u003e \u003cp\u003e \u003cem\u003eRNA extraction.\u003c/em\u003e RNA was extracted following procedures described in M\u0026ucirc;ller \u003cem\u003eet al.\u003c/em\u003e, 2017 \u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInstantaneous β-lactamase activity determination along the day in LD cultures.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. baumannii\u003c/em\u003e Ab825 cells were grown overnight in LB at 37 \u0026ordm;C in the dark, and then inoculated in fresh new LB media at a DO\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.05. The bacteria were then grown under 12L:12D photoperiod for 4 days at 23\u0026ordm;C. At 7 am and 7 pm of the 3rd and 4th days (LD3 and LD4), samples were retrieved and processed using a nitrocephin-based colorimetric method for β-lactamase activity detection following the manufacturer recommendations (Amplite Colorimetric Beta-Lactamase Activity, AAT Bioquest). The reactions were incubated at room temperature, with the plate protected from light, and after 60 minutes absorbances at 490, 380, and 600 nm were determined using in a microplate reader (Bio Tek Instruments EPOCH2T). A 490/380 ratio was calculated using these values and normalized to the OD\u003csub\u003e600\u003c/sub\u003e corresponding to the time point for each sample. Four replicates of the experiment were carried out, one of them only measured on the third day.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e For the analysis of antibiotic susceptibility, the effect of measurement time on the mean of ratio/OD was analyzed using a mixed effects model with replicates included as a random effect. Residuals of the fitted model confirmed that necessary assumptions were met. Post-hoc pairwise mean comparisons were conducted using the Kenward-Roger correction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eConceptualization: V.P., B.E.P.M., M.L.M., D.A.G. and M.A.M. Formal analysis: V.P., B.E.P.M., M.L.M., N.A., M.L.L., J.I.F., M.B.A., D.A.G. and M.A.M. Funding acquisition: M.A.M and D.A.G. Investigation: V.P., B.E.P.M., M.L.M., M.L.L., J.I.F., M.B.A., G.D.V., M.F., D.A.G. and M.A.M. Methodology: V.P., B.E.P.M., M.L.M., N.A., D.A.G. and M.A.M. Project administration: M.A.M. Visualization: V.P., M.L.M., J.I.F., M.B.A. and M.A.M. (Writing\u0026mdash;original draft: M.A.M and D.A.G. Writing\u0026mdash;review \u0026amp; editing: V.P., B.E.P.M., M.L.M., J.I.F., M.B.A., G.D.V., M.F., D.A.G. and M.A.M.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from the Agencia Nacional de Promoci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica to MAM (PICT 2019\u0026thinsp;\u0026minus;\u0026thinsp;01484) and DG (PICT 2021\u0026thinsp;\u0026minus;\u0026thinsp;1051). MAM, BPM and DG are career investigators of CONICET, while VP and NA are fellows from the same institution. We thank Dr. Adri\u0026aacute;n E. Granada (Universitat Medizin, Berlin, Germany) for his kind assistance using pyBoat and data analyses.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSwan, J. A., Golden, S. S., LiWang, A. \u0026amp; Partch, C. L. 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Interestingly, it senses and responds to blue light, which modulates global aspects of its physiology including the pathogenicity. We hypothesized that light could serve as a signal to synchronize the bacterial physiology to the host\u0026rsquo;s behavior, or to the environment. At environmental temperatures, light regulation is mainly governed by the BLUF-type photoreceptor BlsA. In this work, we identified the existence of daily rhythms in \u003cem\u003eblsA\u003c/em\u003e expression displaying a robust response to light, as well as endogenous circadian rhythms in \u003cem\u003eA. baumannii\u003c/em\u003e. In fact, we show that \u003cem\u003eblsA\u003c/em\u003e gene expression can be synchronized to 24-hour blue light-dark cycles, which immediately resynchronizes after a phase shift due to a longer night. Upon release to constant darkness, bacterial populations present free-running oscillations with a period close to 24 hours. Furthermore, our data indicate that BlsA is involved in synchronization to the zeitgeber during light-dark cycles. Importantly, β-lactamase activity varied along the day in cultures under light-dark period, establishing a new paradigm. Our work contributes to the developing field of circadian clocks in bacterial human pathogens, which could impact the microorganisms\u0026rsquo; lifestyle and pathogenicity.\u003c/p\u003e","manuscriptTitle":"Circadian rhythms in the critical human pathogen Acinetobacter baumannii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-04 05:25:50","doi":"10.21203/rs.3.rs-5277866/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"307b46b6-aeb1-4d78-bead-d5182074f48d","owner":[],"postedDate":"November 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39228389,"name":"Biological sciences/Microbiology/Bacteria/Bacterial physiology"},{"id":39228390,"name":"Biological sciences/Microbiology/Pathogens"}],"tags":[],"updatedAt":"2025-10-01T07:06:49+00:00","versionOfRecord":{"articleIdentity":"rs-5277866","link":"https://doi.org/10.1038/s42003-025-08732-2","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-09-30 04:00:00","publishedOnDateReadable":"September 30th, 2025"},"versionCreatedAt":"2024-11-04 05:25:50","video":"","vorDoi":"10.1038/s42003-025-08732-2","vorDoiUrl":"https://doi.org/10.1038/s42003-025-08732-2","workflowStages":[]},"version":"v1","identity":"rs-5277866","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5277866","identity":"rs-5277866","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}