Casein kinase 2 (CK2) is highly activated in Alzheimer disease (AD) and is associated with neurofibrillary tangles formation. Phosphorylated SET, a potent PP2A inhibitor, mediates tau hyperphosphorylation in AD. However, whether CK2 phosphorylates SET and regulates tau pathological phosphorylation in AD remains unclear. Here, we show that CK2 phosphorylating SET at Ser9 induced tau hyperphosphorylation in AD. We found that either Aβ treatment or tau overexpression stimulated CK2 activation leading to SET Ser9 hyperphosphorylation in neurons and animal models, while inhibition of CK2 by TBB abolished this event. Overexpression of CK2 in mouse hippocampus via virus injection induced cognitive deficit associated with SET Ser9 hyperphosphorylation. Injection of SET Ser9 phosphorylation mimetic mutant induced tau pathology and behavior impairments. Conversely co-injection of non-phosphorylated SET S9A with CK2 abolished the CK2 overexpression-induced AD pathology and cognitive deficit. Together, our data demonstrate that CK2 phosphorylates SET at Ser9 leading to SET cytoplasmic translocation and inhibition of PP2A resulting in tau pathology and cognitive impairments. Casein kinase 2 (CK2) is highly ubiquitous, evolutionarily conserved in eukaryotic cells . The CK22PP2A), also known as SET, is increased  is compromised in the AD brains , while tncreased . SET is ncreased , where increased , modulatncreased . In the ncreased . Our prencreased . CK2 co-ncreased  while SEncreased , indicatSupplementary Table S1.Site-directed mutations were constructed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Site-directed mutagenesis was carried out to mutate Ser9 of SET to either alanine (S9A) or glutamic acid (S9E), which mimicked the non-phosphorylated or phosphorylated condition of SET respectively. All mutants were generated by PCR and cloned in a His-tagged pcDNA3.1 (-) vector respectively in Xho I and Kpn I restriction sites. We transfected cells with plasmid coding for CK2α, which corresponds to catalytic alpha subunit. All plasmids were sequenced and prepared using an endotoxin-free plasmid extraction kit (Tiangen). AAV2-hTau (full-length 441- amino acid human 4 repeat tau), AAV2-pCAG-SET WT, AAV2-pCAG-SET S9A, AAV2-pCAG-SET S9E, and AAV2-pCAG-CK2 were constructed and packaged by Obio Technology  Co., Ltd. Lipofectamine 2000 transfection reagents were from Invitrogen. Bicinchoninic acid (BCA) protein detection kit was from Pierce . Reagents for cell culture were from Gibco BRL . Antibodies employed in this study are listed in 3×Tg AD mice (PS1m146v/APPswe/TauP301L) were purchased from the Jackson Laboratory. APP/PS1 mice were from the Model Animal Research Center of Nanjing University. Male C57BL/6 mice  were supplied by the Experimental Animal Central of Wuhan University. All the animals were housed in an air-conditioned room  with free access to food and water. The behavioral tests were performed on their active hours.PP2A activity in the cell extracts and brain lysates were measured using the phosphatase kit V2460 according to the manufacturer’s procedure (Promega).Fear conditioning was carried out as described previously . Mice weThe standard Morris water maze (MWM) procedure was used with minor modifications as described previously . The watThe mice were anesthetized by chloral hydrate and perfused intracardially with 400 mL normal saline containing 0.5% sodium nitrite, followed by 400 mL 4% formaldehyde solution and the Golgi dye solution containing 5% chloral hydrate, 4% formaldehyde, and 5% potassium dichromate. After being perfused, the brains were dissected into 5-mm × 5-mm sections and transferred to a vial containing Golgi dye solution for 3 days in dark, then immersed in solution containing 1% silver nitrate for another 3 days. The brains were serially sectioned into 40-μm-thick sections using a vibrating microtome . The number of spines was counted manually from the two-dimensional projections. Classification of mushroom spines was performed as described previously .CK2 activity in the cell extracts and brain lysates were measured using the CK2 kinase Assay/Inhibitor Screening kit CY-1170 according to the manufacturer’s procedure (Cyclex).Primary hippocampus neurons were prepared from E17 to E18. Hippocampus were dissected and gently minced in Hank’s buffered saline solution, then suspended in 0.25% (vol/vol) trypsin solution at 37°C for 15 min. Neurons were cultured in neurobasal medium supplemented with 2% (vol/vol) B-27 and 1× GlutaMAX for AAV infection. All cell culture reagents were purchased from Thermo Fisher Scientific.2 at 37°C. HEK293/tau cells were seeded into 6-well plates 1 day before transfection, performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instruction. Forty-eight hours after transfection with SET [wild type (WT)] or mutants respectively, cells were rinsed twice in ice-cold PBS (pH 7.5) and lysed with buffer containing 50 mM Tris-Cl, pH 8.0, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.02% sodium azide, 100 mg/mL phenylmethysulfonyl fluoride, and 10 mg/mL protease inhibitors  followed by sonication for 5 s on ice. After centrifugation at 12,000 g for 5 min at 4°C, supernatants were fetched out and added with equal volume of 2 Laemmli sample buffer. Samples were boiled for 10 min before electrophoresis. Protein concentration was estimated by BCA kit .HEK293 cells were stably transfected with the longest human tau (tau441) (HEK293/tau). The cells were cultured in Dulbecco’s modified Eagle’s medium  in the presence of 200 mg/mL G418 containing 10% fetal bovine serum, and in a humidified incubator aerated with 95% air and 5% CO2, 10% (vol/vol) glycerol, 1:100 PMSF, 1:1,000 protease inhibitor mixture containing 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride, aprotinin, bestatin, leupeptin, E-64, and pepstatin A. The proteins in the extracts were separated by SDS/PAGE and analyzed by Western blotting using antibodies. Samples of HEK293/tau cell extracts were similarly analyzed. Immunoreactive bands were visualized with the Odyssey Infrared Imaging System (LI-COR Biosciences) and quantitatively analyzed by ImageJ software.Mice brain tissues, cortex, and hippocampus, were quickly dissected out and homogenized on ice to generate 12% (w/v) homogenate in buffer containing 50 mM Tris⋅HCl pH 8.0, 150 mM NaCl, 1% (vol/vol) Triton X-100, 1 mM EDTA, 1 mM MgClCultured cells were fixed in 4% (vol/vol) paraformaldehyde for 15 min and permeabilized in phosphate buffer containing 0.5% Triton X-100 (PBST). Non-specific binding was blocked by incubation in PBST buffer containing 0.1% Triton X-100 and 5% (wt/vol) BSA for 1 h. The primary antibodies against pSET (1:100), pSTAT1 (1:500), or CK2 (1:500) were then applied in blocking solution and incubated at 4°C overnight. The secondary antibodies conjugated to Alexa-Fluor 488/548 were added to the coverslip for 1 h at room temperature, and then Hoechst  for 30 min. The coverslips were washed and mounted onto slides. Images were acquired using a laser two-photon confocal microscope .2PO4, 24 mM NaHCO3, 6 mM MgSO4, 0.5 mM CaCl2, and 10 mM D-glucose. Coronal slices (350-μm thick) containing the dorsal hippocampus were cut at 4–5°C in the slicing solution using a Leica VT1000S vibratome and then transferred to an incubation chamber filled with oxygenated slicing solution in a 30°C water bath for 1 h before recording. Slices were laid down in a chamber with an 8 × 8 microelectrode array in the bottom planar  and kept submerged in artificial cerebrospinal fluid  with a platinum ring glued by a nylon silk. Signals were acquired using the MED64 System . The fEPSPs in CA1 neurons were recorded by stimulating the Schaeffer fibers from DG. LTP was induced by applying three trains of high-frequency stimulation .Mice (2 month-old) were used for all our electrophysiology experiments. Mice were deeply anesthetized as mentioned above. When all pedal reflexes were abolished, brains were removed and placed in ice-cold oxygenated slicing solution containing the following: 225 mM sucrose, 3 mM KCl, 1.25 mM NaHBilateral hippocampus DG zone of mice were injected with AAV respectively as described previously . The injt-test was used to determine the different means among the groups. The level of significance was set at P < 0.05.Data were expressed as means ± standard deviation (SD) and analyzed using commercial software . The two-way analysis of variance or one-way analysis of variance, or a Student’s Figure 1A) and 3xTg AD  mouse models from 3 to 6 month age. Immunoblotting analysis and quantification support that SET Ser9 again was highly phosphorylated in 6-month-old mice compared with 3-month-old in both animal models . In WT C57 mice, no difference was observed in CK2 activity and SET phosphorylation from 3 to 6 month age . As a substrate of CK2 . These data implicate that CK2 activation in AD may bring along with SET hyperphosphorylation in an age-dependent manner.CK2 is activated in AD and we have previously showed CK2 phosphorylates SET in HEK293/tau cells , howevere of CK2 , the posFigure 2A). A similar result was observed by infecting primary neuronal culture with an adeno-associated virus type 2 (AAV2) coding for human Tau (AAV2-hTau) . Immunoblotting validated SET Ser9 was highly phosphorylated following treatment with Aβ  and human tau overexpression . Moreover, treatment with TBB, a CK2 inhibitor, significantly decreased the SET Ser9 hyperphosphorylation induced by Aβ or hTau . Positive control STAT1 was also highly phosphorylated in both conditions by CK2 .To test the possibility that CK2 activation causes SET hyperphosphorylation in AD, we treated primary neuronal culture with Aβ and analyzed CK2 kinase activity. We found that Aβ stimulus resulted in a marked increase in CK2 activity . As positive control, STAT1 was highly phosphorylated and strongly localized in the nucleus. Treatment with TBB abolished STAT1 phosphorylation . Hence our data suggest that Aβ or tau can induce CK2 activation and may subsequently lead to SET Ser9 phosphorylation, resulting in its cytoplasmic translocation.SET is a nuclear protein which translocate to the cytoplasm in AD . To demoSupplementary Figure S3), while SET was prominently phosphorylated in CK2 injected mice . The quantification of cells with cytosolic p-SET in cortex and hippocampus further implicated that SET phosphorylation led to its cytoplasmic translocation . Immunoblotting validated that overexpression of CK2 significantly increased SET phosphorylation at Ser9 compared to the control . Next, we performed fear conditioning experiment. We found that overexpression of CK2 strongly incurred the cognitive deficit . Moreover, MWM test also showed that CK2 overexpression triggered the cognitive impairments . However, overexpression of CK2 did not affect the motor activity because the swimming speed remained comparable in control and CK2 overexpressed animals . To demonstrate indeed CK2-triggered cognitive defect is associated with synaptic plasticity deficit, we performed electrophysiology analysis . We found that overexpressed CK2 substantially decreased the fEPSP slope, supporting that CK2 amplification damaged or sabotaged the synaptic plasticity . To further investigate whether CK2 overexpression affects the synapses, we infected primary hippocampal neurons with control AAV2 or AAV2-CK2. In 3 days, we conducted immunofluorescence staining, and found that overexpression of CK2 decreased the total dendritic length and the average number of dendritic branches. Noticeably, the spine density and mushroom type were reduced by CK2 overexpression as well . To support this observation, we also performed immuno-blotting analysis of a panel of synaptic proteins in the hippocampus of CK2 mice and in primary hippocampal neurons. We found that numerous synaptic proteins including synaptophysin, synapsin1, and NR1 were evidently reduced after CK2 infection , and the quantitative data were summarized in Figures K and Supplementary Figure S10B. Thus, our data strongly support that CK2 overexpression and its activation initiates the cognitive defects while impairing synaptic plasticity and synaptogenesis.To demonstrate whether CK2 activation induces any cognitive defects, we injected C57/BL6 mice dentate gyrus with AAV2 coding for CK2 (AAV2-CK2). In 1 month, immunofluorescent analysis showed that SET translocated from nucleus to cytoplasm in hippocampus dentate gyrus zone  or CK2 infected mice . To further investigate whether CK2-induced tau hyperphosphorylation is mediated via SET phosphorylation, HEK293/tau cells were transfected with WT SET and different SET mutants including non-phosphorylation SET S9A and phosphorylation mimetic SET S9E . We found that overexpression of WT SET led to significant reduction in PP2A phosphatase activity, which was further reduced by SET S9E, while non-phosphorylation SET S9A displayed no any inhibitory activity . Next, we monitored tau phosphorylation status with different antibodies in immunoblotting analysis. Tau phosphorylation at Ser199, Ser202/Thr205 (AT8), Thr231, Ser396, and Ser404 sites tightly correlated with PP2A phosphatase activity , and the quantitative data was summarized in Figure 4C.Previous studies have showed that CK2 immunoreactivity is markedly increased in NFTs-bearing hippocampal neurons with strong anti-tau immunolabeling, compared with those without NFTs . In the Supplementary Figures S12D–F). In 1 month, we analyzed PP2A enzymatic activity. As expected, overexpression of SET significantly inhibited PP2A activity. Noticeably, phosphorylation mimetic SET S9E displayed much stronger inhibitory activity, by contrast, SET S9A exhibited no inhibitory activity . Immunoblotting from hippocampal  and cortical  tissues revealed that the phosphorylation of tau also tightly correlated with the phosphatase activity of PP2A. The quantification data were summarized in Figure 4F and Supplementary Figure S13B. These data further demonstrate that SET phosphorylation inhibits PP2A activity and subsequently induces tau hyperphosphorylation.To assess whether SET phosphorylation is responsible for CK2 overexpression-induced cognitive defects, we injected different AAV2  into the hippocampus of C57/BL6 mice . We made similar observations in fear conditioning assay . Electrophysiology also supported that overexpressed WT SET or S9E evidently reduced synaptic plasticity. In contrast, SET S9A displayed no any effect . Golgi staining also strongly supported that overexpressed WT SET or S9E significantly reduced spine density and mushroom type, by contrast, SET S9A had no any effect . The quantification was summarized in Figures 5I,J. Moreover, we also performed immunoblotting analyses with tissues from hippocampus  or cortex  and found that overexpressed WT SET or S9E robustly reduced the synaptic proteins including synaptotagmin, synaptophysin, NR2B, PSD95, and PSD93. Once again, SET S9A had no any effect, indicating that WT SET or its phosphorylation mimetic mutant S9E substantially reduces the synaptic proteins expression, fitting with the Golgi staining observations. The quantified data were summarized in Figure 5L and Supplementary Figure S14B. Overall, our data strongly suggest that SET phosphorylation induces tau hyperphosphorylation via inhibiting PP2A, leading to the loss of synaptic plasticity.Next, we performed the behavioral assay. We employed MWM and fear conditioning assays. MWM demonstrated overexpression of WT SET or S9E significantly induced the cognitive defects. By contrast, SET S9A overexpressed mice remained comparable with WT control . PP2A phosphatase activity assay supported that SET S9A transfected cells displayed much higher phosphatase activity than WT SET . Immunoblotting assay of p-tau tightly correlated with the phosphatase activity . The quantified data were summarized in Figure 6C. Together, these data strongly suggest that phosphorylation of SET at Ser9 is responsible for CK2 activation-induced cognitive defect through blocking PP2A phosphatase activity.To test whether SET phosphorylation by CK2 is really responsible for its overexpression-induced cognitive deficit, we firstly transfected HEK293/tau cells with WT SET or non-phosphorylation SET S9A in the presence of CK2 . In 1 month, we performed phosphatase PP2A activity analysis . As expected, tau phosphorylation tightly correlated with PP2A phosphatase activity in both hippocampus  and cortex . The quantification of the western blot results was summarized in Figure 6F and Supplementary Figure S16B. These data further confirm that phosphorylation of SET at Ser9 is required for CK2-induced tau pathology.To investigate whether SET phosphorylation is indeed responsible for this event, we co-injected AAV2 SET S9A with AAV2 CK2 into the hippocampus of C57/BL6 mice  and fear conditioning assays . Electrophysiology analysis showed that co-injection of non-phosphorylation SET S9A with CK2 significantly increased the fEPSP slope than WT SET co-injection with CK2 , supporting non-phosphorylation of SET at Ser9 attenuates the loss of the synaptic plasticity. Notably, Golgi staining indicated that SET S9A animals revealed substantially more dendritic spines and mushroom type spines compared to WT SET co-injected ones in the presence of CK2 , supporting that blocking phosphorylation of SET at Ser9 inhibits the CK2-induced synaptic plasticity impairment and the resulting cognitive defect. The quantified data were summarized in Figures 7I,J. Immunoblotting also fitted with this observation as the synaptotagmin, synapsin, PSD95, and PSD93 were clearly increased in SET S9A samples compared with WT SET , supporting that the blocking phosphorylation of SET reverses the synaptic defect induced by CK2.Next, we performed MWM and fear conditioning cognitive functional assays. Again, non-phosphorylation SET S9A completely reversed CK2-induced defects in both MWM (In the current study, we provide extensive evidence demonstrating that CK2 activation by Aβ or tau can induce SET Ser9 phosphorylation, leading to its cytoplasmic translocation, where it blocks PP2A activity, resulting in tau hyperphosphorylation in AD mouse model. Overexpressed phosphorylation mimetic SET S9E mimics the pathology and behavior deficit induced by CK2 strongly supporting that SET Ser9 phosphorylation is responsible for the CK2-mediated pathological effects. On the other hand, we provide additional evidence by co-injecting CK2 virus and non-phosphorylated SET S9A. We found that non-phosphorylated SET reversed CK2 overexpression-induced pathology and cognitive defects. Together, our data strongly support the notion that CK2-mediated phosphorylation of SET induces tau payhology in AD.SET is a nuclear protein , which hOur findings show that CK2 phosphorylates Ser9 on SET leading to its cytoplasmic translocation and inhibition of PP2A resulting in tau phosphorylation in AD. This finding provides a novel molecular mechanism for tau pathology in AD development.All animal experiments were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology and performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.XW designed the research. QZ, YX, YW, YS, KZ, YM, FH, MW, DK, and QW performed the research. BZ, RL, J-ZW, KY, and XW analyzed the data. KY and XW wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cis-autophosphorylation in the activation-loop and trans-autophosphorylation at several S/T sites along the protein are required for HIPK2 activation, subcellular localization, and subsequent posttranslational modifications. The specific function of a few of these autophosphorylations has been recently clarified; however, most of the sites found phosphorylated by mass spectrometry in human and/or mouse HIPK2 are still uncharacterized. In the process of studying HIPK2 in human colorectal cancers, we identified a mutation (T566P) in a site we previously found autophosphorylated in mouse Hipk2. Biochemical and functional characterization of this site showed that compared to wild type (wt) HIPK2, HIPK2-T566P maintains nuclear-speckle localization and has only a mild reduction in kinase and growth arresting activities upon overexpression. Next, we assessed cell response following UV-irradiation or treatment with doxorubicin, two well-known HIPK2 activators, by evaluating cell number and viability, p53-Ser46 phosphorylation, p21 induction, and caspase cleavage. Interestingly, cells expressing HIPK2-T566P mutant did not respond to UV-irradiation, while behaved similarly to wt HIPK2 upon doxorubicin-treatment. Evaluation of HIPK2-T566 phosphorylation status by a T566-phospho-specific antibody showed constitutive phosphorylation in unstressed cells, which was maintained after doxorubicin-treatment but inhibited by UV-irradiation. Taken together, these data show that HIPK2-T566 phosphorylation contributes to UV-induced HIPK2 activity but it is dispensable for doxorubicin response.HIPK2 is a Y-regulated S/T kinase involved in various cellular processes, including cell-fate decision during development and DNA damage response.  HIPK2 (Homeodomain-Interacting Protein Kinase 2) is an evolutionarily conserved tyrosine-regulated serine/threonine kinase. HIPK2 phosphorylates a large body of proteins belonging to different signaling pathways involved in the control of development, cell response to DNA damage and hypoxia, differentiation, and cytokinesis [reviewed in 1-4].In physiological conditions, HIPK2 localizes mainly in nuclear speckles, including PML-bodies, and only a small fraction is found in the nucleoplasm or cytosol . In unsti.e., NP_073577.3 and NP_034563.2 for human and mouse HIPK2, respectively) and, when required, add in brackets the aminoacid number used in the relative references. Both human and mouse HIPK2 have been shown to become catalytically active by their cis-autophosphorylation of the activation loop at Y361 (Y354) and S364 (S357) . T566 was not found phosphorylated in the human HIPK2 by the same analyses . Since nTo verify this differential contribution, U2OS cells were transfected with GFP-tagged wt HIPK2 or the T566P mutant; the GFP-empty vector was transfected as negative control. The three transfected populations were treated with ADR, UV-irradiated, or maintained in non-treated conditions. As expected from the above reported data Figure , EdU incThe involvement of HIPK2 in a multitude of biological processes and its participation in many different signaling pathways is made possible by PTMs that increase its functional diversity. In this light, the finding that a specific HIPK2 autophosphorylation site, T566, diversely contributes to UV- and doxorubicin-mediated DDR may not be surprising. However, thus far, HIPK2 activation and function has been indiscriminately studied upon UV, IR or chemotherapeutic drug treatments. For example, HIPK2 autophosphorylation at T880/S882 and Pin1 binding have been studied upon doxorubicin treatment ; c-Abl-mTarget enrichment was performed using in-solution technology , and the resulting target libraries were sequenced by Illumina sequencing technology (HiSeq2000). Raw image files were processed by Illumina basecalling software (CASAVA 1.7) using default parameters. Paired-end reads were aligned to the human genome (UCSC GRCh37/hg19) with the Burrows-Wheeler aligner (BWA v. 0.7.10) . PresumeThe human osteosarcoma U2OS cells were cultured in DMEM supplemented with 10% (v/v) FBS and antibiotics (Invitrogen). The patient-derived colon spheroids were kindly provided by Prof R. De Maria and cultured in a serum-free medium supplemented with 20 ng/ml EGF and 10 ng/ml FGF-2 (PrePro –Tech) . For DDRRNA interference was obtained by HIPK2-specific Stealth RNAi sequences and Stealth RNAi-negative control (UNC) (Invitrogen), as previously described . Cells wTotal RNA was isolated using the RNeasy mini Kit (Qiagen). cDNA was synthesized using a M-MLTV RTase and amplified with GoTaq DNA polymerase (Promega). The following primers were employed (NCBI RefSeq NM_022740.4):HIPK2-fw: ggctgaccggcgggagtt; HIPK2-rev: ggtcaggccgggcacaaatct.PCR-amplifications were performed in duplicate on two different RNA preparations.The pEGFP-HIPK2-FL vector, encoding for a N-terminal GFP-tagged wild type HIPK2-FL protein  was mutaT566P: cacggtgaaccagagcaaaccccctttcatcac;K565T: acggtgaaccagagcacaacccctttcatcacg.The obtained pEGFP-HIPK2-T566P and pEGFP-HIPK2-K565T vectors were validated by direct sequencing and transfected in U2OS cells by electroporation  using a Gene Pulser (Bio-Rad) or by Lipofectamine (Invitrogen), according to manufacturer's instructions.32P]-ATP, as described . When required, to reduce protein phosphorylation, the total cell extracts were incubated at 30°C for 30 min in the presence of 20 U/ml calf intestinal alkaline phosphatase (Sigma). After centrifugation, GFP-fusion proteins were purified from supernatant by overnight incubation with anti-GFP Ab-sepharose beads (Abcam) at 4°C and used as enzymatic source. Western blot (WB) analysis with a different anti-GFP Ab  was employed to quantify purified proteins. For kinase assay, recombinant proteins were incubated with recombinant p53, p63, and MBP for 30 min at 30°C in a kinase buffer in the presence of [γescribed . The phoA rabbit polyclonal Ab recognizing the phosphorylated HIPK2-T566 site, anti-p-HIPK2 (Thr566) Ab was raised against a phospho-peptide representing the human HIPK2 region surrounding T566 and affinity purified with the immunizing phosphorylated peptide after negative absorption with the non-phosphorylated peptide. Peptide design, Ab production and purification were all performed by the custom antibody service of Thermofisher.Total cell extracts were prepared, separated, transferred onto nitrocellulose membrane, and immunodecorated as described . The folCells were incubated in the presence of 10 μM EdU labeling solution for 3 hrs, fixed in 2% formaldehyde, permeabilized with 0.25% TritonX 100, blocked with 5% BSA an developed according to manufacturing instructions . Nuclei were counterstained with 1X Hoechst® 33342. At least 500 cells per sample were counted. Fluorescent signals were recorded by Olympus BX53 microscope (Olympus Life Science) and images were captured with a ProgRes MFCOOL camera (Jenoptik) at a magnification of 40X.For comparison between two independent groups, the Student's t-test was used.


Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL) and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways.Cycle inhibiting factors (Cifs) are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including  Burkholderia pseudomallei and enteropathogenic and enterohemorrhagic Escherichia coli [1/S and G2/M phase transitions. It has been shown that the cell cycle arrest is induced as a result of stabilization of cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1 (hereafter referred to as p21 and p27) [Cycle inhibiting factors (Cifs) are a group of bacterial virulence proteins that are secreted by the Type III Secretion System of some Gram-negative bacterial pathogens including hia coli ,2. Upon and p27) –6. Celluand p27) . Cif hasCRLs constitute the largest family of E3 ubiquitin ligases, primarily due to their modular structure. There are six different Cullin homologous proteins, with each forming a scaffold onto which different E3 ligase complexes assemble ,11. CRLsRecent studies have revealed that Cif functions as a deamidase that targets Nedd8 on Gln40, thus converting it into a glutamate ,13. ConsHere we show that in addition to Nedd8 deamidation, Cif also has CRL independent effects. Thus, we found that Cif induces a potent and selective activation of the pro-survival mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. We sought to characterize the mechanism through which Cif activates ERK. We show that Cif-mediated ERK activation is dependent on its deamidase activity but independent of CRL inhibition, suggesting that the MAPK/ERK pathway is a novel target of Cif that is different from CRL. Our results show that Cif targets the SOS1-Grb2 complex in the MAPK/ERK pathway and modifies the phosphorylation status of the Ras guanine nucleotide exchange factor, SOS1, specifically in the region of SOS1 containing the CDC25-homology and proline-rich domains. We suggest that Cif-mediated ERK activation functions to counter the proapoptotic effects of CRL inhibition.2 tissue culture incubator. Transient transfections were performed using Genejuice transfection reagent (Novagen) in accordance with the manufacturer’s directions for sub-confluent cells. MLN4924 was a gift from Millennium Pharmaceuticals, Inc.HEK293T cells were cultured in Dulbecco’s modified eagle medium (DMEM) (Invitrogen) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Hyclone), 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen) in a humidified 37°C, 5% COB. pseudomallei Cif plasmids bearing an N-terminal 2X FLAG or V5 epitope tag sequence used were previously described [B. pseudomallei Cif C156S catalytic mutant (V5-Cif C156S) was generated via site-directed mutagenesis using forward primer C CGG ACT TTC GGC CA-3’5’-GAT GAC GCC CGT GT and reverse primer G ACA CGG GCG TCA TC-3’5’-TGG CCG AAA GTC CG, and cloned into pcDNA3.1 with 5’ KpnI and 3’ XbaI and verified by DNA sequencing. FLAG-BimEL plasmid was a kind gift from Dr. Ong Sin Tiong . pBabe-Puro-MEK-DD and pBabe-Puro-B-RAF-V600E were gifts from Dr. William Hahn (Dana Farber Cancer Institute) (Addgene plasmid # 15268 and # 15269 respectively) [The FLAG- or V5-escribed . B. pseuctively) . The FLA3VO4, 20 mM Sodium β-Glycerophosphate, 10 mM Sodium Pyrophosphate, 0.5% Triton X-100, Roche protease inhibitor cocktail and 0.1% β-Mercaptoethanol. Lysates were precleared by centrifugation before use for Western blotting. Equal amounts of protein were loaded for Western blot analysis. All the following antibodies used were obtained from Cell Signaling Technology: anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-Mcl-1, anti-phospho-Bad (Ser112), anti-Bad, anti-phospho-MEK1/2, anti-MEK1/2, anti-Grb2, anti-phospho-Akt (Thr308), anti-Akt, anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-calnexin, anti-phospho-p70 S6K1 (Thr389), anti-p70S6K1, except for anti-p27 (BD Biosciences), anti-HIF-1α (BD Transduction Laoratories), anti-GAPDH , anti-Glut1 (Abcam), anti-α-tubulin (Molecular Probes), anti-β-actin (Sigma), anti-FLAG M2 (Sigma), anti-V5 (Serotec) and anti-HA (Roche).Cells were washed with ice-cold Phosphate-Buffered Saline (PBS) and then lysed in Triton X-100-containing lysis buffer. The composition of the lysis buffer was as follows: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na20 μl of anti-FLAG M2 agarose beads (Sigma) were used for immunoprecipitation. 500 μl of precleared lysate from cells transfected with FLAG-mSOS1 + empty vector or FLAG-mSOS1 + V5-Cif in 60-mm tissue culture dishes was added to the agarose beads. Untransfected cell lysate was added to control beads. The samples were tumbled for 1 hour at 4°C, and the beads were then washed four times in ice-cold NP40 lysis buffer  and once in ice-cold buffer containing 50 mM Tris (pH 7.5). The immunoprecipitated mSOS1 proteins were then eluted and denatured in 2X Laemmli sample buffer (Biorad) containing 5% β-Mercaptoethanol and subjected to SDS-PAGE and Western blotting.et al., manuscript in preparation). Therefore, we sought to determine the exact molecular target and mechanism of action of Cif in regulating ERK activity. We first determined if Cif exhibits a preference for activating the ERK pathway compared to the p38 or stress-activated protein kinase/c-Jun amino terminal kinase (SAPK/JNK) MAPK pathways. Ectopic expression of Cif using a FLAG- or V5-epitope tagged Cif plasmid in HEK293T cells resulted in a marked induction of ERK1/2 phosphorylation both , Bim long (BimL), and Bim extra long (BimEL) [EL has been reported to be the most abundant isoform. This isoform has also been shown to contain an ERK1/2 docking domain and ERK1/2 phosphorylation sites. Phosphorylation of BimEL by ERK1/2 targets it to the proteasome for degradation. Therefore, to test whether Cif counters the effect of the proapoptotic protein Bim, we expressed FLAG-tagged BimEL in the presence or absence of Cif and measured the phosphorylation of BimEL by western blot. We also used a known activator of ERK MAPK, active MEK (MEK-DD), as a positive control, as well as the MEK1/2 inhibitor U0126 as a negative control. As shown in EL was detected when FLAG-BimEL was co-expressed with Cif, compared to the empty vector control in the first lane. A similar band shift was also observed when FLAG-BimEL was co-expressed with MEK-DD. Cif- and MEK-DD-induced BimEL phosphorylation was not observed when ERK1/2 activation by Cif and MEK-DD was prevented through treatment with U0126  . BimEL hth U0126 . This inrine 112 . HoweverCif could potentially induce ERK1/2 phosphorylation via two mechanisms—by inhibiting MAPK phosphatases or by activating ERK or upstream MAP kinases. To determine if Cif induces ERK activation by inhibiting MAPK phosphatases, we performed a chase experiment whereby we followed the rate of ERK dephosphorylation in the presence of the MEK inhibitor U0126. U0126 blocks the activity of phosphorylated MEK, thus rendering MEK unable to phosphorylate its downstream substrate ERK1/2. In the experiment in To elucidate the target of Cif in the MAPK/ERK pathway, we took a systematic bottom-up approach. Thus, we examined if Cif directly activates a candidate kinase or a kinase that is upstream in the pathway by using a panel of kinase inhibitors. We found that when cells were treated with the MEK inhibitor U0126, Cif-dependent ERK activation was completely prevented . U0126 bWe next determined whether Cif directly activates MEK or another kinase or signaling intermediate that is further upstream of MEK. We treated cells with the Raf inhibitor BAY 43–9006, which inhibits the kinase activity of C-Raf and B-Raf and thus blocks MEK and ERK phosphorylation. As shown in Therefore, to determine if Cif directly activates Raf, we used a dominant negative Ras mutant, S17N HRas, as an approach to inhibit Ras activity. It is generally accepted that S17N HRas exerts its effects by sequestering upstream activators and simultaneously being impaired in activating downstream effectors . As a reThe dominant negative Ras functions by sequestering the upstream Grb2-SOS1 complex. This suggests that ERK activation by Cif is dependent on a functional Grb2-SOS1 complex. In response to RTK activation, the Grb2 protein normally recruits SOS1 to the plasma membrane. Upon plasma membrane translocation, SOS1 can activate plasma membrane-bound Ras by functioning as a guanine nucleotide exchange factor (GEF) for Ras. Therefore, we next tested whether Cif requires plasma membrane translocation of the Grb2-SOS1 complex to activate ERK. To test this, we used dominant negative mutants of Grb2 that prevent the recruitment of SOS to the plasma membrane ,22. The Our results in Our results suggest that Cif exerts its effects on the MAPK/ERK pathway downstream of the RTKs, and that it potentially acts at the Grb2-SOS1 complex. Hence, we sought to characterize the potential mechanism through which Cif modifies the Grb2-SOS1 complex to activate ERK MAPK. Grb2-dependent recruitment of SOS1 to RTKs is required to activate the GEF activity in SOS1 to catalyze GDP to GTP exchange in Ras . We hencThe Ras guanine nucleotide exchange factor SOS1 has a multi-domain structure. The domain organization of SOS1 comprises of a histone fold domain, a Dbl homology (DH)-Pleckstrin homology (PH) domain, a Ras exchanger motif (REM), a CDC25-homology (CDC25-H) domain with GEF activity, and a proline-rich C-terminal region that binds Grb2 and links SOS1 to activated RTKs. The catalytic REM and CDC25-H domains govern the GEF activity of SOS1 on its target Ras protein. The histone fold domain and the DH-PH domain have been suggested to exert an autoinhibitory effect on the catalytic REM and CDC25-H domains ,25. It iCif has previously been shown to inhibit CRL function . CRL inhvia a novel mechanism that may involve SOS1 phosphorylation. Cif appears to induce the phosphorylation of a region in SOS1 that comprises the CDC25-H and proline-rich domains. The CDC25-H domain, together with the REM domain, mediates Ras nucleotide exchange. The C-terminal region has been suggested to exert a negative regulatory effect on the activity of human SOS1 [Our studies investigating the various MAPK/ERK pathway signaling intermediates and our finding of a potential Cif-induced SOS1 posttranslational modification suggest that Cif acts at the level of SOS1. Cif is known to have deamidase activity . Howeverman SOS1 ,27,28. TIn future studies, it will be important to identify the phosphorylated amino acids in SOS1 and to characterize the mechanism through which Cif induces SOS1 phosphorylation. Cif-induced SOS1 phosphorylation may be due to deamidation of an upstream regulator of SOS1, such as a protein kinase. Alternatively, Cif may also deamidate a particular glutamine residue in SOS1, which in turn would prime the phosphorylation of SOS1 by an upstream protein kinase.Finally, our results raise the interesting possibility of the existence of an upstream regulator of SOS1 that can lead to a marked activation of ERK signaling. Hence, another important question is to elucidate how SOS1 phosphorylation results in increased GEF activity. So far, SOS1 phosphorylation by different protein kinases has been found to mainly play a negative regulatory role on MAPK/ERK signaling ,29. ThusB. pseudomallei is currently not well characterized, since not all B. pseudomallei isolates possess Cif. It was originally proposed that Cif promotes the intracellular bacterial replication by inhibiting the turnover of epithelial host cells [et al. [Yersinia pseudotuberculosis infection, Cif plays a role to inhibit the activity of perforin-2, a mediator of the innate immune response of host cells. The authors found that upon Y. pseudotuberculosis infection, perforin-2 becomes activated through monoubiquitination. Perforin-2 ubiquitination is mediated via the CRL SCFβ-TrCP and this results in the translocation of perforin-2 to cellular membranes, in particular the plasma and endosomal membranes. Upon translocation, perforin-2 then exerts its bactericidal activity by inducing lysis of plasma membrane-bound or endosome-encapsulated bacteria. Cif mediated CRL inhibition prevents perforin-2 monoubiquitination and activation [in vivo Cif increases Y. pseudotuberculosis virulence in wild type but not perforin-2 deficient mice. This indicates that inhibiting the perforin-2 dependent host response is the primary function of Cif in Y. pseudotuberculosis.Whether Cif promotes the pathogenicity of st cells . This ef [et al. , it was tivation . ImportaY. pseudotuberculosis infection is unlikely to be of major relevance during the infection with Cif-expressing B. pseudomallei. This is because B. pseudomallei can readily escape from the endosomes into the host cytosol [B. pseudomallei infection. Our study suggests an alternative mechanism through which Cif may potentially exert its cellular effect. Prolonged CRL inhibition is known to induce cellular apoptosis. This is partly due to prevention of the degradation of key cell cycle regulatory proteins, including the important DNA replication licensing factor CDT1. Failure to degrade CDT1 leads to DNA re-replication and induction of cellular apoptosis [EL in an ERK1/2-dependent manner. ERK1/2-dependent phosphorylation is well known to induce proteasome-dependent BimEL protein degradation. Surprisingly, despite inducing marked BimEL phosphorylation, Cif reduced the BimEL steady state protein levels only slightly. It may be necessary to carry out half-life measurements of endogenous BimEL protein to detect Cif-induced changes in BimEL protein stability. We also observed that Cif increased the expression of the anti-apoptotic protein Mcl-1 and the phosphorylation of Bad, leading to inhibition of its pro-apoptotic function (see B. pseudomallei virulence would be of interest.However, the described mechanism in  cytosol , thus avpoptosis . Given ttion see . HoweverB. pseudomallei virulence factor Cif to activate MAPK/ERK signaling. The elucidation of the exact molecular mechanism is expected to give important new insights into the regulation of the MAPK/ERK signaling pathway. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian cellular signaling pathways.In conclusion, we have identified a novel deamidation-dependent mechanism of action of the


We denote the Transpolar Drift regime as decisive for the fate of methane released on the Siberian shelves.Methane sources and sinks in the Arctic are poorly quantified. In particular, methane emissions from the Arctic Ocean and the potential sink capacity are still under debate. In this context sea ice impact on and the intense cycling of methane between sea ice and Polar surface water (PSW) becomes pivotal. We report on methane super- and under-saturation in PSW in the Eurasian Basin (EB), strongly linked to sea ice-ocean interactions. In the southern EB under-saturation in PSW is caused by both inflow of warm Atlantic water and short-time contact with sea ice. By comparison in the northern EB long-time sea ice-PSW contact triggered by freezing and melting events induces a methane excess. We reveal the Ttranspolar Drift Stream as crucial for methane transport and show that inter-annual shifts in sea ice drift patterns generate inter-annually patchy methane excess in PSW. Using backward trajectories combined with δ However, estimations of methane emissions reveal still large disparities in the Arctic methane budget: bottom-up estimations of all methane emissions are larger than the top-down atmospheric inversions, based on methane atmospheric observations2.Arctic methane sources are considered to contribute to Arctic amplification of global warming as significant methane emissions into the atmosphere may generate positive feedbacks to global warming3, are mostly based on sea-air flux calculations during ice-free conditions5 and ref. therein. Using this method, large uncertainties in estimations might result from detecting source-released methane in sea water which does not finally transit the sea-air interface on the shelves. Instead of efflux on shelves, methane might be trapped in sea ice or in dense shelf water formed during ice formation and subsequently transported by shelf outflow towards the interior Arctic Ocean. Although these pathways are not well constrained, a positive relation between increasing emissions and sea ice decline is reported as feedback to the Arctic amplification of global warming6.Emissions estimated from marine sources, mainly localized on the shallow shelves7, methane is over-saturated beneath multi-year sea ice8 and observed to be taken up in fast ice by freezing9. In particular, the mismatch between annual sea-air flux estimates and below-sea ice concentrations in Siberian shelf seas on the one hand5 and methane release from sea ice into PSW in the interior Arctic on the other10 strongly point to unconsidered shelf-ocean interactions.There is growing evidence that sea ice is crucial to Arctic methane cycling: atmospheric concentrations are higher over open leads11. Unlike the Beaufort Gyre, that may keep sea ice for several years, the TDS transports mainly first or second year sea ice (FYI & SYI). To test our hypothesis we combined oceanographic and geochemical data sets in the Eurasian Arctic with trajectories of sea ice drift. We show that sea-ice drift pattern impacts methane cycling: Certain regions of sea-ice formation on the shelf seas and the drift duration are crucial for the amount of shelf-sourced methane finally released. This affects the extent of methane super-saturation in PSW.As both, the methane uptake- and methane release-locations, are linked by drifting sea ice we hypothesize a “conveyer belt” for transport of Siberian shelf-sourced methane through the Eurasian Arctic within the large Transpolar Drift Stream (TDS). The TDS is one of the two large systems of wind driven sea ice drift and near-surface currents in the Arctic Ocean transporting sea ice from the Siberian coast and shelf across the Arctic Basin into the Fram Strait13.We detected different levels of methane saturation related to the atmospheric equilibrium in ice covered Polar surface water (PSW) in the Eurasian Basin (EB) Fig. . PSW com14 and using an atmospheric mole fraction of 1.88, the monthly mean from August 2007, 2011 and 2015 .The methane saturations were calculated by applying the equilibrium concentration of methane in sea water with the atmosphere as function of temperature and salinity15. When entering the Arctic Ocean the sea water temperature drops to less than 3 °C  which corresponds to a nearly 20% enhanced solubility capacity, i.e. a saturation concentration of 3.3 nM. On top, melted ice reduces the salinity to 34.3 while the temperature drops to the freezing point resulting in a saturation concentration of 3.8 nM which finally accounts for an enhanced solubility capacity of nearly 30%. This saturation concentration designates the highest saturation concentration reachable by cooling, which means that this value (3.8 nM) also limits the range where the methane saturation level is mainly cooling-triggered.The methane saturation concentration in northern North Atlantic amounts 2.7 nM, i.e. sea water with temperature of 10 °C and a salinity of 35When achieved, further enhancements in saturation concentrations are caused by freshening during sea ice melt or fresh water inflow, i.e. in that range the methane saturation level is freshening-triggered. We calculated the highest equilibrium concentration of 3.94 nM when salinity drops to 27.3.Summarized, the methane saturation concentration calculated as a function of sea water temperature and salinity allows separating the increasing saturation capacity in ice covered sea water to a mainly cooling-induced and a mainly freshening-induced branch Fig. . The coo15. Methane is mainly under- to slightly super-saturated and mostly localized along the cooling branch  enters the Arctic Ocean. Coming from the south, inflowing water is cooled down by ~10 °C to near-freezing temperatures during the journey through the Nordic seas and the Fram Straitnch Fig. .Figure 216 and in 2007 thick sea ice covered the southern EB.Towards the sea ice covered surface water the under-saturation is decreasing although the saturation concentration is increasing by cooling and freshening. As sea water which is covered for a longer time by sea ice is less under-saturated, methane release from melting sea ice is most likely to reduce the under-saturation Fig. . Althoug17 to the wind driven sea ice drift (from north-east)18 results in just a short contact of sea water with the sea ice cover on top. In this regard, the “non-common history” of sea ice and PSW advected along the southern EB focuses the view to the duration of sea water-sea ice contact for the level of saturation generated in PSW.However, the effect of sea ice-released methane to enhance the saturation level remains small in this region as the opposite direction of the ocean current along the Svalbard and Barents Sea continental margin (from south-west)5 km2 of sea ice per winter from the Laptev Sea towards the Fram Strait20. Both, the wind-driven sea ice drift and near-surface currents are thought to move in the same direction, i.e. from northeast to southwest21. During that common journey brine release and haline convection induced by freezing and melting create a strong sea ice-ocean coupling22. Hence the spatial coherence creates a first-year to multiyear sea ice-water contact resulting in a “common history” of sea ice and PSW underneath. In that context methane surplus in PSW clearly appears to be sea-ice-sourced and freezing events as most important for methane release. By comparison, in 2015 we detected just a moderate super-saturation which spreads from the cooling to the freshening branch, i.e. sea ice-released methane is added during freezing and melt events. The different scales of super saturation between 2011 and 2015 at the 60°E transect reveal pronounced inter-annual heterogeneities in methane excess. These perplexing circumstances point to a strong impact of the sea ice type and the duration of sea ice coverage on methane super-saturation in PSW. The variations might be: (I) source –triggered i.e. different sea ice types with different methane amounts incorporated therein, (II) related to varying amounts of methane finally released from sea ice into the PSW and (III) coupled to differences in preservation of methane excess over a time period of several seasons to a year in PSW.The regional contrast to the northern EB, where the PSW remains uninfluenced by AW inflow, is remarkable. We detected in two different years various levels in methane supersaturation ranging from the cooling to the freshening branch Figs  and 3. I(I)We use trajectories from sea ice crossing the 60°E section in 2011 and 2015 to compare drift pattern, age of sea ice and shelf regions of ice formation and find pronounced differences between both years Fig. .Figure 418O isotopic composition in sea ice corroborates its formation in the outer shelf regime of the Laptev Sea . After formation, sea ice was advected north by prevailing offshore winds until it crossed the 60°E section. The δSea Fig. . In that4%, was formed in polynya regions near the coast between October and December. The δ18O values of this sea ice show significant influence of meteoric water and corroborate sea ice formation in the shallow polynya regions 12. Hence to a certain degree the depth of winter mixing reflects the intensity of winter freezing events. Indeed, the extreme methane super-saturation in 2011 between 85° and 87.5°N was localized in the region with the deepest winter mixed layer depth in that year indicating strong freezing events26. This observation is corroborated by the stable oxygen isotope composition (δ18O) which shows generally negative fractions of sea-ice meltwater (fSIM) in the winter mixed layer and thus reflects the net-influence of sea-ice production with subsequent brine release27. Positive sea ice meltwater fractions with fSIM of ~2–3% are available just in the upper ~20 m of the water column in 2011 In addition, sea-ice melt has various effects on keeping methane excess in PSW over a time period of several seasons: When sea ice starts to melt, methane release from sea ice is ongoing until brine is drained. Hence, during that early stage of sea-ice melt methane excess further increases in PSW. However, during a later stage of sea-ice melt, i.e. when brine drainage ceases but sea ice melt is ongoing, freshwater is added to the PSW and starts to dilute the methane excess in PSW.26. Thus freshening by sea ice melt and dilution of the methane excess remain small in PSW. Indeed the large methane surplus in 2011 is localized on the cooling branch, i.e. the dilution effect by freshening during sea ice melt remains low. By comparison, in 2015 methane spreads also along the freshening branch, i.e. in that year the melt effect influences the methane excess in PSW. The decreased salinity corroborates the ongoing sea ice melt . By comparison, drift scenario 2 reflect transport of methane-charged sea ice from the shallow Laptev Sea Polynya regions. Sea ice taken up in these regions and transported within the transpolar Drift system induces hotspots of methane super-saturation of up to 104% in PSW when methane-release occurs, in particular during freezing events. Extrapolating the hotspot size to an area of 100 km2 and the top 60 m 3.4 Tg methane is estimated to be released from sea ice from just a small area. The comparable dimensions of methane budgets trapped in sea ice from large and small areas respectively highlight the significance of sea ice drift patterns for storing methane in PSW. A cascade of feedback processes initiated by trapping methane in sea ice on shelves during sea ice formation and released by freezing and melting events, refers to seasonal methane storage in sea ice. This combines remote sources with the locations where discharge occurs, while both, the atmosphere or the ocean, may act as final sink. Remarkable in this regard is that methane transport within sea ice away from the Laptev Sea shelf coincides with a lower methane surplus in shelf water during the sea ice free season29 compared to other Arctic shelf regions5.Drift scenario 1 shows transport of freeze up-sea ice formed off shore on the Laptev Sea shelf. Coming that way, methane-discharge from drifting sea ice creates super saturation up to 200% in PSW underneath the TDS. A budget calculation with just an average 150% saturation results in 3.8 Tg methane trapped in the top 30 m of PSW in the Transpolar Drift area  system with a Carousel Water Sampler . Salinity was calibrated using discrete samples from the rosette, and processed with an on-board Salinometer . Details of the measurements and processing are described in Schauer 18O signature of seawater were collected from up to six different depths during the up cast at each CTD station with 10 L Niskin bottles mounted on a rosette sampler. The intakes for the CTD sensors were mounted at the bottom of the frame to avoid disturbance by the frame reaching the sensors during downcast. The bottles were mounted at a similar level or higher than the sensors. As the bottles sample over a vertical distance of about 1 m (length of the Niskin bottles) the disturbance to the sensors during up-cast and corresponding bottle sensor values is considered negligible.Water samples for measurements of methane concentration and δ18O signature were taken from ice cores in 2011 and 2015. Sea ice thickness along sampled transects was ~1.3 m to ~2.3 m. The ice was cut into 5 to 10 cm slices, thawed and the water stored for measurements. All water samples from ice cores and water column were analyzed for the oxygen isotope ratios 18O/16O at the Stable Isotope Laboratory . All isotope measurements were performed using the classical CO2-water equilibrium method35. The overall measurement precision for all δ18Oanalysis was ±0.04‰ or better. The 18O/16O ratio is given n respect to V-SMOW in the δ-notation36.Sea ice samples for δ37. The degassing line consists of a sample bottle mounted inside an ultrasonic bath with removable valve connections to a gas burette and a water reservoir bottle. The top of the burette is equipped with a septum port and a gas sample bulb. A vacuum (<20 mbar) is generated within the degassing line before degassing the sample. The sample bottle is then connected and the dissolved gas is driven out of the sample by ultrasonic energy, alternating (5 s on and 10 s off) for about 5 min. The extracted gas is exposed to atmospheric pressure and the volume reduction is compensated by the water from the reservoir bottle. The gas volume is determined on the burette and the gas can then be taken trough the septum for the GC analyses37. Aliquots were measured with the head space method for inter calibration. For this the water was immediately filled from the Niskin bottles into 120 ml serum bottles. Bottles were sealed with gastight butyl rubber stoppers and crimped with Aluminium caps. A 5 ml headspace was created by addition of N2 gas. The sample bottles were brought to lab temperature (20 °C) while shaking in a horizontal shaker. A subsample of 150 μl from the headspace gas was then injected into the Gas Chromatograph (GC). In 2015 we mainly used the head space method for determining the methane concentration. In 2011 and 2015 we used the GC Agilent GC7890A and in 2007 the GC Chrompack 9003 each with a flame ionization detector (FID). For gas chromatographic separation we used a packed column (Porapac Q 80/100 mesh). The GC oven was operated isothermally (60 °C) and the FID was held at 200 °C. Two sets of standard gas mixtures were used for calibration. The standard deviation of duplicate analyses was 5%. This overall error is almost exclusively due to the gas extraction procedure, the GC precision had an error of 1%.Methane concentrations were analysed within a few hours after sampling. The dissolved gases were extracted from the water by vacuum-ultrasonic treatment18O based calculation of water mass fractionation is based on S/δ18O mass balances of marine water, meteoric or river water and sea-ice meltwater38. The calculations used in this manuscript follow the calculations used in Bauch et al., 2011 and are briefly summarized here:The δ18O show a first order linear correlation due to the mixture of ~0‰ δ18O marine water with significant amounts of isotopically depleted meteoric water. Meteoric water consists of river runoff and local precipitation, with similar isotopic composition due to their common source and is referred to as river water within this study. The deviations from the linear correlation are caused by sea-ice processes. In the southern Eurasian Basin the contributing water masses are river water, sea-ice meltwater and Atlantic-derived waters that can be separated by simple 3-component mass balance calculations39. The marine source in the northern and eastern Eurasian Basin and in the North American basins (Makarov and Canadian basins) is expected to be a mixture of Atlantic and Pacific-derived waters42 and a 4-component mass balance has to be applied. In this study a 4-component N/P-based mass balance is applied. The mass balance is governed by the following equations:fa is the fraction of Atlantic water, fp the fraction of Pacific-derived water, fSIM the fraction of sea ice meltwater, and fr is the fraction of river water. S, O and P with the corresponding subscript are the endmember values and measured values of salinity, δ18O and the phosphate value of each endmember or the measured sample. Fixed phosphate endmember values are assigned to river water and sea-ice meltwater  endmembers for the Pacific and Atlantic fractions from the “pure Atlantic water line” defined for our study  for each sample. Due to inaccuracies in end-members and measurements, N/P-based calculations may also produce slightly negative fractions fp of Pacific-derived waters. These however, remain relatively small also within the Atlantic regime  and are still within the uncertainty (~10%) of the method42.Salinity and δfSIM reflects the amount of water removed by sea-ice formation, and the absolute value is proportional to the subsequent addition of brines to the remaining water. Therefore we refer to negative fractions of sea-ice meltwater also as sea-ice derived brine influence or just brine influence. The sea-ice meltwater fraction does not include meltwater from ice formed from river water; this is river water previously transported by ice and is identified by its δ18O and salinity signature, and it is accounted for in fr accordingly. All fractions are net values reconstructed from the δ18O, salinity and the nutrient signature of each sample and are the result of time integrated effects on the sample volume over the residence time of the water.A negative sea-ice meltwater fraction 43. To track sea ice, two different sets of ice drift products were used: During summer month (June – August), the Polar Pathfinder Sea Ice Motion product provided by the National Snow and Ice Data Center (NSIDC) given on a 25 km grid44 was applied. During the rest of the year, tracking is forced with sea ice motion data provided by the Center for Satellite Exploitation and Research (CERSAT) at the Institute Francais de Recherche pour d’Exploitation de la Mer (IFREMER). Motion data are available with a grid size of 62.5 km, using time intervals of 3 days for the period between September and May45. Sea ice concentration data used in this study are obtained from the NSIDC. Following Krumpen et al.20 the tracking procedure works as follows: Using motion and concentration data, a specific ice area is tracked backwards until: (a) the ice reaches a position next to a coastline, (b) the ice concentration at a specific location reaches a threshold value of >15% when ice parcels are considered lost, or (c) the tracking time exceeds 4 years.Sea ice trajectories were used to follow pathways of sea ice and identify source areas by tracking the sampled ice backward using a combination of ice drift and concentration data obtained from low resolution passive microwave satellites. Ice motion data used in this study are provided by different institutions and have been widely used in sea ice studieshttps://www.pangaea.de/?t=Oceans&q=Bauch%2C+Dorothea.Data available from Pangaea repository at


Staphylococcus aureus, Streptococcus pneumoniae, Pseudonomas aeruginosa, and Aspergillus brasiliensis in the disc-diffusion assay. The new EO blend significantly inhibited the growth of the four microorganisms. The zones of inhibition of new EO blend were greater than the air freshener and similar to the liquid soap and body spray, with the exception of Str. pneumoniae in which the body spray provided greater inhibitory zone. The new EO blend and the single oils, with the exception of peppermint, equally inhibited the growth of S. aureus and Str. pneumoniae suggesting no additive effect. P. aeruginosa and A. brasiliensis showed variable susceptibility to all EOs except for no susceptibility to orange and limonene. No difference was found between (−) and (+)-limonene; whereas, (+)-menthol showed greater effect than (−)-menthol. In conclusion, blending the EO of orange, patchouli, peppermint, and clary sage was beneficial in inhibiting the growth of S. aureus, Str. pneumoniae, P. aeruginosa, and A. brasiliensis providing a natural antimicrobial fragrance option over synthetics fragrances used in soaps, body sprays, and air fresheners.Plant essential oils (EOs) are known to inhibit the growth of bacteria and fungi. Whether these antimicrobial effects are comparable to synthetic household products is less clear. Furthermore, limited research is available on the potential additive effect of blending EOs. In this investigation, a new EO blend containing orange, patchouli, peppermint, and clary sage was compared to its individual single oils and to three household products–air freshener, liquid soap, and body spray–for their ability to inhibit the growth of  •A new essential oil blend inhibited the growth of four common environmental pathogens.•The antimicrobial activity of a natural essential oil blend was similar or better to three synthetic products.•Peppermint essential oil was the main ingredient in the essential oil blend that provided the antimicrobial activity. Plant EOs have been used as natural fragrances by applying to the skin (functioning as a body spray) or diffusing into a room (functioning as an air freshener). In addition to the benefit of their natural pleasant scent, many plant EOs also have antimicrobial effects S. aureus in food S. aureus and E. coliDespite extensive research on antimicrobial effects of single EOs P. aeruginosa, S. aureus, Str. pneumoniae, and A. brasiliensis. A secondary objective was to compare the antimicrobial activity of the new EO blend with three household products – an air freshener, a body spray, and a liquid soap – that contained benzalkonium chloride, limonene and linalool, or neither. Lastly, in order to guide a potential improvement in formulation, studies were also performed to understand which component of the novel EO blend was responsible for any antimicrobial effect. For this set of experiments, the individual single EOs and major constituents contained in the new EO blend were assessed for their ability to inhibit the growth of the microorganisms.A novel EO blend containing the EOs of orange, patchouli, peppermint, and clary sage, was formulated with the goal to achieve both, a high pleasant scent and an antimicrobial effect. In the current study, the primary objective was to evaluate the potential antimicrobial effect of this new EO blend against the common environment pathogens 22.1Citrus sinensis), patchouli (Pogostemon cablin), peppermint leaf (Mentha piperita), and clary sage , marketed as TForce™, was obtained from 4Life® Holdings, LLC . TForce also contains a small amount of proteins derived from cow colostrum and egg yolk that are not expected to contribute to any outcome measure assessed herein. The single natural EOs of orange, patchouli, peppermint, and clary sage were obtained from The Lebermuth Company, Inc. . The single constituents (S)(−)-limonene and (R)(+)-limonene were obtained from Sigma-Aldrich Corp . (+)-Menthol crystals were obtained from Alfa Aesar , and (−)-menthol crystals were obtained from Tokyo Chemical Industry Co., LTD . The synthetic cosmetics were purchased at regular stores: Glade spray air freshener aerosol Soho Social Citrus & Mimosa flower (lot number P365 686376) by Johnson & Son, Inc. , Noir Tease scented body mist spray (lot number 6323K2B2) by Victoria's Secret , and Soft on Skin Lemon & Verbena antibacterial liquid soap (lot number GN16242L607) by Lysol® .Ten different samples were tested for their ability to inhibit the growth of the microorganisms described below. A new EO blend of the natural EOs of orange peel . In brief, a gas chromatography coupled with a flame ionization detector (GC/FID) was used with the following analytical conditions: polar column: VB1; apolar column: DBWAX; dimensions: length = 60 m, diameter = 0.32 mm, stationary phase = 0.25 μm, vector gas = hydrogen; flow: 2 ml/min; division ratio: 1:100; split injection: 0.1  μl; injection temperature:250 °C; detection temperature: 260 °C; temperature programme: 60 °C for 15min, and 2 °C/min until 250 °C and 250 °C for 20 min.2.32SO4 and concentrating it by soft distillation with Vigreux column. GCMS separation was made using capillary column containing a chiral phase. The beta-cyclodextrins make it possible to separate the enantiomers and positional isomers. The recognition of the molecules was done by comparing the mass spectra and the retention time to a table of reference peaks obtained from standard materials. The enantiomeric proportions were calculated directly based on the area of the peaks.The chiral method was adapted from the German standard §64 LFBG L00.00–106 and performed by Eurofins Scientific . Briefly, analysis was conducted by first extracting the volatiles from samples and subsequently analyzing them by chiral gas chromatography mass spectrometry (GCMS). Extraction was performed by SDE (Simultaneous Solvent Distillation Extraction), followed by drying the extract using Na2.4Pseudonomas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Streptococcus pneumoniae ATCC 29514, and Aspergillus brasiliensis ATCC 16404 were obtained from and maintained in growth media at the Microbiology Division at Nelson Laboratories . These microorganisms were selected for this study because they are common environmental organisms and their growths are inhibited by essential oils The pathogens 2.56 CFU/mL with sterile distilled water. A. brasiliensis was grown in Sabouraud dextrose broth at 25 °C for 5 days and diluted with sterile distilled water to 104–105 CFU/mL. Diluted cultures were cooled and poured into each sterile plastic petri dish (85 mm diameter). An adaptation of Kirby-Bauer method  was used Bacteria were grown in soybean-casein digest broth at 35 °C for 24 h and diluted to 102.6p < 0.05).The zone of inhibition was measured using calibrated calipers sensitive to 0.01 mm. When zones were absent, the value zero was entered. In cases of complete plate inhibition, the value considered was the size of the plate (85 mm). Statistical analyses were conducted using Statistica software . The mean zone of inhibition ± standard deviation (SD) of triplicate measurements was analyzed using one-way ANOVA followed by Newman-Keuls post-hoc test for determination of significance among groups. Difference among groups were considered significant if the probability of type I error was <5%  and (−) limonene; whereas, it was able to differentiate linalyl acetate and menthol. Results revealed that all menthol and linalyl acetate in the new EO blend are R isomers.3.43.4.1P. aeruginosa in comparison to water control (p < 0.05); although this antimicrobial activity was significantly lower than the positive control phenol (p < 0.05). The new EO blend inhibition of P. aeruginosa's growth was similar to the inhibition provided by the body spray and liquid soap (p > 0.05). However, greater inhibition occurred with new EO blend, liquid soap, and body spray than with the air freshener (p < 0.05). Surprisingly, the single EO of orange, and its main constituent limonene (both isomers), did not inhibit the growth of P. aeruginosa (p > 0.05). On the other hand, the single EO of patchouli, peppermint, and clary sage inhibited the growth of P. aeruginosa to a similar or lesser extent than new EO blend (p < 0.05). However, despite growth inhibition provided by the peppermint oil, neither isomer of menthol inhibited P. aeruginosa's growth (p > 0.05).Data presented in 3.4.2P. aeruginosa, the new EO blend inhibited the growth of S. aureus in comparison to water control (p < 0.05) (p > 0.05). In contrast, new EO blend had a greater zone of inhibition than the air freshener (p < 0.05). All of the single EOs in new EO blend, orange, patchouli, peppermint, and clary sage, significantly inhibited the growth of S. aureus when compared to water control (p < 0.05). However, a greater zone of inhibition was found with the peppermint EO (p < 0.05). Furthermore, both isomers of limonene oil significantly inhibited the growth of S. aureus when compared to water control (p < 0.05) with no difference between the (+) and (−) isomer. Both menthol isomers also inhibited the growth of S. aureus, with a greater effect of (+)-menthol, but smaller effect than peppermint EO (p < 0.05).Similar to data on  < 0.05) . Similar3.4.3Str. pneunomiae (p < 0.05) (p < 0.05). The single EOs of orange and patchouli provided similar inhibition than the new EO blend. The peppermint and clary sage single EOs inhibited Str. pneunomiae's growth to a greater extent than the other EOs (p < 0.05). Both, (+)-limonene and (−)-limonene also inhibited Str. pneunomiae's growth to a similar extent than the other EOs. Lastly, both menthol isomers also inhibited the growth of Str. pneumoniae, with a greater effect of (+)-menthol, but smaller effect than peppermint EO (p < 0.05).Likewise, the new EO blend inhibited the growth of the Gram-positive bacteria  < 0.05) . Similar3.4.4A. brasiliensis when compared to water control (p < 0.05) (p < 0.05). Surprisingly, no inhibition of A. brasiliensis occurred with orange EO, (+)-limonene, and (−)-limonene. Patchouli EO presented a small zone of inhibition, lower than with the new EO blend (p < 0.05); whereas, peppermint and clary sage EOs provided a greater zone of inhibition than the new EO blend (p < 0.05). Only the (+)-menthol isomer inhibited A. brasiliensis's growth, although this effect was significantly lower than the zone provided by peppermint EO (p < 0.05).Lastly, the new EO blend also inhibited the growth of  < 0.05) . This an4P. aeruginosa, S. aureus, Str. pneumoniae, and A. brasiliensis in the disc-diffusion assay. These antimicrobial effects of new EO blend were consistently greater than the growth inhibition provided by an air freshener, and similar to a body spray and liquid soap . Furthermore, the main ingredient in new EO blend, orange EO, does not appear to be the main contributor to new EO blend's effect against these microorganisms.The current study demonstrates that a new EO blend containing the EOs of orange, patchouli, peppermint, and clary sage, inhibits the growth of P. aeruginosa and S. aureus cultures in wounds of rats S. aureus and E. coli when combined with lemon EO in an in vitro assay S. aureus and Aspergillus P. aeruginosa, S. aureus, Str. pneumoniae, and A. brasiliensis. To our knowledge, no antimicrobial ingredient is present in the common air freshener used in the current study in which results demonstrate no growth inhibition against P. aeruginosa, S. aureus, and A. brasiliensis. However, a small but significant antimicrobial effect was found against Str. pneumoniae.Previous studies have reported the antimicrobial effect of synthetic cosmetics or hygiene products. For example, benzalkonium chloride, the antibacterial ingredient in the liquid soap used in the current study, decreased the growth of Streptococcus when used with soap P. aeruginosa. In the current study, P. aeruginosa was inhibited by peppermint and patchouli EOs, but not by clary sage, orange or its constituent limonene suggesting that the patchouli and peppermint in the new EO blend are responsible for its effect against P. aeruginosa. Similar findings have been previously reported. Das et al. (2011) P. aeruginosa in the disc-diffusion assay. Furthermore, Sokovic et al. (2010) P. aeruginosa with peppermint and menthol oils using the disc-diffusion assay. In contrast, no inhibition of P. aeruginosa was found in the disc-diffusion assay with clary sage or limonene oils Peppermint and patchouli EOs, but not orange and clary sage EOs, play a role in the inhibitory effect of the new EO blend against A. brasiliensis by the new EO blend does not seem to be mediated by its main ingredient, orange EO. In the current study, A. brasiliensis was inhibited mainly by peppermint and clary sage EOs. The zone of inhibition provided by patchouli EO was small in comparison to the other oils and due to its content in the new EO blend being low (less than 1.00% of patchouli oil's constituents), patchouli oil is likely not playing a significant role in the inhibition of A. brasiliensis. Previous studies reported similar results with no inhibitory activity of A. brasiliensis with limonene or orange oils A. brasiliensis has also been reported with patchouli oil Similarly, the growth inhibition of S. aureus and Str. pneumoniae. The new EO blend and its individual single oils, as well as the constituents limonene and menthol, inhibited the growth of S. aureus and Str. pneumoniae, although not to a similar extent, suggesting that all of the oils contributed to the antibacterial effect of the new EO blend. Particularly, S. aureus showed greater susceptibility to peppermint EO, and Str. pneumoniae showed greater susceptibility to peppermint and clary sage EOs, suggesting that these EOs might have played a greater role in the new EO blend's antimicrobial effect against these two bacteria. These data are in agreement with previous reports in which good antimicrobial effects against S. aureus and Str. pneumoniae were found with orange On the other hand, all of the oils inhibited the Gram-positive bacteria P. aeruginosa and A. brasiliensis); patchouli EO alone (against A. brasiliensis); peppermint EO alone (against P. aeruginosa); and clary sage EO alone (against P. aeruginosa). Despite of the fact that some single EOs provided greater zones of inhibition than the new EO blend, only the new EO blend and the peppermint single EO inhibited all of the microorganisms tested. It is possible that the greater susceptibility of S. aureus, Str. pneumoniae, and A. brasiliensis to peppermint EO versus the new EO blend is due to the fact that the peppermint EO contains more of its main constituent menthol  provided greater antimicrobial effect than: orange EO alone  and (−)-limonene isomers, but (+)-menthol was more effective. These results are in accordance with Aggarwal et al. (2002) 5P. aeruginosa, S. aureus, Str. pneumoniae, and A. brasiliensis than an air freshener, and similar inhibition to a liquid soap, and a body spray . These antimicrobial effects of new EO blend were likely not provided by the orange essential oil, seen that orange essential oil and its main component limonene were not effective or less effective than the EO blend in inhibiting the four microorganisms. Blending these four essential oils provided a wider antimicrobial benefit than observed by its individual single oils. However, further investigation might elucidate whether reducing the amount of orange and increasing the amount of peppermint essential oil improve the antimicrobial effects of this blend.The current study demonstrated that a blend of EOs of orange, patchouli, peppermint, and clary sage, provides greater growth inhibition of EO.PVB, BV, and DV are currently employees of 4Life Holdings, LLC.This study was funded by 4Life Holdings, LLC.PVB designed the study, interpreted the data, wrote the manuscript and made substantial contribution to conception and data analysis. BV performed data analysis and made substantial contribution to conception, design and revision of the manuscript. DV conceptualized and designed the study, and made substantial contribution to data analysis and interpretation of data. All authors read and approved the final manuscript.


Marinomonas primoryensis is an extremely long, 1.5 MDa protein consisting of five different regions. The fourth region, a 34 kDa domain, is the only part that confers ice binding. Bioinformatic studies suggest that this IBP serves as an adhesin that attaches the bacteria to ice to keep it near the top of the water column, where oxygen and nutrients are available. Using temperature-controlled cells and a microfluidic apparatus, we show that M. primoryensis adheres to ice and is only released when melting occurs. Binding is dependent on the mobility of the bacterium and the functionality of the IBP domain. A polyclonal antibody raised against the IBP region blocks bacterial ice adhesion. This concept may be the basis for blocking biofilm formation in other bacteria, including pathogens. Currently, this IBP is the only known example of an adhesin that has evolved to bind ice.Ice-binding proteins (IBPs) are typically small, soluble proteins produced by cold-adapted organisms to help them avoid ice damage by either resisting or tolerating freezing. By contrast, the IBP of the Antarctic bacterium  IBPs adsorb to ice surfaces to serve different biological roles ,2. SmallIn comparison with fish, some cold-adapted insects produce ‘hyperactive’ AFPs that can inhibit ice growth by more than 5° due in part to differences in ice plane affinity . IBPs inMarinomonas primoryensis (MpIBP) is an exceptionally large protein that consists of five distinct structural regions with a total mass of approximately 1.5 MDa [MpIBP_RIV), which is 2% of the whole protein weight, has ice-binding activity [MpIBP_RIV is a single, calcium-dependent 34 kDa domain composed of 13 repeats of approximately 19 amino acids that folds into a repetitive β-solenoid structure, as shown by X-ray crystallography [−1MpIBP_RIV is over 2° [MpIBP_RIV in vitro, without the rest of the protein [MpIBP is due to region II (MpIBP_RII), composed of approximately 120 tandem repeats of a 104-amino-acid extender domain. In total, the protein is estimated to be 0.6 µm long if fully folded and extended [MpIBP is not secreted into the medium [IBPs differ not only in their natural function but also in their sequences and structures . They ar 1.5 MDa ,18. Onlyactivity . MpIBP_Rlography . A flat, over 2° ,18, and  over 2° . The strextended . Unlike e medium .MpIBP and the fact that 98% of its mass is not related to antifreeze activity led to the notion that this protein serves a distinct function from other IBPs. Bioinformatic analyses suggest that MpIBP is an adhesin and is a type of biofilm-associated protein [MpIBP out of the cell but leaving it bound to the cell surface  [MpIBP_RIV was prepared as described previously [Dry media and agar were purchased from BD Difco. Other materials were purchased from Sigma-Aldrich, unless specified otherwise. Polyclonal antibodies were raised in rabbits immunized with recombinant (4xR_II) . The 4xR(4xR_II) . As a coeviously .2.2.Marinomonas primoryensis were cultured from frozen glycerol stocks of Antarctic isolates [600 nm) of 0.3–0.5. To check the effect of EGTA on M. primoryensis, 3 µl of 250 mM EGTA solution (pH 8) was added to 50 µl aliquots of bacterial culture to make a final concentration of 15 mM EGTA. The high concentration was necessary to chelate all calcium ions in the medium. Alternatively, the bacterial culture was centrifuged for 5 min at 2000g and the pellet was suspended in 20 mM Tris-HCl (pH 8), 500 mM NaCl and 5 mM EGTA. This bacterial suspension was incubated for 0.5–2 h at 4°C before observation. Pseudomonas borealis, P. syringae and the P. syringae cit7del mutant were kindly provided by Virginia Walker, Queen's University, Kingston, Ontario, Canada. These strains and P. fluorescens  were grown in 3% or 10% tryptic soy broth  for 2–4 days at 4°C without shaking. Other bacterial strains used as controls were gifts from several laboratories. A detailed list of those strains with recommended growth conditions from the donors are given in the electronic supporting material.isolates , and wer2.3.M. primoryensis was pelleted for 5 min at 4500g and washed twice with 0.1 M sodium cacodylate (pH 7.4) supplemented with 5 mM CaCl2 (CaCo buffer). The bacteria were fixed in 3% paraformaldehyde , PA, USA) and 2% glutaraldehyde  in CaCo buffer for 24 h at 4°C and then washed three times in 3 ml CaCo buffer. The washed bacteria were resuspended in 100 µl CaCo buffer and 1 : 100 diluted bacteria in CaCo buffer were immobilized on polylysine-coated Si wafers (50 µl for each 5 × 5 mm wafer) for 24 h at 4°C. The coated chips were then fixed in 1% osmium tetraoxide solution  in CaCo buffer for 1 h at room temperature followed by three washes in 5 ml CaCo buffer. The samples were then dehydrated by two 5 min incubations in increasing amounts of ethanol  followed by two 10 min washes in 100% ethanol. The dehydrated samples were dried by a critical point drying system  and visualized by ultra-high-resolution scanning electron microscopy .A culture of 2.4.MpIBP_RIV antibody, anti-MpIBP_RII antibody or pre-immunized serum, and incubated at 4°C overnight. Samples were then diluted fivefold in 0.5× marine broth, and ice-nucleating agent  was added (2–5 µg ml−1). In the experiments including AFPs, the samples were also supplemented with 0.1 µM Tenebrio molitor AFP (TmAFP) to help stabilize the ice crystal. The samples were injected into the sample holder and handled as in the ice morphology experiments previously described [We used a custom-made nanolitre osmometer system to observe the behaviour of bacteria in a solution containing an ice crystal. This system allows delicate manipulation of the temperature of an aqueous droplet, approximately 200 µm in diameter with a precision of a few millikelvin. A detailed description of the system has been given elsewhere ,26. In aescribed . Briefly2.5.600 nm of approximately 0.02, corresponding to 1–2 × 107 colony forming units per millilitre (determined by colony counting). Using this concentration, we obtained images with 250–400 bacteria per frame. Higher concentrations resulted in too many bacteria, which were difficult to distinguish and count. Ice crystals were typically followed from 15–20 µm to a diameter of 60 µm, for a period of approximately 10 min. If local melting was observed during this period, the experiment was discarded. This is because local melting resulted in release of bacteria from the surface. On the other hand, if the crystal was growing too fast it exceeded our field of view too quickly and there was not sufficient time for the bacteria to bind. To meet these conditions, we kept the ice growth rates in the range of 0.01–0.04 µm s−1 (diameter) by subtle temperature changes with millikelvin resolution. The experiment was stopped when the crystal projected area reached approximately 3400 µm2 (radius of approx. 33 µm), corresponding to 47% of the frame so that in all images we were able to see more than 50% of solution area . In this way we avoided bias due to too little solution area in the frame. The crystal volume at the end was at least 100-fold smaller than the whole droplet (less than 0.1 nl in droplets of approximately 10–20 nl). Snapshots were taken every minute, and bacteria were manually counted in the solution and on the crystal surface with the aid of electronic registration using ImageJ software [MpIBP_RIV serum or pre-immune rabbit serum was used to make sure that there was no artefact of the serum influencing the results.To count the number of bacteria bound to ice we used low bacterial concentrations, with an ODsoftware . In thes2.6.M. primoryensis were injected into the microfluidic device. Once the device was filled with bacteria the flow was stopped and the bacteria next to the ice front were observed using a Nikon Eclipse Ti inverted microscope equipped with an Intenslight C-HGFIE fibre illuminator . Experiments were recorded using a Neo sCMOS camera .We used a custom-designed temperature-controlled cell that allows local growth of ice by formation of a temperature gradient, as shown in 2.7.M. primoryensis were centrifuged for 5 min at 5000g. The pellet was resuspended in 1.5 ml of buffer containing 10 mM Tris-HCl (pH 8.0), 140 mM NaCl and 1 mM CaCl2 (buffer Ca+) and washed twice by repeating the centrifugation and resuspension process. The pellet was then resuspended in 4% paraformaldehyde (EMS) in buffer Ca+ for 10 min for fixation, and washed twice as above. After the second wash, the pellet was resuspended in 150 µl of buffer Ca+. The fixed bacteria were immobilized by placing 15 µl aliquots on 10 × 10 mm glass coverslips coated with poly-l-lysine. After three washes with the same buffer without calcium (buffer Ca−), the samples were blocked with blocking buffer, 10% goat serum  in buffer Ca−, overnight at 4°C. The following day the blocking solution was removed and a solution of 1 : 500 anti-MpIBP_RIV antibody in blocking buffer+0.1% Triton 100 was placed on the coverslips for 1 h at room temperature. As a control, we used serum from the same rabbit before immunization. The coverslips were washed three times in Ca− buffer and 1 : 1000 Alexa Fluor®488-AffiniPure Goat Anti-Rabbit IgG (H + L)  was added. After incubation for 1 h, the coverslips were washed in double-distilled water and mounted on glass slides using immune-mount reagent . Images were taken using an Olympus dp71 camera connected to an Olympus ix51 fluorescence microscope.Cultures (5–10 ml) of 3.3.1.Marinomonas primoryensis observed by electron microscopy are typically 2–5 µm long with a single polar flagellum  and the MCF device . Although a low percentage of bacteria survived freezing at approximately −30°C (less than 10%), we found that freezing survival was dramatically improved (more than 90%) by the addition of an ice-nucleating agent (Snomax) that elevated the freezing temperature to around −10°C.lagellum . Althouga present a single ice crystal grown in solution containing a diluted M. primoryensis culture. The crystal at the beginning is clear and smooth, with only a single bacterium bound to its surface (indicated by the arrow). In the figure, only a few bacteria are visible around the crystal due to the low cell number and the limited focus depth, but their presence is clear in the movie. After 5 min, a few tens of bacteria are already concentrated on the surface of the crystal, and after 20 min much of the crystal is covered by a layer of bacteria. As the whole sample contains approximately 10–20 nl of solution, with just a few hundred bacteria, it seems that most of them have bound to the crystal by this time.The electronic supplementary material, movie S1, and figure 4M. primoryensis is crucial for finding and binding ice. Non-motile bacteria that move by Brownian motion in a drop more than 100 µm in diameter are unlikely to encounter an ice crystal that occupies 1% of the fluid volume. A particle 1 µm in diameter moving by Brownian diffusion in water at 0°C will take more than 6 h to travel a distance of 100 µm [a priori that the binding of M. primoryensis to ice is due to its IBP. We cultured other psychrophilic bacteria and motile marine bacteria to test their affinity for ice . Of these bacteria, only the Pseudomonas species could swim at close to 0°C. These include P. borealis [P. syringae (INP+) [P. syringae cit7del mutant (INP−), which lacks ice-nucleating activity [, and P. fluorescens, which was suggested to express both INP and AFP [Pseudomonas borealis freely swim in the solution but do not bind the ice or sense it in any visible way . Bacteria that appear to be on the ice surface in figure 4c are either swimming above the crystal or are non-motile bacteria entrapped by the growing ice, or floating on top of it. Similar results were obtained with INP− P. syringae (cit7del mutant) and with P. fluorescens (not shown). Pseudomonas borealis and P. syringae were reported to have some IR inhibition activity, and ice-shaping activity was noted for P. borealis [MpIBP in role and activity. We did not observe ice shaping by P. borealis, possibly due to the low concentration of bacteria used in our study (figure 4c).The motility of f 100 µm . Still, borealis ,32 and Pe (INP+) , both ofactivity , and P.  and AFP . Pseudomborealis . It is cTmAFP to the solution, we were able to control ice growth better and inspect stable ice crystals for long time periods. Although there is competition between the free TmAFP molecules and the bacteria, the free protein molecules did not evidently interfere with the binding of the bacteria to ice. The bacteria continued to bind the crystal for over an hour, covering some planes completely. The preferential binding to certain ice faces may be due to the position of the crystal, floating with one plane in contact with the air–water interface, where it is less accessible. Another possibility is that this plane is significantly less preferred for binding by MpIBP, relative to the other covered planes. It might also be that adsorbed TmAFP molecules hinder access of the M. primoryensis to it, although at these low protein concentrations (corresponding to TH of less than 0.1°C) the distance between TmAFP molecules should be enough for bacteria to bind [TmAFP with spruce budworm antifreeze protein (sbwAFP) or snow flea antifreeze protein (sfAFP) did not change the ability of the bacteria to cover the crystal from all directions. When an ice crystal covered with bacteria was melted, as shown in the electronic supplementary material, movie S4, the bacteria released to the solution began to swim as before. This process was repeated several times with the same sample giving the same outcome.The electronic supplementary material, figure S1 , presents time-lapse snapshots of ice crystals grown in the presence of small amounts (up to 1 µM) of AFP in addition to the bacteria. By adding free  to bind . In many3.2.M. primoryensis using microfluidics in the MCF apparatus , where we could grow an ice crystal in pure water in the middle of the main chamber . At these cell concentrations, the amount of MpIBP on the ice crystal is sufficient to prevent growth in particular directions and cause ice shaping into a rounded hexagon. However, the concentrations of bacteria in solution are insufficient to stop the ice from growing.Ice crystals in water or solutions without active IBPs grow as flat discs where the two flat surfaces are the basal planes connected by unfaceted prism surfaces. The crystal shape is altered by active IBPs in various manners depending on their binding plane specificity even at low concentrations. Figure 63.5.b, we caught a crystal rotating during growth just a few seconds after lowering the temperature below the melting point, which allowed us to visualize both the basal and prism planes just after they were formed. It is clear from these images that most of the bacteria are located on the basal planes, while a few are bound to the non-basal surfaces. After longer periods of incubation, the whole crystal is covered with bacteria in all directions. Figure 6c demonstrates preferential binding to the basal plane of a large ice crystal grown in the MCF device. We noted that bacteria bound to the basal plane are more static than those on the other planes, where they tend to slide along the ice front.Flat crystals obtained at low concentrations of AFP tend to float with their large, basal planes parallel to the field of view. In figure 63.6.M. primoryensis bound to its surface. As the ice grows, the bacteria move towards the advancing front without being incorporated into the crystal, but many individuals concentrate between grain boundaries. At high supercooling, bacteria can be trapped by fast-growing ice, forming dense patches at the interface with the polydimethylsiloxane walls of the MCF . The electronic supplementary material, movie S6, and figures -MpIBP_RIV antibodies blocked the ability of M. primoryensis to bind ice. Addition of anti-4xR_II antibodies had no apparent effect on the ice binding (c). This emphasizes that MpIBP_RIV is the domain responsible for adhesion to ice. To further verify this point, we tested the effect of the anti-sera on the TH and ice-shaping activity of recombinant MpIBP_RIV. At 5 µM and 33% serum, we did not see a difference between the TH of MpIBP_RIV in the presence of non-immunized serum, anti-MpIBP_RIV antibody or anti-MpIBP_RII antibody. However, at one-fifth of this MpIBP_RIV concentration (1 µM) and 97% serum the sample containing anti-MpIBP_RIV serum had no TH activity and almost no ice shaping, whereas the samples containing anti-4xRII or the non-immunized serum had a TH of approximately 1°C. This indicates that the anti-MpIBP_RIV antibodies disrupt the ice-binding site (IBS) of MpIBP_RIV and do not interfere indirectly with the ability of the bacteria to bind ice. The antibodies by themselves have no apparent TH or ice-shaping activity when tested without MpIBP_RIV. Addition of the metal chelators EDTA or EGTA, the latter of which is more specific to Ca2+ ions, resulted in severe damage to the motility of the bacteria and it was difficult to compare the ice-binding properties of non-swimming bacteria with those of swimming ones.To check if  binding c. This eM. primoryensis to bind ice by counting the free bacteria in solution relative to surface-bound bacteria over time. M. primoryensis bound to a single ice crystal and M. primoryensis free in solution. At time zero, there are few individuals on the ice surface. After only 3 min, half of the visible bacteria were already bound to the ice crystal. After 10 min approximately 90% of the visible bacteria were bound to the ice. Longer accumulation times were observed for lower bacteria concentrations and shorter times for dense populations (approx. 2 min for twofold concentration and 6 min for one-fifth). Addition of anti-MpIBP_RIV serum to the solution reduced the attachment to ice to practically zero. The analysis was conducted on diluted bacteria solutions to be able to count the bound bacteria over 10 min of binding before the ice gets either too large or too crowded whereupon it becomes difficult to discriminate a single bacterium.We analysed the time it takes for a population of 4.Marinomonas primoryensis were first isolated from coastal sea ice in the sea of Japan [MpIBP showed that this protein is not secreted into the medium [MpIBP might have a different role in Nature from the DUF3494 bacterial IBPs, which seem to control the structure of ice surrounding the microorganism [of Japan  and thenof Japan . Using ae medium , unlike e medium . These forganism . Other iorganism ,41 or hiMpIBP is located on the outer surface of the bacteria  and extender domains (region II) of 5.MpIBP is an ice adhesin that serves to attach M. primoryensis to ice. When bound to its host bacterium, MpIBP does not significantly affect the freezing or melting points of the ice. This remarkable adaptation of the adhesin principle used by bacteria to bind to many kinds of surfaces is currently the only example where an adhesin has evolved to bind ice. We show that a polyclonal antibody directed against the ice-binding domain of MpIBP can completely prevent bacterial adhesion to its substrate. Such bacterial adhesion inhibitors are potentially useful for blocking bacterial attachment to other surfaces where biofilm formation is harmful.Our findings provide direct evidence that