Escherichia coli O157:H7 is one of the pathogenic bacteria causing foodborne disease. The use of lytic bacteriophages can be a good solution to overcome the disease. This study is aimed at isolating lytic bacteriophages from environmental sewage with E. coli O157:H7 bacterial cells. The sample used in this study was eight bacteriophages, and the technique used in identifying E. coli O157:H7 carriers of the stx1 and stx2 genes was PCR. The double layer plaque technique was used to classify bacteriophages. Plaque morphology, host specificity, and electron micrograph were used to identify the bacteriophages. The result obtained plaque morphology as a clear zone with the largest diameter size of 3.5 mm. Lytic bacteriophage could infect E. coli O157:H7 at the highest titer of 10 × 108 PFU/mL. Bacteriophages have been identified as Siphoviridae and Myoviridae. Phage 3, phage 4, and phage 8 could infect Atypical Diarrheagenic E. coli 1 (aDEC1) due to their host specificity. The Friedman statistical tests indicate that lytic bacteriophage can significantly lyse E. coli O157:H7 (p = 0.012). The lysis of E. coli O157:H7 by phage 1, phage 2, phage 3, and phage 5 bacteriophages was statistically significant, according to Conover's posthoc test (p < 0.05). The conclusion obtained from this study is that lytic bacteriophages from environmental sewage could lyse E. coli O157:H7. Therefore, it could be an alternative biocontrol agent against E. coli O157:H7 that contaminates food causing foodborne disease. Escherichia coli O157:H7 is suspected of causing 63.000 instances of foodborne illness, 2.100 hospitalizations, 20 deaths, and a $ 271 million economic burden [E. coli O157:H7 was existed in the feces of asymptomatic children attending elementary school in Surabaya, East Java, Indonesia [Foodborne diseases are diseases caused by consuming food and or drinks contaminated by various microorganisms or pathogenic microbes (foodborne pathogens) . Foodborc burden , 5. E. cndonesia –10. Therndonesia .E. coli O157:H7 could cause disease by toxin releasing called shiga-like toxin (stx), which could result in food poisoning [E. coli causes more than 2.5 million diseases worldwide each year, resulting 269 deaths [oisoning . Shiga t9 deaths . Lytic b9 deaths  and as a9 deaths . Because9 deaths . In this9 deaths . Althoug9 deaths .Bacteriophages are mostly composed of nucleic acid, which is nontoxic. Therefore, it is beneficial as it has a low impact on the environment with relatively affordable costs. As a bacteriophage is used for a biocontrol agent, the interaction between bacteriophage proteins with biology or environmental components, animals, and the immune responses of a person which could be increased for potential negative effects needs to be considered  .E. coli O157:H7 isolate stored in Tryptic Soy Broth Media  with 15% glycerol at -20°C was obtained from the Microbiology Laboratory, Soetomo General Academic Hospital, Surabaya, East Java, Indonesia. Stock culture of E. coli O157:H7 was subcultured on Mac Conkey Agar plate media , incubated at 37°C for 24 hours and stored at low temperature.Stock culture of Bacterial DNA extraction referred to Geneaid 2017) procedural [17 proced2. PCR was carried out in a gene thermal cycler . The optimized cycle program of denaturation, annealing, and extension temperatures was as follows: 1 cycle of 2 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C; and 1 cycle of 5 min at 72°C.PCR was performed using 2 mL of template in a 20 mL volume of the PCR PreMix (Promega corporation). The PCR mixture consisted of 1 U thermostable DNA polymerase, 250 mM dNTP, 50 mM Tris-HCl (pH 8.3), 40 mM KCl, and 1.5 mM MgClstx1 uses forward CAGTTAATGTGGTGGCGAAGG and reverse CACCAGACAATGTAACCGCTG of 1221 bp and Stx2 forward ATCCTATTCCCGGGAGTTACG-3 and reverse GCGTCATCGTATACACAGGAGC of 1247 bp [Primer of shiga toxin genes of The samples used for bacteriophage isolation were taken from traditional market sewage, abbatoir, and Kalimas River of Surabaya, Indonesia . The 4.5 mL filtrate was then mixed with 0.5 mL of E. coli O157:H7 at exponential phase culture (McFarland 0.5 ~ 1.5 × 108 CFU/mL) and added 5 mL of Nutrient Broth. Next, the mixture was incubated for 24 hours in a waterbath at 37°C. It was then centrifuged at 3000 rpm at 4°C for 15 minutes. The supernatant was taken by a syringe and filtered using 0.22 μm millipore membrane . The supernatant that has been filtered was inserted into a sterile tube and stored at low temperature [Sample filtration used the modified Thung et al. method, as much as 1 mL of liquid waste sample was diluted into 9 mL of Nutrient Broth  media, centrifuged at 3000 rpm for 20 minutes, and then the supernatant was filtered using 0.45 perature .μL of lytic bacteriophage stock were diluted into the Ringer buffer with serial 10-fold dilutions. Then, each serial dilution of lytic bacteriophages was taken as much as 100 μL and each was mixed with 100 μL of E. coli O157:H7 bacteria at exponential phase culture (McFarland 0.5) into a new sterile Eppendorp tube and incubated at 37°C for 30 minutes. Soft agar consisting 70% nutrient agar media that was previously made was then warmed at 56°C. After that, the previous mixture was added by pouring technique on nutrient agar media. Incubation was carried out at 37°C for 24 hours [Bacteriophage stocks were then inoculated using the modified method of Bonilla et al. by the double layer plaque technique. A total of 100 24 hours .E. coli O157:H7 bacterial culture at exponential phase (McFarland 0.5) and incubated for 24 hours, then centrifuged at 3000 rpm at 4°C for 20 minutes. The bacteriophage filtrate was then filtered using 0.22 μm millipore membrane .As lytic bacteriophages were observed, the purification of lytic bacteriophages was carried out by the plaque formed using Pasteur pipettes and enriched, so that more plaques would produce. Each plaque was transferred to 10 mL of E. coli O157:H7 bacteria. Then, centrifugation was done at 3000 rpm at 4°C for 20 minutes for two replication times. The supernatant was filtered using a 0.22 μm millipore filter membrane  and then stored as bacteriophage stock at low temperature (4°C) [The result was in the form of a bacteriophage filtrate, and then it was carried out in an NA plate media. The plaque formed was then removed and inserted into the Ringer buffer. The bacteriophage suspension was vortexed and left about 5 to 10 minutes at room temperature, and so then the bacteriophages could attach the re (4°C) .E. coli O157:H7 bacteria. Then, centrifugation was done at 3000 rpm at 4°C for 20 minutes for two replication times. The supernatant was filtered using a 0.22 μm millipore filter membrane  and then stored as bacteriophage stock at low temperature (4°C) [A modified method of Thung et al. of bacteriophage propagation was used. The plaque formed previously was then removed and inserted into the Ringer buffer (4 : 1). The bacteriophage suspension was vortexed and left about 5 to 10 minutes at room temperature, and so then the bacteriophages could attach the re (4°C) .E. coli O157:H7 bacteria cultured in Nutrient Broth medium  at exponential phase (McFarland 0.5) were centrifuged at 3000 rpm at 4°C for 20 minutes. The pellets formed were each infected with 100 μL of lytic bacteriophages. Each mixture was incubated at 37°C for 30 minutes, and the mixture was then added to 10 mL of Nutrient Broth medium and incubated for 24 hours at 37°C. Then, each of them was centrifuged at 3000 rpm at 4°C for 20 minutes. The supernatant formed was taken with a syringe and filtered with a 0.22 μm filter membrane . Each supernatant that had been filtered was inserted into a sterile tube and stored as a bacteriophage stock at low temperature (4°C) [Bacteriophage propagation was using the modified method of Bonilla et al., and a total of 10 mL of μL from each dilution of the bacteriophage isolate was taken and transferred to 100 μL of the E. coli O157:H7 bacterial culture, after incubated for 24 hours on Nutrient Broth medium . The suspension was incubated for 30 minutes at 37°C. A total of 7 mL of soft agar that was previously made at 56°C were mixed. After that, each one of the suspensions was poured onto NA plate media  and incubated at 37°C for 24 hours. After incubation, we then observed the plaque formed and expressed as PFU/mL [Bacteriophage quantification was measured by counting the amount of plaque formed in NA media plates as plaque forming units/mL (PFU/mL). Each lytic bacteriophage stock was diluted by 10-fold serial, and then 100 μL of bacteriophage were dropped onto a 400 mesh grid and left for 30 seconds. On carbon-coated grids, bacteriophage samples were negatively stained with 5 μL of 2% (w/v) uranyl acetate. The grids were viewed using a JEM-1010 TEM  [The Eijkman Institute in Jakarta, Indonesia, used transmission electron microscopy to identify the shape of the bacteriophage. A total of 10 , Japan) .E. coli O157:H7 by bacteriophages was using the modified method of Mirzaei and Nilsson and Aryal. One milliliter of E. coli O157:H7 bacteria that had been grown on MacConkey Agar plates was transferred onto Nutrient Broth media  at exponential phase (McFarland 0.5), and each was infected with 1 mL of bacteriophage stock. Each mixture was then incubated for 2 hours, 4 hours, 6 hours, and 8 hours. The opacity assessment of McFarland densitometry was carried out to calculate the number of live bacteria [Lysis of bacteria , 26.Atypical Diarrheagenic E. coli isolates   and keep storage clinical storage Salmonella paratyphi A, Salmonella paratyphi B, and Shigella flexneri isolates . Atypical Diarrheagenic E. coli , S. paratyphi A, S. paratyphi B, and S. flexneri isolates were stored in Tryptic Soy Broth Media with 15% glycerol at -20°C.The bacteria used for the host specificity test of lytic bacteriophages were DEC1, aDEC2, aDEC3, aDEC4, and aDEC5 was inoculated into Mac Conkey Agar plate media  and incubated at 37°C for 24 hours. A positive result gives rise to a pink to rose red colonies, such as its ability to ferment lactose [S. paratyphi A, S. paratyphi B, and S. flexneri isolates were inoculated into Salmonella Shigella Agar plate media  and incubated at 37°C for 24 hours. A positive result gave rise to colorless for S. paratyphi A and S. flexneri, shown as colorless with black centers for S. paratyphi B as its ability to produce hydrogen sulfide (H2S) [Stock culture of a lactose , 28   , Sulfate Indol Motility/SIM , Simmons Citrate Agar/SCA , and Urea broth  [oid, UK)   by exponential phase (McFarland 0.5), mixed with 100 μL of lytic bacteriophages stock, and diluted into the Ringer buffer with serial 10-fold dilutions. Then, each serial dilution of lytic bacteriophage was taken as much as 100 μL into a new sterile Eppendorf tube and incubated at 37°C for 30 minutes for the lytic bacteriophages to attach. A total of 7 mL of soft agar that was previously made at 56°C was mixed by pouring technique on Nutrient Agar plate media, and incubation was carried out at 37°C for 24 hours. Then, the plaque formed was observed and counted [In cultures of  counted .Statistical analysis used in this study was a nonparametric test in the form of the Friedman test. A significant result was continued with Conover's posthoc.E. coli O157:H7 isolates carrying the stx1 and stx2 genes using the PCR technique show that only the stx1 gene was borne by E. coli O157:H7 isolates, as shown in The results of molecular identification of stx2 gene, this study discovered the stx1 gene formed by E. coli O157:H7 to the present in a 1220 bp band approximately. This gene was also found in the feces of children in Southern Iran [E. coli O157:H7 must also be monitored as a foodborne pathogen.In contrast to the ern Iran  and humaern Iran . As a reThe samples were collected from the environmental sources as shown in the following table:E. coli O157:H7 carrying shiga toxin gene cultures grown in the double layer plaque indicate the presence of lytic bacteriophages  and the accessibility of the bacteriophage to the target bacteria , 39. Preeceptors , 41. Theeceptors .The concentrations of the purified bacteriophages were then calculated based on the amount of plaque formed. The amount of plaque formed was then calculated in plaque forming units (PFU/mL), which is a measure of the amount of virus infective per volume of fluid. In addition, the lytic bacteriophage concentration was calculated as seen in 3 to 10 × 108 PFU/mL. The number of bacteriophages previously detected was >108 PFU/mL of Bacillus cereus bacteriophage [15 PFU/mL of Klebsiella pneumoniae bacteriophage [12 PFU/mL of E. coli bacteriophage [16 PFU/mL [6 PFU/mL for T4 bacteriophage [11 PFU/mL [10 PFU/mL [10 PFU/mL of E. coli bacteriophage [Salmonella spp. bacteriophages with titers of 5 × 108 PFU/mL [7 PFU/mL [Bacteriophage quantification was measured by counting the amount of plaque formed, and the concentration obtained was of 5 × 10riophage , 1015 PFriophage , 5 × 101riophage , 1.2 × 16 PFU/mL , and 5 ×riophage , and 1.31 PFU/mL , 2.1 × 10 PFU/mL , and 2.6riophage . Marti e8 PFU/mL . Bao et 7 PFU/mL .8 and 1011 PFU/mL, this could be due to the optimum plating condition, and other parameters such as the buffer in which the bacteriophages were suspended, the incubation media [2+ ions which can stabilize the fragile interface of the virion with its receptors [3H5NaO3, 3 g NaCl, 0.15 g KCl, and 0.1 g CaCl2.2H2O, while other studies used SM buffer consisting of 5.8 g NaCl, 2 g MgSO4·7H2O, 50 mM Tris-Cl (pH 7.5), and 5 mL gelatine in 1 L H2O [Other studies have shown similar results with bacteriophage titres between 10on media , Ca2+ ioeceptors , 41, andeceptors , 50. Thi 1 L H2O , 51, 52.The morphology of bacteriophages, such as the head and tail shapes, was discovered to be identical to the order of Caudovirales , 54, basBacteriophages have a head and tail that vary in size, as seen in Myoviridae were bacteriophages with a long contractile tail, while Siphoviridae had a long noncontractile tail [Siphoviridae bacteriophage ranged from 50 to 133.3 nm, while Myoviridae bacteriophage had a head diameter of 83.3 to 100 nm. The tail length of Siphoviridae bacteriophages ranged from 100 to 288 nm, while the tail length of Myoviridae bacteriophages ranged from 83.3 to 100 nm. The tail diameter of Siphoviridae bacteriophages ranged from 6.3 to 17.4 nm, while the tail length of Myoviridae bacteriophages ranged from 10 to 16.7 nm  at 37°C at an incubation interval of 0 hour, 2 hours, 4 hours, 6 hours, and 8 hours. The lysis of E. coli O157:H7 is as seen on One mL of E. coli O157:H7 can be seen. In comparison to the amount of E. coli O157:H7 in the control, which was 1.5 × 108 CFU/mL without lytic bacteriophage injection, the lowest value of E. coli O157; H7 obtained was 3.7 × 107 CFU/mL, with a maximum of 1.2 × 108 CFU/mL (phage 1). The following table shows the reduction in E. coli O157:H7 caused by lytic bacteriophage:The ability of LBP to lyse E. coli O157:H7 by 75% . Based on these characteristics, more research is needed on the lysis of bacteriophages with a higher number of Siphoviridae and Myoviridae bacteriophages, as well as a bigger number of E. coli O157:H7 bacterium.At first glance, the The host's specificity is depicted in aDEC1 , with estimating a large effect size of Kendall's W by 0.993 [Statistical analysis using JASP 0.14.1.0 programme described a nonparametric test. The Friedman test conducted a significant effect in the lyse of by 0.993  , and LBP7 (p = 0.022), whereas phage 2 had a statistically significant difference in E. coli O157:H7 amount compared to phage 3 (p = 0.036), phage 6 (p = 0.014), and phage 7 (p = 0.014), according to Conover's posthoc test. In addition, phage 5 had a significantly different impact in the lyse of E. coli O157:H7 relative to phage 6 (p = 0.036) and phage 7 (p = 0.036). Phage 6 had a significantly different effect in the lyse of E. coli O157:H7 compared to phage 8 (p = 0.057), and phage 7 had a significantly different effect in the lyse of E. coli O157:H7 compared to phage 8 (p = 0.057)  see .E. coli O157:H7 was shown to be lower than on phage 1, the number of E. coli O157:H7 detected by phage 3, phage 6, and phage 7 was statistically lower than phage 2, and the number of E. coli O157:H7 detected by phage 6 and phage 7 produced a lower number of E. coli O157:H7 than phage 5.On phage 6 and phage 7, the amount of E. coli O157:H7 carrying the stx1 gene at the highest titer of 10 × 108 PFU/mL. Bacteriophages have been identified as Siphoviridae and Myoviridae by electron micrograph.Lytic bacteriophage identified from environmental sewage in Surabaya, Indonesia, shows plaque morphology as a clear zone with the largest diameter size of 3.5 mm, and lytic bacteriophage could infect Atypical Diarrheagenic E. coli 1 (aDEC1) due to their host specificity. The Friedman statistical tests indicate that lytic bacteriophage can significantly lyse E. coli O157:H7 (p = 0.012). The lysis of E. coli O157:H7 by phage 1, phage 2, phage 3, and phage 5 bacteriophages was statistically significant, according to Conover's posthoc test (p < 0.05). The conclusion obtained from this study is that lytic bacteriophages from environmental sewage could lyse E. coli O157:H7. Therefore, it could be an alternative biocontrol agent against E. coli O157:H7 that contaminates food causing foodborne disease.It was studied that phage 3, phage 4, and phage 8 could infect


Saccharomycescerevisiae, the Rpd3L complex contains a histone deacetylase, Rpd3, and the DNA binding proteins, Ume6 and Ash1, and acts as a transcriptional repressor or activator. We previously showed that RPD3 and UME6 are required for the activation of PDR5, which encodes a major efflux pump, and pleiotropic drug resistance (PDR) in ρ0/− cells, which lack mitochondrial DNA. However, there are inconsistent reports regarding whether RPD3 and UME6 are required for Pdr5-mediated PDR in ρ+ cells with mitochondrial DNA. Since PDR5 expression or PDR in the ρ+ cells of the rpd3Δ and ume6Δ mutants have primarily been examined using fermentable media, mixed cultures of ρ+ and ρ0/− cells could be used. Therefore, we examined whether RPD3 and UME6 are required for basal and drug-induced PDR5 transcription and PDR in ρ+ cells using fermentable and nonfermentable media. UME6 suppresses the basal transcription levels of the ABC transporters, including PDR5, and drug resistance in ρ+ cells independent of the carbon source used in the growth medium. In contrast, RPD3 is required for drug resistance but did not interfere with the basal PDR5 mRNA levels. UME6 is also required for the cycloheximide-induced transcription of PDR5 in nonfermentable media but not in fermentable media.In  Saccharomyces cerevisiae, ATP-binding cassette (ABC) transporters, such as Pdr5, Snq2, and Yor1, efflux a variety of functionally and structurally unrelated compounds, such as fluconazole and cycloheximide from cells [S. cerevisiae [PDR5, SNQ2, YOR1, PDR10, and PDR15 are regulated by the paralogous Zn2Cys6 transcription factors, Pdr1 and/or Pdr3 [PDR5, SNQ2, YOR1, PDR10, and PDR15 [PDR3 also has two PDREs in its promoter region that are recognized by Pdr3 and Pdr1 [PDR3 is subject to positive transcriptional autoregulation by Pdr3 and transcriptional regulation by Pdr1, via these two PDREs [PDR5 and SNQ2 and PDR are reduced by PDR1 deletion in ρ+ cells, which contains mitochondrial DNA, compared to PDR3 deletion [PDR1 and PDR3 substantially reduces the basal expression of their target genes, including PDR5, and causes hypersensitivity to multiple drugs compared with the disruption of either PDR1 or PDR3 [PDR1 and PDR3 have functionally overlapping roles in PDR.In the yeast om cells ,4,5. Therevisiae ,7. The A/or Pdr3 ,9. Pdr1 nd PDR15 ,12. PDR3and Pdr1 . Thus, Pwo PDREs . The basPDR3, PDR5, SNQ2, YOR1, PDR10, and PDR15, and thereby induce the PDR [PDR1 and PDR3 [PDR5, SNQ2, YOR1, PDR10, or PDR15 mRNA levels were found to be upregulated by the compounds such as cycloheximide, benomyl, fluphenazine, 4-nitroquinolineN-oxide, cantharidin, 2,4-dichlorophenol, or polyoxyethylene-9-laurylether [PDR5, SNQ2, and YOR1 after exposure to drugs or chemical compounds [0 cells lacking mitochondrial DNA in S. cerevisiae and Candida glabrata, resulting in the enhanced expression of multidrug resistance genes, including PDR5 and CgCdr1, and the elevated PDR. Notably, Pdr3, but not Pdr1, is required for the induction of PDR5 expression in ρ0 cells [Gain-of-function mutations, such as pdr1-3 and pdr3-7 in Pdr1 and Pdr3, lead to the overexpression of  the PDR ,14,15,16 the PDR . On the and PDR3 ,19,20. Hrylether ,22. Pdr1ompounds ,22,23. Tρ0 cells .S. cerevisiae, there are two Rpd3 complexes, Rpd3S (0.6 MDa) and Rpd3L (1.2 MDa). Both complexes share Rpd3, Sin3, and Ume1 [HO promoter by Ash1 to specifically inhibit its expression [In and Ume1 . Rpd3 isand Ume1 ,27. Inteand Ume1 ,28. For and Ume1 . Alternapression . In addipression  and the pression . RPD3 and UME6 are required for the activation of PDR5 transcription and drug resistance by retrograde signaling in the ρ0 cells of S. cerevisiae [+ cells in the rpd3Δ, but not the ume6Δ and ash1Δ strains, displayed sensitivity to cycloheximide at the minimum inhibitory concentration [RPD3 reduces basal PDR5 transcription levels in ρ+ cells [+ cells of the rpd3Δ strains of Candida albicans and S. cerevisiae is not due to reduced PDR5 mRNA levels but instead due to diminished Hsp90-dependent antifungal drug resistance [UME6 in ρ+ cells conferred sensitivity to atorvastatin, cycloheximide, and benomyl but not to ketoconazole, fluconazole, and oligomycin via spot dilution assay [UME6 does not reduce Pdr5 expression by western blot analysis [RPD3 and UME6 are required for PDR5-mediated PDR in ρ+ cells.We previously showed that revisiae . On the ntration . They alρ+ cells . Howeversistance . In addion assay . The autanalysis . Thus, t+ cells grown in yeast extract peptone dextrose (YPD) culture medium containing glucose as the carbon source are used when drug susceptibility and basal PDR5 expression were examined. Since ρ+ cells grown in YPD medium spontaneously generate ρ0/− cells that lack mitochondrial DNA at a rate of 0.1–1% per generation, the above studies could have used mixed cultures of ρ+ and ρ0/− cells [0/− cells can grow on YPD culture medium containing glucose as a fermentable carbon source but cannot grow on yeast extract peptone glycerol (YPG) medium containing glycerol as a nonfermentable carbon source. Thus, we can examine the effect of RPD3 and UME6 deletion on PDR5 transcription and PDR in pure ρ+ cells grown on YPG culture medium. Therefore, in this study, we examined whether RPD3 and UME6 are required for basal and drug-induced PDR5 transcription and drug resistance in the ρ+ cells of S. cerevisiae grown in YPG medium. Furthermore, the results obtained in YPG medium were compared with those obtained from ρ+ cells grown in YPD medium. As a result, we show that UME6 suppresses the basal transcription levels of the ABC transporters, including PDR5, and drug resistance in ρ+ cells independent of the carbon source used in the growth medium. In contrast, we also show that RPD3 is required for drug resistance but did not interfere with the basal PDR5 mRNA levels.However, in the above studies, ρ/− cells ,37. ρ0/−UME6, RPD3, PDR3, ASH1, or GAT3 deletions, open reading frames of the genes were replaced with KanMX or bleMX6 gene cassettes by PCR-mediated one-step gene disruption in the FY1679-28C background [0 cells and obtain ρ+ cells. The FY1679-28C  strain was used as the wild-type strain . To consckground . The strYeast cells were grown in YPD medium  or YPG medium  at 30 °C with shaking.+ cells from each yeast strain were aerobically grown to the logarithmic phase (an OD600 of 0.6–0.9) at 30 °C in YPG or YPD media in triplicate. Five microliters of 10-fold serial dilutions of the YPG cultures containing the same number of cells was spotted on YPG plates with or without 3 μg/mL fluconazole  (or 0.1 μg/mL cycloheximide (Wako)) and incubated at 30 °C for 21 days. Five microliters of 10-fold serial dilutions of the YPD cultures containing the same number of cells was spotted on YPD plates with or not without 30 μg/mL fluconazole  (or 0.5 μg/mL cycloheximide (Wako)) and incubated at 30 °C for 21 days.The relative resistance of each yeast strain to fluconazole or cycloheximide was estimated with a spot dilution assay using YPG or YPD media ,41. ρ+ cKanMX gene cassette was cocultivated with the other gene deletion mutant that had been replaced with the bleMX6 gene cassette [+ cells from each mutant corresponding to 0.1 OD600 initial concentration were cocultivated in 10 mL of YPG or YPD media with or without 30 μg/mL fluconazole (or 0.5 μg/mL cycloheximide) [One gene deletion mutant replaced with the cassette . ρ+ celleximide) . The alieximide) ,42. The eximide) ,42. + cells from each yeast strain were grown to the logarithmic phase (an OD600 of 7–9) in YPG or YPD media in duplicate. The cultures were diluted to an OD600 of 0.1 and grown to an OD600 of 0.4–0.8 [ρ 0.4–0.8 ,43,44. A 0.4–0.8 ,43,44. T+ cells from the wild-type and ume6∆꞉꞉bleMX6 strains were grown to an OD600 of 7–9 in YPG or YPD media, diluted to an OD600 of 0.1, and grown to an OD600 of 0.4–0.8 in duplicate [ρuplicate ,43,44. Auplicate ,43,44. T® Premix Ex Taq II (TaKaRa) in a Step One Real-time PCR system (Applied Biosystems). A minus reverse transcriptase control was used as the negative control. Serial dilutions of control cDNA were prepared to produce a standard curve for each primer pair. The primers used for qRT-PCR are listed in ACT1, which was used as an endogenous control. The normalized levels are shown relative to samples from the wild-type or cycloheximide-untreated wild-type strains, which were set to 1.Reverse transcription of total RNA was performed using the PrimeScript 1st strand cDNA Synthesis Kit (TaKaRa). SYBR Green qRT-PCR for cDNA from the individual duplicate samples was performed using the TB Greenume6∆ strain at each time point in Figure 2 was normalized to that at 0 h. A paired t-test was used for statistical analysis in Figure 2. An unpaired Student’s t-test was used for statistical analysis in Figures 3 and 4. Statistical significance was indicated as p < 0.05.The survival rate of the RPD3 and UME6 are responsible for PDR in ρ+ cells of S. cerevisiae, a spot dilution assay using YPG medium containing the nonfermentable carbon source glycerol was carried out to determine the resistance or susceptibility in the ρ+ cells of the rpd3∆ and ume6∆ mutants to the PDR substrates fluconazole and cycloheximide. ρ+ cells of the wild-type strain required a lower concentration of fluconazole and cycloheximide for growth inhibition in YPG medium than in YPD medium, which is consistent with previous reports [+ cells of the ume6∆꞉꞉bleMX6 mutant were slightly more tolerant to fluconazole and cycloheximide than those in the wild-type, ash1∆꞉꞉bleMX6, pdr3∆꞉꞉bleMX6, and gat3∆꞉꞉bleMX6 strains . As a result, there were no significant differences in fluconazole and cycloheximide resistance between the wild-type, ume6∆꞉꞉bleMX6, ash1∆꞉꞉bleMX6, pdr3∆꞉꞉bleMX6, and gat3∆꞉꞉bleMX6 strains in YPD plates  (+ cells of the ume6∆꞉꞉KanMX strain were eliminated from the cocultures of the ume6∆꞉꞉KanMX and ash1∆꞉꞉bleMX6 strains in YPG medium over time in the absence of cycloheximide but selected in the presence of 0.5 μg/mL cycloheximide (data not shown). In addition, the ρ+ cells of the ume6∆꞉꞉KanMX strain were eliminated from the coculture over time in the absence of fluconazole but not in the presence of fluconazole when cocultivated with gat3∆꞉꞉bleMX6 in YPG medium with or without 30 μg/mL fluconazole (+ cells of the gat3∆꞉꞉KanMX and ume6∆꞉꞉bleMX6 mutants cocultivated in YPG medium with or without 30 μg/mL fluconazole (data not shown).In the spot dilution assay above, the ρ strains . To deteon assay , and the < 0.05) A. Similaconazole B. Furtheume6∆ is dependent on the carbon source used in the growth medium. Rather than using the coculture grown in YPG medium, the survival rate of each strain using the coculture grown in YPD medium was estimated in the same way. Consequently, the ρ+ cells of the ume6∆꞉꞉KanMX strain were eliminated from the coculture over time in the absence of cycloheximide but not in the presence of 0.5 μg/mL cycloheximide when cocultivated with the ash1∆꞉꞉bleMX6 mutant in YPD medium (p < 0.05) (+ cells of the gat3∆꞉꞉KanMX and ume6∆꞉꞉bleMX6 strains cocultivated in YPD medium with or without 30 μg/mL fluconazole (or 0.5 μg/mL cycloheximide) (data not shown). These results indicate that, in contrast to previous reports, UME6 acts as a silencer rather than an enhancer of drug resistance in ρ+ cells of S. cerevisiae independent of the carbon source used in the growth medium.We next examined whether the higher cycloheximide and fluconazole resistance in  < 0.05) C. Furthe+ cells of the rpd3∆ strain in the spot dilution assay, while increased drug resistance in the ρ+ cells of the ume6∆ strain was found in the spot dilution and cocultivation assays. These results suggested that the transcriptional levels of ABC transporters such as PDR5 are reduced in the ρ+ cells of the rpd3∆ mutant and increased in the ρ+ cells of the ume6∆ mutant. Thus, we investigated the mRNA levels of ABC transporters PDR5, PDR10, SNQ2, and YOR1 and their transcriptional regulators PDR1 and PDR3 in the ρ+ cells of the wild-type, ume6∆, rpd3∆, pdr3∆, ash1∆, and gat3∆ strains grown in YPG medium to the logarithmic phase by real-time RT-PCR. PDR1, PDR3, PDR5, SNQ2, and YOR1 but not PDR10 expression was significantly induced in the ume6∆ strain compared with the wild-type strain (p < 0.05), while RPS9B expression, used as a control, was downregulated in the ume6∆ strain compared with the wild-type strain   (Decreased drug resistance was found in the ρe strain A. The tr < 0.05) A. The laG plates A. Therefeduction ,45. In c < 0.05) A. PDR5, PDR10, SNQ2, and YOR1 and their transcriptional regulators PDR1 and PDR3 in ρ+ cells of the wild-type, ume6∆, rpd3∆, pdr3∆, and ash1∆ strains grown to the logarithmic phase in YPD medium in the same way. In contrast to the results obtained by real-time RT-PCR using YPG medium, only the basal PDR5 and PDR10 mRNA levels were significantly upregulated in the ume6∆ strain compared with the wild-type strain in YPD medium (p < 0.05)   B. In addG medium A,B. Howe > 0.05) B. TherefNA level . In addiD medium B.+ cells of the ume6∆ mutant in YPG and YPD media using real-time RT-PCR. In YPG and YPD media, the ABC transporter mRNA levels in the ume6∆ strain at 0 min were higher than those in the wild-type strain at 0 min (p < 0.05), which is consistent with the results in UME6 is required for the repression of basal transcription of the ABC transporters, including PDR5, in ρ+ cells, independent of the media used. In addition, the degree of upregulation of PDR5 transcription in the ume6∆ strain at 0 min was relatively lower in YPD medium than in YPG medium, which is consistent with the results in PDR10, in the wild-type strain were significantly higher at 45 min than at 0 min in YPG and YPD media (p < 0.05)    A,B. In c > 0.05) A. In YPD < 0.05) B. These UME6 suppresses the basal transcription levels of the ABC transporters, including PDR5, and drug resistance in the ρ+ cells of S. cerevisiae independent of the carbon source used in the growth medium while RPD3 does not interfere with basal PDR5 transcription but contributes to drug resistance. The results found for RPD3 in this study were consistent with previous reports [UME6 suppresses basal PDR5 mRNA levels and drug resistance in the ρ+ cells of S. cerevisiae. ρ+ cells of the ume6∆ strain conferred more resistance to fluconazole and cycloheximide than those of the wild-type strain in the spot dilution assay using YPG plates but not using YPD plates. In addition, although the ρ+ cells of the ume6∆ mutant were eliminated from the coculture over time in both YPG and YPD media without cycloheximide, these cells accounted for the majority of the coculture over time in YPG medium containing 0.5 μg/mL cycloheximide , Med13 (Srb9), the cyclin-dependent kinase Cdk8 (Srb10), and cyclin C (Srb11). The Cdk8 subcomplex has both a negative and a positive role in gene transcription [PDR5 expression in ρ0 cells but not in ρ+ cells, indicating a difference in the regulatory machinery of PDR5 transcription between ρ+ and ρ0 cells [PDR5 by Ume6 between ρ0 and ρ+ cells. The transcriptional mediator complex serves as a link between sequence-specific transcription factors and the RNA polymerase II machinery . There acription ,50,51. Pcription . Deletioρ0 cells . This diUME6 is required for the intact enhancement of PDR5 and YOR1 transcription after cycloheximide exposure in YPG medium and for intact cycloheximide-induced transcription of SNQ2, but not PDR5, in YPD medium. This result suggested that basal transcriptional repression of the ABC transporters, including PDR5, by UME6 is lifted in response to drug exposure in YPG medium. However, it is unknown why the intact induction of PDR5, but not SNQ2, in the ume6∆ mutant occurs after cycloheximide exposure in YPD medium (PDR5 transcription or intact drug-induced PDR5 upregulation in the ume6∆ mutant is more effective for drug resistance in YPD medium? Despite intact cycloheximide-induced transcription of PDR5 in the ume6∆ strain in YPD medium, there was no difference in cycloheximide resistance between the ρ+ cells of the wild-type and ume6∆ strains in the spot dilution assay using YPD plates, while a little difference in the cocultivation assay using YPD plates. In addition, upregulation of basal PDR5 transcription levels in the ume6∆ strain compared to the wild-type strain was relatively lower in YPD medium than in YPG medium. Therefore, the upregulation of basal PDR5 transcription in the ume6∆ strain may be more important for drug resistance than intact transcriptional upregulation of PDR5 in the ume6∆ strain following cycloheximide exposure. This study indicated that D medium B. This mRPD3 and UME6 in basal and drug-induced transcription of the ABC transporters, including PDR5, and PDR in the ρ+ cells of S. cerevisiae. Using spot dilution and cocultivation assays and YPG and YPD media, we have shown that RPD3 contributes to drug resistance in ρ+ cells, while in contrast to the previous reports, UME6 contributes to the suppression of drug resistance in ρ+ cells, independent of the carbon source used in the growth medium. In addition, using a real-time PCR assay, we have shown that RPD3 does not interfere with the basal PDR5 mRNA level in ρ+ cells, while UME6 is involved in the suppression of basal transcription of some ABC transporters, including PDR5, in ρ+ cells regardless of the carbon source utilized. We have also described that UME6 contributes to an intact transcriptional enhancement of PDR5 in cycloheximide-exposed ρ+ cells in YPG medium but not in YPD medium. This and our previous work will provide useful knowledge on yeast multidrug resistance via the transcriptional regulation of efflux genes by UME6 in ρ+ and ρ0 cells.This study investigated the roles of


Aliivibrio wodanis 06/09/139 harbors two QS systems, the main LuxS/LuxPQ system and an N-acyl homoserine lactone (AHL)-mediated AinS/AinR system and a master QS regulator, LitR. To explore the QS and survival strategies, we performed genome analysis and gene expression profiling on A. wodanis and two QS mutants (ΔainS and ΔlitR) at two cell densities (OD600 2.0 and 6.0) and temperatures (6 and 12°C). Genome analysis of A. wodanis revealed two CRISPR systems, one without a cas loci (CRISPR system 1) and a type I-F CRISPR system (CRISPR system 2). Our analysis also identified three main T6SS clusters  and four auxiliary clusters, as well about 80 potential Type VI secretion effectors (T6SEs). When comparing the wildtype transcriptome data at different cell densities and temperatures, 13–18% of the genes were differentially expressed. The CRISPR system 2 was cell density and temperature-independent, whereas the CRISPR system 1 was temperature-dependent and cell density-independent. The primary and auxiliary clusters of T6SSs were both cell density and temperature-dependent. In the ΔlitR and ΔainS mutants, several CRISPR and T6SS related genes were differentially expressed. Deletion of litR resulted in decreased expression of CRISPR system 1 and increased expression of CRISPR system 2. The T6SS1 and T6SS2 gene clusters were less expressed while the T6SS3 cluster was highly expressed in ΔlitR. Moreover, in ΔlitR, the hcp1 gene was strongly activated at 6°C compared to 12°C. AinS positively affected the csy genes in the CRISPR system 2 but did not affect the CRISPR arrays. Although AinS did not significantly affect the expression of T6SSs, the hallmark genes of T6SS (hcp and vgrG) were AinS-dependent. The work demonstrates that T6SSs and CRISPR systems in A. wodanis are QS dependent and may play an essential role in survival in its natural environment.For bacteria to thrive in an environment with competitors, phages and environmental cues, they use different strategies, including Type VI Secretion Systems (T6SSs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to compete for space. Bacteria often use quorum sensing (QS), to coordinate their behavior as the cell density increases. Like other aliivibrios,  Vibrio species  is a cell density-dependent cell-to-cell communication system in which bacteria produce and respond to signaling molecules called autoinducers (AIs), which subsequently activates the QS transcriptional regulator to control specific functions such as bioluminescence, motility, biofilm, secretion and virulence –5. Multi species . Two QS  wodanis . The Ain wodanis , 8. Thes wodanis . The Lux wodanis , 11. Thecholerae –14. At ltor LitR , 14, 15.of genes .In the environment, bacteria co-exist in communities with multiple competitors, including other bacterial species and phages, and have to respond to various cues such as changes in temperature, nutrient and iron availability, pH, osmolarity and salinity –22. Henccas genes, a leader sequence and a CRISPR array with repeats separated by several spacer sequences  gene cluster and later in many other bacteria  in several vibrios such as olyticus –52. T6SSolyticus –56. In solyticus , 58. Thebacteria –61. The bacteria –64. Sevebacteria –69. T6SSbacteria . For insectively . In addiectively .A. wodanis is a Gram-negative, rod-shaped, non-luminescent and motile bacterium with multiple polar flagella  from Atlantic salmon  during outbreaks of winter ulcer that mainly occurs at a temperature below 8°C  of 6.0  and the other at the cell density close to the end log phase (OD600 6.0) to study the role of cell densities in gene expression. Furthermore, in our recent study, we found that the temperature 6°C which is lower than the winter ulcer disease threshold temperature 8°C, has more impact on AHL production and cytotoxicity in CHSE cell line than 12°C  cell line  to reveal the global gene expression in the wild type and its QS mutant strains ΔainS and ΔlitR.flagella . A. wodaectively . The genplasmids . A. wodaelow 8°C , 75. Infelow 8°C , 76. Winelow 8°C , 77. Alt unclear , 77. Exp in fish . A. woda viscosa . In a co systems . In the ) of 6.0 , 80. MorA. wodanis 06/09/139 used was originally isolated from the head kidney of an Atlantic salmon on the west coast of Norway in 2006  with a total concentration of 1.0% (wt/vol) peptone , 0.5% (wt/vol) yeast extract , 2.5% NaCl (wt/vol)  and 1.5% agar . The pH of the media was adjusted to 7.5. Three biological replicates of pre-cultures of A. wodanis 06/09/139 and the mutants ΔainS and ΔlitR were grown from a single colony in 2 ml of Luria-Bertani Broth (LB2.5) overnight at 12°C, 220 rpm.The  in 2006 . The connt study . A. wodaA. wodanis 06/09/139, ΔainS and ΔlitR were diluted to a start OD600 of 0.01 in LB2.5 in a final volume of 60 ml using a 250 ml baffled flasks. The cultures were grown further in parallel using two Infors HT Multitron incubators set at 6 and 12°C at 220 rpm. The cultures were diluted 1:10 for OD600 measurement and harvested at low cell density (LCD) OD600 of 2.0 and high cell density (HCD) OD600 of 6.0. A small culture volume (1 ml) was harvested and mixed with two volumes of RNAprotect Bacteria reagent . The treated cultures were then incubated for 5 min at room temperature and vortexed for a few seconds before centrifuging at 13,000 rpm for 2 min in a cold Heraeus fresco 21 centrifuge . The pellets were flash-frozen with liquid nitrogen and stored at −80°C until RNA isolation. The total RNA from the cell pellets was isolated using the MasterpureTM complete DNA/RNA purification kit  according to the manufacturer's instructions. The RNA concentration was measured in NanoDropTM 2000c spectrophotometer . Ribosomal RNA (rRNA) was depleted from the total RNA using a Ribo-zero rRNA removal kit for bacteria . The RNA quality before and after rRNA depletion was determined using Agilent 2100 Bioanalyzer .Pre-culture biological triplicates of https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The gene expression levels were determined using EDGE-pro v1.0.1  values, and the cutoff values of ≥ 2.0 or ≤ −2.0 with padj values of < 0.05 were counted as significantly differentially expressed genes (DEGs). The transcriptome profiles of each strain was compared as high cell density against low cell density (tpHCD/LCD), high growth temperature 12°C against low temperature 6°C (tp12°C/6°C) and mutants against wild type (tpΔlitR/WT and tpΔainS/WT). These abbreviations of comparisons are used throughout this paper. The RNA sequence data of wild type A. wodanis, ΔainS and ΔlitR have been deposited in the European Nucleotide Archive (www.ebi.ac.uk/ena) under study accession number PRJEB34433.The mRNA libraries were prepared using the TrueSeq stranded mRNA library kit  and sequenced on Nextseq 500  using 150 cycles mid-output kit and run as a 75 bp paired-end reads at Norwegian Sequencing Center. The image analysis and base calling were performed using Illumina's RTA software version 2.4.6. Reads with low base call quality were removed using Illumina's default chastity criteria. The quality of the raw sequencing data was controlled using FastQC version 0.11.5 (o v1.0.1  and DESeo v1.0.1  with deflitR/WT and tpΔainS/WT) at different cell densities and temperatures against the reference organism A. wodanis 06/09/139  found when wild type was compared against cell densities (tpHCD/LCD) and temperatures (tp12°C/6°C) and also when mutants were compared against wild type (tpΔ6/09/139 .A. wodanis 06/09/139 genome was identified using CRISPRCasFinder (A. wodanis using the CRISPR finder tool (A. wodanis were identified using the phage search tool (PHASTER)  , 88.A. wodanis 06/09/139 were identified using SecReT6 (A. wodanis 06/09/139 in this study follows the naming of V. cholerae T6SS (A. wodanis were predicted using the Bastion 6 tool (The T6SS gene clusters in  SecReT6  and a ho SecReT6 . The namrae T6SS . Potentin 6 tool .A. wodanis 06/09/139 produces one AHL (3OHC10-HSL) and encodes genes for QS (A. wodanis 06/09/139 was found to affect various phenotypes that are possibly linked to the winter ulcer disease development (A. wodanis by comparing wild type and its ΔlitR and ΔainS mutants at low (OD600 of 2.0) and high cell density (OD600 of 6.0) and two different temperatures, 6 and 12°C.We have previously shown that s for QS , 79. Morelopment . Genome A. wodanis and mutant strains ΔlitR and ΔainS at different cell densities and temperatures are listed in A. wodanis was above 90% in all samples suggesting that the transcriptome data were sufficient for further analysis.The transcriptome profiles of n = 4,282) of A. wodanis were differentially expressed. Increasing the growth temperature to 12°C lowered this number of DEGs to 15%. Among the tested tpHCD/LCD conditions of wildtype, ΔlitR and ΔainS  was observed in the ΔlitR mutant tpHCD/LCD at 12°C. The ainS mutant tpHCD/LCD at 6°C and 12°C showed 15 and 18% DEGs, respectively. Further, the DEGs in tpHCD/LCD (WT_12°C) and tpHCD/LCD (WT_6°C) were sorted using KEGG BRITE and KEGG pathway mapper using A. wodanis 06/09/139 as a reference organism. The functional families with at least 15 DEGs were enzymes, transporters, secretion system, bacterial motility proteins and ribosome as shown in n = 5) with FC values ≥ 2.0 were also mapped into the functional families prokaryotic defense system, which includes the CRISPR related genes. The KEGG pathway mapping revealed that the DEGs (n ≥ 30) in the wild type tpHCD/LCD were involved in metabolic pathways, microbial metabolism in diverse environments, biosynthesis of secondary metabolites, ABC transporters, biosynthesis of antibiotics, two-component system, carbon metabolism and the ribosome. The numbers of DEGs involved in these pathways were similar in wild type tpHCD/LCD at both temperatures except in the ribosome pathway. At tpHCD/LCD (6°C), 42 DEGs mapped into the ribosome pathway, whereas only one DEG mapped at tpHCD/LCD (12°C)  .A. wodanis genes at HCD and LCD, respectively (ainS mutant (tp12°C/6°C), 10 and 14% of total genes were differentially expressed at HCD and LCD, respectively. Compared to the wild type and the ΔainS mutant, 31% of the total genes in the ΔlitR mutant were differentially expressed (tp12°C/6°C) at LCD, whereas at HCD, 4% of the total genes were differentially expressed. Further, the DEGs in wild type (tp12°C/6°C) with FC values ≥ 2.0 and ≤−2.0 were sorted into KEGG BRITE and KEGG pathway mapper. The functional families with DEGs of ≥ 15 were enzymes, transporters, ribosome, ribosome biogenesis and non-coding RNAs (n < 10) in wild type (tp12°C/6°C) mapped into families such as secretion system and prokaryotic defense system. The KEGG pathway mapping revealed that the DEGs (n ≥ 30) in the WT(tp12°C/6°C) were involved in pathways such as metabolic pathways, microbial metabolism in diverse environments, biosynthesis of secondary metabolites, ABC transporters, biosynthesis of antibiotics, two-component system, carbon metabolism, purine metabolism, Aminoacyl-tRNA biosynthesis and ribosome. The numbers of DEGs involved in these pathways were higher at tp12°C/6°C (LCD) than at tp12°C/6°C (HCD). For example, 43 DEGs at LCD mapped to the ribosome pathway while only 6 DEGs mapped at HCD (Comparison of the transcriptome profiles of the wild type grown at 12°C and 6°C (tp12°C/6°C) revealed DEGs, accounting for 13 and 17% of the total 4282 ectively . In the ing RNAs . Few DEGd at HCD .litR mutant to the wild type tpΔlitR/WT at HCD, 3 and 4% of the total genes were differentially expressed at 12 and 6°C, respectively. In the tpΔlitR/WT (HCD_6°C), 4% of the total genes were differentially expressed. In tpΔlitR/WT (LCD_12°C), 5% of total genes whereas in tpΔlitR/WT (LCD_ 6°C), 11% of the total genes were found to be differentially expressed. The number of DEGs was two times higher at 6°C and LCD compared to 12°C and HCD (ainS mutant with the wild type (tpΔainS/WT) at HCD, 0.7 and 0.4% of the total genes were differentially expressed at 12 and 6°C, respectively. In tpΔainS/WT, during LCD and 12°C, 0.9% of the total genes and at LCD and 6°C, 0.4% of the total genes were found to be differentially expressed. The DEGs in ΔlitR and ΔainS mutants were sorted using KEGG BRITE (A. wodanis 06/09/139 (n ≥ 5) in the ΔlitR and ΔainS mutants compared to wild type with FC values of ≥ 2.0 and ≤−2.0 were associated with functional families such as enzymes, secretion system, bacterial motility proteins, transporters and prokaryotic defense system. The KEGG pathway mapping revealed that the DEGs in the tpΔlitR/WT and tpΔainS/WT mutants affected several pathways, including metabolic pathways, two-component system, biosynthesis of secondary metabolites, Quorum sensing and bacterial chemotaxis  and master QS regulator gene litR (AWOD_I_0419) showed FC values of 2.01 and 2.24, at 6°C, respectively, while no significant differences were observed at 12°C.Only a few genes known to be involved in the s tested . When coqrr sRNA gene (AWOD_I_sRNA_054) was significantly higher with FC value of 2.92 at LCD, whereas no difference in its expression was observed at HCD. Temperature alone did not significantly impact other genes of the QS system at either HCD or LCD (When the wild type profile at high temperature was compared at low temperature (tp12°C/6°C), the D or LCD .litR mutant relative to the wild type (tpΔlitR/WT), only the FC value of ainS at LCD and 12°C was significant , although slightly lower than the cutoff FC value used in this study. The deletion of litR did not affect other QS related genes in A. wodanis under the conditions tested. On the other hand, when comparing the ΔainS mutant profile to the wildtype (tpΔainS/WT), the litR expression was significantly lower at all conditions examined. The FC values of litR were notably lower at HCD compared to LCD. At HCD, the FC values for litR at 6°C and 12°C were −2.53 and −2.87, respectively. At LCD, the FC values at 6°C and 12°C were −1.93 and −2.04, respectively, indicating that the expression of litR in the ΔainS mutant increases with increased density and independent of the temperature. Furthermore, the phosphorelay protein-encoding gene luxU (AWOD_I_0921) expression was significantly higher in the ΔainS mutant at HCD (FC = 2.81) and LCD (FC = 2.17) at 6°C, whereas at 12°C no differences were observed. Hence, the luxU expression in the ΔainS mutant was slightly higher at HCD compared to LCD and its expression was affected only at low temperature.Comparing the profile of the ΔA. wodanis using CRISPRCas finder  at HCD than at LCD after growth at 6°C. No significant differences in FC values of the cas genes were observed in wild type (tpHCD/LCD) at 12°C , suggesting temperature difference influences CRISPR system 1. However, temperature had no effect on the CRISPR system 2.cas genes of CRISPR system 2 were strongly reduced in the ΔlitR mutant at all conditions examined. The FC values of the cas genes in the ΔlitR mutant increased with increasing cell density at both temperatures. Notably, in the ΔlitR mutant at 6°C, the FC values of csy3 and csy1 were twice as high at HCD (FC = − approx. 10.0) compared to LCD (FC = − approx. 5.0). At LCD, the genes csy1, csy2, csy3, and csy4 had lower FC values at 6°C compared to 12°C, whereas the cas1 and cas3 showed higher FC values  and LCD and 6°C . However, at HCD, the expression of the CRISPR array of system 2 was not significantly different. The data also showed that the CRISPR array of system 1 was highly expressed at HCD and 12°C, whereas the FC values were not significantly different at HCD and 6°C or at LCD at 6°C and 12°C. In the ΔainS mutant compared to wild type, genes such as csy2, csy3, and csy4 showed significantly lower expression at two experimental conditions, LCD, 12°C and HCD, 6°C. No significant differences were observed in expressions of the CRISPR array of system 1 and 2 in the ΔainS mutant.Further, the expression data showed that the expression of C values . HoweverA. wodanis was found to harbor three T6SSs, each encoding the conserved core and accessory T6SS genes. T6SS1 was located on chromosome 1, whereas the T6SS2 and T6SS3 were on chromosome 2. The T6SS1 gene clusters (AWOD_I_0981-0995) were composed of 3 operons with 15 consecutive genes, whereas the T6SS2 gene clusters consisted of 1 operon with 15 consecutive genes (AWOD_II_0111-0125) and T6SS3 gene clusters with 2 operons with 20 consecutive genes (AWOD_II_1008-1027) as shown in vasABDEFGHJKLQRS, valine-glycine repeat protein G (vgrG) and Hemolysin Coregulated protein (hcp) and the accessory genes vasCIUVX , Aux-2 (AWOD_II_0126-0141), Aux-3 (AWOD_II_1028-1032) and Aux-4 (AWOD_II_1054-1060). The auxiliary clusters were located together with the core T6SS genes encoding Proline-alanine-alanine-arginine repeats (PAAR), VgrG and Hcp. The Aux-1 cluster does not encode Hcp, and Aux-4 does not encode a PAAR protein. Four genes encoding adaptor proteins of the DUF4123 family (unknown function) were identified adjacent to vgrG1, vgrG2 and vgrG4, whereas no adaptor protein-encoding gene was present adjacent to vgrG3.vasCIUVX , 92, 93.ectively –96. The haperone , 98. ThevasR1Q1J1) and T6SS2 (vasH1I1J1K1L1) were highly expressed at both 6 and 12°C, compared to LCD. No differential expression was observed for the T6SS3  of T6SS2 were differentially expressed at HCD, while at LCD, only vasQ2 was differentially expressed. As for the auxiliary clusters, only the genes encoding membrane protein and DUF4123 (AWOD_I_1435 and AWOD_I_1439) of Aux-1, hcp1 of Aux-2 and a putative uncharacterized protein (AWOD_II_1031) of Aux-3 were differentially expressed.The temperature did not affect the expression of the T6SS1 and T6SS3 in the wild type. However, a few genes (litR mutant was compared to wild type (tpΔlitR/WT), the data revealed that the vasR1Q1J1 operon of T6SS1 was differentially expressed at all tested conditions. The FC values of genes vasR1Q1J1 were marginally higher at HCD compared to LCD. Particularly the FC values of vasQ1 gene in ΔlitR mutant were twice as high at HCD compared to LCD at both temperatures. The FC values of vasR1Q1J1 operon in the ΔlitR mutant were significantly higher at 6°C compared to 12°C at both cell densities  compared to high (12°C) temperature. The T6SS3 gene cluster was only differentially expressed at LCD and 6°C with an average FC value of 2.0. In ΔlitR mutant, some genes of Aux-1, Aux-2 and Aux-3 were differentially expressed, whereas the Aux-4 showed no differential expression  protein in the Aux-2 were differentially expressed, where the FC values (FC = −22.75) of hcp1 at HCD and 6°C was approximately thrice compared to LCD (FC = −8.32). Similarly, three times higher expression of hcp1 was observed at 6°C compared to 12°C at both HCD and LCD. About 6 of the 16 total genes in the Aux-2 cluster showed significantly lower expression at LCD and 6°C. The Aux-3 was highly expressed in the ΔlitR mutant, relative to wild type, at HCD and 12°C and at LCD and 6°C. When comparing the ΔainS mutant with the wild type (tpΔainS/WT), only the expression of vasR1 and vasQ1 genes of the T6SS1 cluster were significantly reduced, whereas no differences in T6SS2 and T6SS3 gene clusters were observed. The vgrG1 of Aux-1 showed lower expression  at LCD and 6°C while the hcp1 of Aux-2 showed significant lower expression (FC < −2.0) at all conditions except at LCD and 12°C. Additionally, the genes AWOD_II_1031 and hcp3 of Aux-3 showed significant higher expression at HCD and 12°C. Other genes of auxiliary clusters in ΔainS mutant did not show significant differential expression.When the Δensities . The genpression . In Aux-We predicted 80 potential effectors proteins using the T6SE prediction tool Bastion 6  91). Of. Of91). In wild type, 24 effector genes were differentially expressed at HCD (tpHCD/LCD) at 6°C, while 19 effectors were differentially expressed at 12°C . These elitR mutant, genes encoding effector molecules with significantly lower expression (FC < −2.0) were putative exported proteins , outer membrane protein (AWOD_I_1120), putative lipoprotein , putative polysaccharide deacetylase (AWOD_I_1338), membrane protein (AWOD_I_1567), amine oxidase (AWOD_II_0852) and phospholipase (AWOD_II_1212). The effector molecules genes differentially expressed in ΔainS mutants included a putative histidine decarboxylase (AWOD_I_1509), and a secreted endonuclease (AWOD_II_0252). Additionally, genes encoding effectors such as a porin-like protein H (AWOD_I_1000), an endonuclease I precursor (AWOD_I_2248) and a 6-phospho-beta-glucosidase (AWOD_I_0029) were differentially expressed in both ΔlitR and ΔainS mutants. The differential expression of effector molecules in ΔlitR mutant was seen more often during LCD and 6°C than at other experimental conditions like HCD and 12°C  and temperatures (tp12°C/6°C), the strongest effect in terms of number of DEGs were at LCD (164 more genes than at HCD) and at 6°C (140 more genes than at 12°C). Furthermore, the functional mapping of DEGs revealed that families such as secretion system, prokaryotic defense system and transporters were highly expressed at HCD compared to LCD. As expected, the gene families related to protein synthesis such as ribosomes, ribosome biogenesis and transfer RNA biogenesis were less expressed at HCD compared to LCD (tpHCD/LCD). Similar cell density-dependent gene expression has been reported in Aliivibrio salmonicida, where about 1,000 genes were differentially expressed in response to increase in cell density  and tpΔlitR/WT (6°C) compared to at HCD and 12°C, suggesting LitR is a global regulator when the temperature is low and the cells are in the early log phase. This is in contrast to A. salmonicida, where the numbers of DEGs in ΔlitR mutant were higher at HCD compared to LCD at 12°C (litR/WT and tpΔainS/WT) showed about 5 to 24 times more DEGs in tpΔlitR/WT compared to tpΔainS/WT at both cell densities and temperatures. This indicates that only a few genes seem to be regulated through the AinS-dependent QS system. This is similar to A. salmonicida, where only about 20 genes were differentially expressed in ΔainS mutant when compared to wild type, whereas in litR mutant compared to wild type, 3 to 10 times more DEGs were found , indicating that the ainS expression is not entirely LitR-dependent. These results confirm our previous work, where we showed that the 3OHC10-HSL production is cell density- and temperature-dependent  in the ΔlitR mutant. This confirms our previous work where we showed that the 3OHC10-HSL production is not completely controlled through LitR  in wild type seems to be cell density-dependent at 6°C, where the cas genes showed increased expression at HCD compared to LCD. However, cell density had no significant effect on the expression of CRISPR arrays of CRISPR systems 1 and 2 regardless of temperatures. Therefore, the CRISPR systems are partially dependent on cell density. Previous studies suggest that bacteria are at higher phage risk at higher cell density densities, and thus protection against phage is important at HCD  to the T6SS system 1 in A. fischeri MJ11, while the T6SS2 shows high similarity (54–92%) to V. cholerae O1E1 T6SS and V. fluvialis T6SS2 (A. wodanis shows similarity (33–84%) to M. viscosa, V. anguillarum, A. salmonicida and Vibrio tapetis . The genes encoding Hcp (AWOD_II_1028 and AWOD_II_1032), which are located close to each other, shows 71% similarity to M. viscosa hcp, MVIS_3030  and negatively affect the expression of T6SS3 gene cluster. This demonstrates that QS regulation of the T6SSs in A. wodanis is very complex. The regulation of T6SS3 by LitR in A. wodanis indicates T6SS3 may play different roles than T6SS1 and T6SS2. Such reciprocal regulation has also been shown in V. parahaemolyticus, where the QS regulator OpaR downregulates T6SS1 and upregulates T6SS2 where T6SS1 functions as anti-bacterial and T6SS2 as anti-eukaryotic , V. alginolyticus (LuxR) and V. anguillarum (VanT) .LitR in m (VanT) , 51, 134ell line . Temperaolyticus , 135. SilitR/WT at 6°C. Ankyrin repeat proteins are known to be involved in pathogenesis by imitating and impeding host function  can be found in the article/AM, EH, HH, and NW conceived and designed the experiments. AM performed the experiments. AM, EH, and NW analyzed the transcriptomics data. AM and NW wrote the manuscript. All authors contributed to reviewing and proof reading the final manuscript.This work was funded by the Research council of Norway (#270068: ELIXIR2) and UiT The Arctic University of Tromsø. The publication charges for this article have been funded by UiT The Arctic University of Tromsø.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To understand the individual functional roles of the Nad1, Nad2, Nad4, and Nad5 subunits in bamboo, each cDNA of these four genes was cloned into the pYES2 vector and expressed in the mitochondria of the yeast Saccharomyces cerevisiae. The mitochondrial targeting peptide mt gene (encoding MT) and the egfp marker gene  were fused at the 5′-terminal and 3′-terminal ends, respectively. The constructed plasmids were then transformed into yeast. RNA transcripts and fusion protein expression were observed in the yeast transformants. Mitochondrial localizations of the MT-Nad1-EGFP, MT-Nad2-EGFP, MT-Nad4-EGFP, and MT-Nad5-EGFP fusion proteins were confirmed by fluorescence microscopy. The ectopically expressed bamboo subunits Nad1, Nad2, Nad4, and Nad5 may function in ion translocation, which was confirmed by growth phenotype assays with the addition of different concentrations of K+, Na+, or H+.Mitochondrial respiratory complex I catalyzes electron transfer from NADH to ubiquinone and pumps protons from the matrix into the intermembrane space. In particular, the complex I subunits Nad1, Nad2, Nad4, and Nad5, which are encoded by the  Mitochondrial complex I EC 1.6.5.3), also called NADH:ubiquinone (UQ) oxidoreductase, is the largest and most complicated enzyme complex in the respiratory electron transfer chain. Mitochondrial complex I catalyzes the transfer of two electrons from NADH to UQ, coupling the translocation of four protons across the inner membrane, thus contributing to the electrochemical potential .6.5.3, a. All of Bambusa oldhamii were encoded by distinct sets of exons . Th. Th48]. + translocation [E. coli were used to adjust the orientation of the Nad4 transmembrane domains [+ translocation [The topology model of Nad1 A suggestlocation . No char domains . Moreovelocation . In Figulocation . Two α-hmt gene) of the ATPase δ subunit from S. cerevisiae [nad1, nad2, nad4, and nad5 genes. This MT peptide should allow Nad1, Nad2, Nad4, and Nad5 to pass through the mitochondrial outer membrane and to be folded into the inner membrane [nad1, nad2, nad4, and nad5 genes were inserted correctly into pYES2-mt-egfp . The constructed plasmids were further confirmed by DNA sequencing and were found to be accurate after comparison with the cDNA sequences of the nad1, nad2, nad4, or nad5 genes (data not shown).The bamboo Nad1, Nad2, Nad4, and Nad5 subunits of mitochondrial complex I are encoded by mtDNA, synthesized by mitochondrial ribosomes, and do not possess MT peptides. In order to direct the bamboo Nad1, Nad2, Nad4, and Nad5 subunits to yeast mitochondria, an MT peptide . The total RNA of yeast transformants was then reverse-transcribed to cDNA. The yeast transformant cDNAs were used as templates for PCR with several pairs of primers: mt-egfp-F and mt-egfp-R/I-nad5-R . The exped in MG A, the 1. in MN1G A, the 1. in MN4G A, the 1. in MN2G B, and th in MN5G B. This sThe total membrane proteins were isolated from the yeast wild-type (WT) and transformants MN1G, MN2G, MN4G, and MN5G. The membrane proteins were analyzed with 12% SDS-PAGE electrophoresis . The MT-In order to investigate whether the fusion proteins were targeted to subcellular mitochondria, the EGFP fluorescence was detected in the yeast transformants MN1G, MN2G, MN4G, or MN5G using a fluorescence microscope. The yeast WT was used as a reference because it did not contain EGFP and did not emit green fluorescence. The yeast transformant containing MT-EGFP (MG) was used as a comparison for EGFP green fluorescence. The cells of the yeast WT and transformants were induced with SC-U medium plus 2% galactose for 0–9 h at 30 °C and then a time course of EGFP fluorescence development was observed with a fluorescence microscope. The images were taken at (A) 0 h, (B) 3 h, (C) 6 h, and (D) 9 h . Bright Since there were some reports showing that Nad1, Nad2, Nad4, or Nad5 subunits may not involve in direct proton translocation ,54,55,56+, Na+, or H+. The yeast transformants MG, NM1G, NM2G, NM4G, and NM5G were grown on SC-U medium (+-containing solutions (0.8 M KCl and 1 M KCl), Na+-containing solutions (0.8 M NaCl and 1 M NaCl), or H+-containing solutions , the identities of nad1, nad4, and nad5 cDNAs from B. oldhamii and P. edulis were still higher than 96.2%. In addition, the nad2 cDNA sequence from B. oldhamii showed 71.9% nucleic acid identity to homologous sequences from other monocotyledons and Arabidopsis.The cDNAs of nad2 and nad5 are divided into five exons, while in the B. oldhamii mitochondrial gene map, nad2 is divided into five exons and nad5 is divided into four exons [After comparison with wheat, rice, and maize mitochondrial genome maps, it was found that mitochondrial genes were highly conserved but there was little shared synteny between these species ,19,57. Fur exons ,20,58. TThe mitochondrial total RNA, including mRNA, was reverse-transcribed into cDNA using random hexamers as primers, which gave better sequence preservation than oligo dTs because plant mitochondrial mRNA would be degraded by a 3′- to 5′-exoribonuclease if the mitochondrial mRNA had been polyadenylated. Additionally, non-AUG start codons have been found to initiate the translation of proteins in mitochondria and chloroplasts ,60.nad1, nad2, nad4, and nad5. These editing sites were found to be non-random and clustered in groups [The alignment of cDNA and mtDNA sequences showed that there may be some C-to-U RNA editing sites in n groups . It was n groups .The cDNA and amino acid sequences of the bamboo Nad1 and Nad4 subunits were compared with the human ND1 (Nad1) and ND4 (Nad4) subunits . The idenad1, nad4, and nad5 cDNA that there were a number of nucleotide differences, such as TTC→TTT, AAA→AAG, ACA→ACT, and TCC→TCA, that seemingly led to no change in amino acid residues during translation, apart from one difference—CAA→AAA  have been located on the internal surface of the membrane in the bacterium P. denitrificans, and these were reported to be involved in binding and reducing UQ [B. oldhamii and P. edulis might be involved in the binding and reduction in UQ. Lim et al. [B. oldhamii and P. edulis with other monocotyledons, there was one difference, with L347 in the transmembrane segment X of Nad4 in bamboo rather than the P347 present in other monocotyledons (in this study) [B. oldhamii and P. edulis.The Nad1, Nad4, and Nad5 amino acid sequences of ucing UQ . It has ucing UQ . The seqm et al.  have stam et al. . It was s study) . In the s study) . A compas study) ,69,70. TS. cerevisiae. A proposed approach is allotopic expression, in which the mtDNA-encoded gene is transferred to the nucleus and the protein is synthesized in the cytosol and subsequently imported into the mitochondria [S. cerevisiae mitochondria and colocalized with the MitoTracker Red mitochondrial marker probe . The importance of Nad1, Nad2, Nad4, and Nad5 in the assembly of mitochondrial complex I has been demonstrated through mutant studies. In mutants of complex I in the green alga Chlamydomonas, the absence of intact Nad1 affected the complete assembly of complex I [Arabidopsis otp43 mutant results in a loss of complex I activity and a decrease in the respiration rate [Chlamydomonas complex I mutants [+/e− stoichiometry, indicating its direct involvement in proton translocation in complex I [The Nad1, Nad2, Nad4, and Nad5 subunits are the core membrane subunits of the mitochondrial complex I . These fomplex I ,77. Furtomplex I  and it hion rate . Nad2 ac mutants . Regardiomplex I . Other somplex I  and thatomplex I .Overall, this study was successful in expressing the bamboo Nad1, Nad2, Nad4, and Nad5 subunits in yeast mitochondria and it has provided a possible pathway for further investigation of these membrane proteins.Bambusaoldhamii and Phyllostachys edulis were obtained from the local market. The leaf sheaths covering the shoots were removed, and only the top 10 cm of the shoot was used. Escherichia coli strain TOP10F’  was used in all recombinant DNA experiments and grown in Luria-Bertanie (LB) broth or on LB agar plates at 37 °C. Saccharomyces cerevisiae INVSc1  was cultured in yeast extract–peptone–dextrose (YPD) medium at 30 °C.Fresh edible bamboo rhizome shoots of N,N,N′,N′-tetraacetic acid (EGTA), 0.1% (w/v) BSA, and approximately 10 mg mitochondrial protein mL−1. The concentration of the crude mitochondrial protein suspension was determined by the Biuret Assay using BSA as a standard [Young bamboo shoots were freshly obtained from a local farm. Mitochondria were isolated according to the method of Chien et al.  and all standard . Isolateg  to remove the supernatant. The pellet was resuspended gently with a mixture containing 250 μL of phenol and 250 μL of RNA extraction solution comprising 200 mM LiCl, 200 mM Tris-HCl (pH 8.0), 20 mM EDTA, and 0.2% (w/v) SDS. The RNA extract was incubated at 80 °C for 5 min. Then, 100 μL of chloroform and isopropanol (24:1) was added to the RNA extract and inversed gently. The mixture was centrifuged at 4 °C for 5 min at 12,000× g. The clear supernatant was transferred to a new Eppendorf tube, topped up with an equal volume of 4 M LiCl, and then incubated at −80 °C for at least 2 h. Then, the mixture was centrifuged at 4 °C for 20 min at 12,000× g and the white pellet settled at the bottom of the Eppendorf. After the supernatant was removed, the pellet was resuspended in 100 μL of sterilized ddH2O. Then, 10 μL of 3 M NaOAC (pH 5.2) and 250 μL of 100% ethanol were added to the suspension. The mixture was incubated at −70 °C for 2 h and then centrifuged at 4 °C for 15 min at 12,000× g. Subsequently, the RNA pellet was washed with 500 μL of 70% ethanol, centrifuged at 4 °C for 5 min at 12,000× g, and dried inside a laminar flow cabinet for 20 min. The RNA pellet was resuspended in 30 μL of sterilized ddH2O and stored at −70 °C.Total mtRNA was isolated by the phenol-chloroform method. About 10 mg of crude mitochondria were centrifuged at 4 °C for 15 min at 15,000× −1 (5 μM) random hexamer and ddH2O to a final volume of 12 μL. The RNA and dT mixtures were incubated at 70 °C for 50 min and then chilled on ice for 1 min. The mixtures were gently mixed with 7 μL of reaction buffer containing 1× FS of PCR buffer (Invitrogen), 2.5 mM MgCl2, and 0.5 mM dNTP. Then, the RNA and dT and reaction buffer mixtures were incubated at 42 °C for 5 min. They were further incubated at 42 °C for 50 min after adding 200 U superscript II reverse transcriptase . The reaction was terminated at 70 °C for 15 min and the obtained cDNA was stored at −20 °C until later use.The RNA and dT mixtures comprised 2 or 5 μg of RNA template combined with 0.5 μL of 1 μg μLnad1, nad2, nad4, and nad5 cDNA genes from B. oldhamii and P. edulis, several pairs of forward and reverse primers  in accordance with monocot mitochondrial DNA (mtDNA) sequences from O. sativa , T. aestivum , and Z. mays . The primers were then used to amplify cDNA fragments by RT-PCR. The RT-PCR products were purified with a Gel Elution Kit  and sent to the Biotechnology Center, National Chung Hsing University (Taiwan) for sequencing. The results were aligned and assembled using BioEdit software  vector as TA-nad1, TA-nad2, TA-nad4, and TA-nad5. The pYES2  vector was used to express the bamboo genes. The sequence coding the mt from the S. cerevisiae ATPase δ subunit (5′ aagcttATGTTACGTTCAATTATTGGAAAGAGTGCATCAAGATCATTGAATTTCGTCGCTAAGCGTTCATATCACCACCATCACCATCACCATCACaagctt 3′) was synthesized . The mt and egfp genes were subsequently ligated into pYES2 (nad1 insert amplified with a designed primer set (HindIII and EcoRI sites by adding 5× DNA ligase reaction buffer and 1 U of T4 DNA ligase (Invitrogen) to yield pYES2-nad1-egfp. pYES2-mt-nad1-egfp was then obtained after the mt gene was subsequently ligated into the plasmid . A 5 μL quantity of the nad4 PCR product and 600 ng μL−1 of linearized pYES2-mt-egfp were added to 14 μL of reaction mixture containing 2 μL of 10× CloneEZ buffer and 1 U μL−1 of CloneEZ enzyme. After gently mixing by pipetting, the reaction mixtures were incubated at 22 °C for 30 min and incubated on ice for 5 min. pYES2-mt-nad4-egfp was then obtained (B. oldhamii nad2 insert was prepared from TA-nad2 clones using BamHI restriction digestion and ligated into multiple cloning sites of the pYES2-mt-egfp plasmid between mt and egfp by adding T4 DNA ligase to yield pYES2-mt-nad2-egfp (B. oldhamii nad5 insert was prepared from the TA-nad5 clone using EcoRI and XmaI restriction digestion and ligated into multiple cloning sites of the pYES2-mt-egfp plasmid between mt and egfp by adding T4 DNA ligase to yield the pYES2-mt-nad5-egfp (The 1.0 kb  primers . The res primers . The PCRto pYES2  followinimer set  was liga plasmid . The nadobtained . The B. ad2-egfp . Similarad5-egfp . All theS. cerevisiae INVSc1 (Invitrogen) was added to 3 mL of YPD medium  and 1 mM EDTA. Then, the cell suspensions were centrifuged for 5 min at 3000× g and the pellets were resuspended with 1–2 mL of 1× LiAc/0.5× TE buffer containing 100 mM LiAc, 5 mM Tris-HCl (pH 7.5), and 0.5 mM EDTA. A 90 μL aliquot of yeast-competent cell suspensions was added to an Eppendorf tube containing 10 μL of glycerol and was quickly stored at −70 °C.Yeast transformation and yeast-competent cell preparation were carried out according to the methods of Gietz et al.  with somD medium  and cultg and the pellet was resuspended in 500 μL of 1× TE buffer. The cell suspension was centrifuged for 10 sec at 15,000× g, and the resulting pellets were resuspended in 100 μL of 1× Tris-EDTA(TE) buffer and then plated onto selective plates.For yeast transformation, the yeast-competent cells were incubated at room temperature for 10 min. A 1 μg aliquot of pYES2-mt-egfp, pYES2-mt-nad1-egfp, pYES2-mt-nad2-egfp, pYES2-mt-nad4-egfp, or pYES2-mt-nad5-egfp plasmids was added to 100 μL of yeast-competent cells and 600 μL of LiAc/PEG/TE buffer containing 100 mM LiAc, 40% PEG-6000, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA, and incubated at 30 °C for 30 min. Then, 70 μL of DMSO was added, mixed, and incubated at 42 °C for 7 min. The mixture was centrifuged for 10 s at 15,000× g and this was followed by washing three times with 1 mL ddH2O. The cells were then resuspended in induction medium  and 10 mM EDTA. The cell suspension was transferred into a 1.5 mL microcentrifuge tube, 40 µL of 10% SDS was added, and then the mixture vortexed for 2 min. An equal volume of phenol was subsequently added to the tube, followed by vortexing and incubation at 65 °C for 4 min. The reaction mixture was rapidly chilled to −80 °C until phenol crystals appeared and was then centrifuged at 12,000× g for 2 min at 4 °C. A 400 μL aliquot of the aqueous phase solution was transferred to a new tube and an equal volume of phenol, chloroform, and isopropanol (25:24:1) was added, vortexed, and centrifuged at 12,000× g for 5 min at 4 °C. Again, a 200 μL volume of the aqueous phase solution was transferred to a new tube and then 20 μL of 3 M NaOAc (pH 5.3) and 50 μL of 95% ethanol were added and precipitated for 2 h at −20 °C. Then, the sample was centrifuged at 12,000× g for 20 min at 4 °C to discard the supernatant. The pellet was washed with 500 μL of 95% ethanol by centrifugation at 12,000× g for 5 min at 4 °C. The precipitated RNA was dried in a laminar flow cabinet for 5–10 min, resuspended with 50 μL of 1× RNA safeguard reagent , and treated with 0.1 U μL−1 DNase I Amp Grade (Invitrogen) per μg of total RNA to remove DNA. The quality of total RNA was checked on a 1% agarose gel using the ratio of OD260 to OD280.Yeast total RNA was prepared according to the method of Schmitt et al.  with modg for 5 min to pellet the unbroken cells and cell debris, and the supernatant was considered to be the total protein fraction. The total protein fractions were centrifuged at 18,000× g for 1 h into sediment total membrane, and the supernatant was considered to be the soluble protein fraction. The pellet consisting of sedimented crude membranes was resuspended in 1 volume of solubilization buffer containing 50 mM Tris-HCl (pH 7.5), 400 mM NaCl, 1% β-mercaptoethanol, 20% glycerol, and 1 mM PMSF, in which the 400 mM NaCl was permitted to strip peripheral membrane proteins. The crude membranes were then harvested by centrifugation at 18,000× g for 1 h. The final pellets containing the crude total membranes were resuspended in solubilization buffer and stored at −20 °C.Yeast cell pellets (0.25–0.5 g per wet weight) were resuspended in 1 mL ice-cold homogenization buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% β-mercaptoethanol, 10% glycerol, and 1 mM phenylmethanesulfonylfluoride (PMSF). Glass beads (1 mm size) were added, and the cell suspension was then vortexed three times for 5 min with 3 min breaks between cycles (4 °C). The glass beads were then sedimented by placing the homogenized suspension on ice. The upper layer suspension was transferred to a fresh pre-cooled Eppendorf tube and centrifuged at 1600× −1) were added as 20 μL aliquots to 1 mL of Quick Start Bradford protein assay dye  and incubated at room temperature for 5 min. Absorbances of each standard and sample were read at 595 nm in a spectrophotometer . The absorbance of the standards versus their concentration was plotted. The concentration of the unknown protein was calculated according to the extinction coefficients.The protein concentration measurement was carried out according to the method of Bradford . BSA sta6 cells mL−1. MitoTracker Red FM was added to the cell suspension to a final concentration of 100 nM and incubated at room temperature for 15–30 min. The fluorescence was observed under a fluorescence microscope . Bright-field microscopy was used to monitor the integrity of the cells and the same cells were photographed in fluorescence with a GFP filter set  and then with the Mito Tracker red filter set .The fluorescence of EGFP was detected to examine whether the MT-EGFP, MT-Nad1-EGFP, MT-Nad2-EGFP, MT-Nad4-EGFP, and MT-Nad5-EGFP proteins were successfully expressed in yeast mitochondria. MitoTracker Red FM  was used as a mitochondrial marker stain. The yeast cells were harvested and resuspended with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4) and 5% glucose at a concentration of 10v/v) methanol in a Hofer TE22 transfer tank  at 4 °C for 90 min. After transfer, the membranes were washed with phosphate-buffered solution (PBS) containing 80 mM Na2HPO4 (pH 7.5), 20 mM NaH2PO4·H2O, and 100 mM NaCl for 5 min. The PVDF membranes were subsequently blocked in PBS buffer containing 5% non-fat milk at 4 °C overnight. The following day, the PVDF membranes were probed with anti-GFP  primary antibodies in PBS containing 1% (v/v) non-fat milk at 4 °C overnight. These steps were followed by three cycles of 15 min washes with PBS plus 0.05% (v/v) Tween 20. Then, the PVDF membranes were probed with anti-rabbit IgG horseradish peroxidase (HRP) conjugate (1:2000 dilution) secondary antibody in PBS containing 1% (v/v) non-fat milk for 1 h at room temperature. After a further three cycles of 15 min washes with PBS plus 0.05% (v/v) Tween 20, the PVDF membranes were detected using DAB solution and a 2.5 mg diaminobenzidine  tablet dissolved in 100 mL of ddH2O with 28 μL of 30% (v/v) H2O2 was added. The PVDF membranes were stained for 5 to 10 min. The reaction was stopped as soon as the specifically stained bands were clearly visible, and the PVDF membrane was washed three times with ddH2O. Finally, the PVDF membrane was scanned with a ScanMarker 9800 XL scanner .The total mitochondrial proteins were first separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) following the method of Laemmli . Then, tg for 5 min and washed with distilled water. The cells were resuspended in pre-warmed dithiothreitol (DTT) buffer (2 mL per gram of wet weight cells) containing 0.1 M Tris-H2SO4 (pH 9.4) and 10 mM DTT, and shaken at 80 rpm at 30 °C for 20 min. The cells were harvested by centrifugation at 3000× g for 5 min and resuspended in Lyticase buffer containing 0.5 mg Lyticase , 1.2 M sorbitol, and 20 mM KH2PO4 (pH 7.4). Then, the cells were harvested by centrifugation at 3000× g for 5 min, resuspended in Lyticase buffer (7 mL per gram of wet weight cells), and shaken at 80 rpm at 30 °C for 3–4 h. The spheroplasts of lytic cells were harvested by centrifuging at 3000× g for 5 min, washed with Lyticase buffer (7 mL per gram of wet weight cells), and resuspended in ice-cold homogenization buffer (6.5 mL per gram of wet weight cells) containing 600 mM sorbitol, 1 mM EGTA, 20 mM 2-(N-morpholino)ethanesulfonic acid (MES)-KOH (pH 6.0), 0.1% BSA, and 0.5 mM PMSF. The cell suspensions from this step were maintained at low temperature to avoid proteolysis. Then, the cell suspensions were transferred into a 100 mL homogenizer to homogenize the spheroplasts with 15 strokes. The homogenate was diluted twofold with homogenization buffer and centrifuged at 1500× g for 5 min at 4 °C into pellet cell debris and nuclei. The supernatant was centrifuged at 4000× g for 5 min at 4 °C to discard the pellet. The collected supernatant was centrifuged at 12,000× g for 15 min. Finally, the crude mitochondrial pellet was resuspended in a solubilization buffer containing 0.6 M sorbitol, 1 mM EGTA, and 20 mM HEPES-KOH (pH 7.4). The mitochondrial protein concentration was determined using the method of Bradford [−1, was stored at −80 °C.The yeast mitochondria were isolated according to the method of Meisinger et al.  with somBradford . The samS. cerevisiae transformants were grown overnight to saturation in liquid SC-U medium plus 2% glucose, washed with ddH2O, and resuspended in SC-U medium plus 2% galactose to induce the expression of MT-Nad1-EGFP, MT-Nad2-EGFP, MT-Nad4-EGFP, or MT-Nad5-EGFP fusion proteins. Tenfold serial dilutions of cell suspension (10−1 to 10−5) were prepared and spotted in 3 μL aliquots onto SC-U medium plus 2% galactose agar plates. In the salt-dependent experiment, 100 mM LiCl, 600 mM NaCl, or 800 mM KCl were added [re added . In the


PDGFRα, PDGFRβ, FGFR1 or JAK2 represent the biomarkers of WHO-defined “myeloid and lymphoid neoplasms with eosinophilia”. The heterogeneous nature of genomic aberrations and the promiscuity of fusion partners, may limit the diagnostic accuracy of current cytogenetics approaches. To address such technical challenges, we exploited a nanopore-based sequencing assay to screen patients with primary Eosinophilia. The comprehensive sequencing approach described here enables the identification of genomic fusion in 60 h, starting from DNA purified from whole blood.Eosinophilia represents a group of diseases with heterogeneous pathobiology and clinical phenotypes. Among the alterations found in primary Eosinophilia, gene fusions involving The online version contains supplementary material available at 10.1186/s40364-021-00337-1. To the Editor,PDGFRα, PDGFRβ, FGFR1 or PCM1-JAK2 (MLN-Eo) is defined by an absolute, persistent, eosinophil count (AEC) ≥1500/uL [PDGFRα, PDGFRβ, FGFR1 or the PCM1-JAK2 fusion are employed for FISH analysis to identify the most recurrent translocations. However, owing to the promiscuous nature of the fusion events [PDGFRα/β, FGFR1 and JAK2 in unamplified DNA samples [the 2016-WHO category of myeloid and lymphoid neoplasms with eosinophilia and abnormalities of n events . On the n events . In this9/L . All subjects were negative for JAK2V617F, MPLW515 or CALRexon9 mutation.To the purposes of the study, we sequenced 12 samples from patients with Eosinophilia  whose familiar or secondary origin were excluded and who had stored samples collected at presentation . Full set of clinical and cytogenetic data were available for all the patients  g PDGFRα  revealed2q12 g PDPDGFRβ and 6,108 bp  to ETV6; more specifically, the clustering of chimeric reads predicted the fusion breakpoint between intron 10 of PDGFRβ  and intron 4 of ETV6 . The translocation found in sample #5 was originated by the fusion between PDGFRβ intron 9  and CCDC88C intron 24 . The chimeric read spanned 32,847 bp, where 22,736 bp were aligned to PDGFRβ and 9,111 bp to CCDC88C.Sequencing data were further analysed by Sniffles. In samples #4 and #5, chimeric reads with multiple alignment pointing were detected at a t and a t(q32q32), respectively. The chimeric reads in sample #4 spanned from 9,394 bp to 52,545 bp, of which at least 810 bp  were aligned to FGFR1 sequence  fused to ZMYM2 ; the fusion breakpoint was located at nucleotides 6,754 and 102,102 of FGFR1 and ZMYM2, respectively. In sample #7, the two chimeric reads  were aligned to FGFR1  and to ZMYM2 . The fusion breakpoint was detected between nucleotides 21,209 of FGFR1 and 89,374 of ZMYM2.In samples #6 and #7 we found, respectively, 3 and 2 chimeric reads predicting for a t. The chimeric reads in sample #6  were composed by the PCM1-JAK2 fusion was detected in any samples of the cohort.No 2 coefficient:1) independently performed on the same samples of eosinophils collected at diagnosis. We show here that long-reads analysis facilitated the identification of the exact breakpoints of gene fusion in the 7 mutated patients, an information not provided by conventional cytogenetic approaches. The described pipeline allows to complete simultaneous genomic search for rearrangements of PDGFRα/β, FGFR1 and JAK2 in 60 h from blood sample collection, at an affordable cost, currently estimated at 500 Euros per sample. Finally, the NS long-reads sequencing of DNA enables the identification of possible unknown fusion partners by the alignment of the chimeric sequences to a reference genome.The NS screening results were in full agreement with FISH analysis (Pearson’s RAdditional file 1.