A homologous recombination-based strategy was used to disrupt C. glabraraORFs witha cassette containing the nat1gene, which codes for nourseothricin acetyltransferase and imparts resistance to nourseothricin. Briefly, 5’-and 3’-UTR region (nearly 500-700 bp) of the gene to be deleted were amplified by PCR using wild-type genomic DNA as template. Both 5’-and 3’-UTR amplified products were fused to one half each ofthenat1gene amplified from theplasmid(pRK625). The two nat1-amplified fragments share about 300-350 bp complimentary region. To obtain fusion products, primers were designed insucha way thatthereverse primer for 5’-UTR and the forward primer for 3’-UTRof the gene of interestshare 20 bp complimentary region with the forwardprimer for 5’nat1fragment andthereverse primer for 3’nat1fragment amplification, respectively. The fused PCR products were co-transformed in to the wild-type strain and transformants were plated on YPD-agar plates. Plates were incubated at 30°C for 16 h to allow the homologous recombination between nat1fragments,and 5’-and 3’-UTR atthegenomic loci. Post incubation,cells were replica plated ontoYPD-agar plate supplemented with 200 μg/ml nourseothricin and incubated for another 24 h. Nourseothricin-resistant colonies were purified and verified for gene disruption viahomologous recombination by PCR using appropriate set of primers

Generation of C. glabratadeletion strains

For Yeast colony PCR, yeast cells were subjected to zymolyase (MP Biomedicals, 0832092) digestion to obtain thespheroplast. To perform zymolyase digestion, a digestion cocktail was prepared in 1XPBS consisting of zymolyase (2.5mg/ml) and sorbitol (1.2 M). The cocktail was dispensed in 0.2ml PCR tubes in 10 μl aliquots anda tip-full of yeast cells wasadded to these tubes. Tubes were incubated at 37°C for 2-3 h and 1 μl of digested mixture was used as a template in a PCR reaction

Yeast colony PCR

To perform restriction digestion of plasmid DNA and PCR-amplified DNA products, restriction enzymes were procured from NEW ENGLAND Biolabs(NEB). Restriction digestion was set in 50 μl reaction volume with appropriate buffer and 1X BSA. For ligation of DNA fragments obtained after restriction digestion, T4 DNA Ligase enzyme (NEB, M0202M) was used. All ligation reactions were set in 20 μl reaction volume containing 1X ligase buffer, 3-10 units of DNA ligase enzyme and vector to insert molar ratio of 1:3. The ligation mixture waseitherincubated at 16°C for 16h or at room temperature for 2-3 h. Post incubation, ligation reaction was inhibited by heatingtubes at 65°C for 15-20 min.2-5 μl of ligation mixture was used to transform ultra-competent E. coliDH5αcells

Restriction digestion and ligation

To extract DNA from agarose gels,the QIAquick® gel extraction kit (QIAGEN, 28706) was used. For purification of PCR amplified DNA products,the QIAquick® PCR purification kit (QIAGEN, 28106) was used. Clean-up of enzymatic reactions was performedusing the MinElute® Reaction Cleanup kit (QIAGEN, 28204). Allprotocolswere followed as per manufacturer’s instruction

Gel extraction, PCR purification and Reaction clean-up

Bacterial plasmid DNA was isolatedusing the QIAprep® spin Miniprep kit (QIAGEN, 27106). 10 ml of LB medium supplemented with appropriate antibioticswas inoculated withasingle bacterial colony and incubated at 37°C for 12-16 h. Cultures were spun down at 8,000 rpm for 5 min, supernatant was discarded and the cell pellet was processed to isolate plasmid DNA as per instructions given in the kit. DNA was eluted either in nuclease-free water or in elution buffer provided in the kit and stored at -20°C until use

Plasmid isolation

were chosen for qPCR. For all qPCR reactions,0.4 μl of cDNA template was used in a 20 μl reaction volume. Reactionswere performed anddata wereanalysed in ABI7500 real-time qPCR machine. Amplified products were run on 2% agarose gel to confirm amplification ofthecorrect size product. CTvalues of respective products were normalized with corresponding CTvalue of the housekeeping gene CgACT1. Relative change in expression was determined by comparative CTmethod,also referred as 2-∆∆CTmethod, utilizing following equation.Fold change upon treatment=2-∆∆CT∆∆CT=∆CT Treated-∆CT Untreated∆CTTreated= CTvalue for gene of interest upon treatment-CTvalue of internal control (CgACT1)upon treatment∆CTUntreated= CTvalue for gene of interest without treatment-CTvalue of internal control (CgACT1)without treatmentThe reaction cycling conditions were as follows1)95°C for 10 min (initial activation)2)95°C for 15 sec (denaturation)3)55°C for 30 sec (annealing)4)72°C for40 sec (extension)5)Go to step 2 (40 cycles)6)72°C for 10 min (final extension)

MESA GREEN qPCR mastermix (RT-SY2X-03+WOULR) supplied by Eurogentech was used in all qPCR experiments. Primers for real-time qPCR experiments were designed by using the Primer3 plus software to obtain 120-200 bp amplification products. Standardization of optimaltemplate and primer concentrationconditionswas done in a PCR reaction and concentrations resulting in good amplification withoutprimer dimers

Quantitative Real-time PCR(qPCR)

Reverse transcriptase “Superscript III” (Invitrogen, 18080-051) was used to perform cDNA synthesis. Briefly, 500 ng of DNase I-digested RNA was incubated with 1 μl of 10 mM dNTP and 50 μM oligo(dT) at 65°C for 5 min in a 10 μl reaction mixture followed by cooling on ice for 5 min. Post incubation, 10 μl of cDNA synthesis mixture was added which contained 2 μl of 10XRT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, 1 μl of RNase out (40 units) and 1μl of Superscript III (200 units). Tubes were incubated at 50°C for 1 h and thereaction was terminated at 85°C for 5 min. The quality of synthesized cDNA was checked by using it as a template in a PCR reaction to amplify the housekeeping gene CgACT1. Amplification of CgACT1was indicative of proper cDNA synthesis

Complementary DNA (cDNA) synthesis

Deoxyribonuclease I (DNase I) enzyme (Invitrogen) was used to remove the DNA contamination from RNA samples,if any. Briefly,1 μg of RNA was subjected to DNase I digestion by using 1 U of DNase I in a 10 μl reaction mixture which contained 1X DNase I buffer and appropriate volume of water. The reaction mixture was incubated at room temperature for 15 min. Post incubation,to inhibit DNase I enzyme activity,1 μl of 25 mM EDTA was added to the reaction mixture and tubes were heated at 65°C for 10 min. DNase I-digested RNA samples were used as template to perform PCR for the amplification of CgACT1geneandabsence of amplification product was used as criterion toconfirmproper DNase I digestion and lackof DNAcontaminationin the RNA sample

DNase I digestion

microcentrifuge tube. For precipitation of RNA, 1/10thvolume of 3 M sodium acetate (pH 5.3) and 2.5 volume of 100% ice-coldethanol was added. In order to facilitate precipitation, tubes werekept at -20°C for 20 min. Tubes were centrifuged at 13,000 rpm for 10 min in a refrigerated centrifuge. The RNA pellet was washed with 70% ethanol,resuspendedin 100-200 μl of nuclease-free water and stored at -20°C untiluse.Care was taken to keep allreagents and tubes on ice to maintain the cold temperature throughout theRNA extractionprocess

All reagents required for RNA extraction were preparedin DEPC-treated water. RNasecontamination from non-autoclavable items wasremoved by wiping them with RNaseZap® (Ambion). Total RNA from yeast cells was extractedusing acid phenolextractionmethod. Briefly, yeast cells were grown underappropriate conditions and at suitabletime points,cells were harvested by centrifugation at 4,000 rpm for 5 min. The cell pellet was washed twice with ice-cold DEPC-treated water, resuspended in 350 μl of AE buffer and transferred toa1.5 ml microcentrifuge tube. To this,40 μl of 10% SDS and 400 μl of acid phenol (pH 4.3) was added. The cell suspension was mixed well by vortexing thrice, short pulsesof10 seconds each,and incubated at 65°C for 15 min with continuous agitation at 800 rpm. Post incubation, cells were kepton ice for 5 min and centrifuged at 13,000 rpm in a refrigerated centrifuge set at 4°C for 10 min. After centrifugation, aqueous layer was transferred to a new1.5 ml microcentrifuge tube and 400 μl of chloroform was added. Tubes were mixed well by gentlyinverting them 4-5 times and centrifuged at 13,000 rpm for 10 min. The aqueous layer was separated and transferred to a new1.5 ml

Total RNA isolation

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Spheroplast lysis method

Yeast genomic DNA was isolated by mechanically lysing the yeast cells. Briefly, 10 ml of overnight grown yeast culture was transferred toa 15 ml centrifuge tube andcells were spun down at 4,000 rpm for 5 min. Media was decanted and cells were washed with 10 ml sterile water. Washedcells were resuspended in 500 μl of Buffer A and transferred to a 1.5 ml microcentrifuge tube. Tubes were incubated at 65°C for 15 min. Post incubation,500 μl of PCI (25:24:1) solution was added. To this, 0.5 g of 0.5 mm glass beads were added and cells were lysed mechanically in a bead-beatinghomogenizer (MP Biomedicals,FastPrep®-24) thrice, 45 seceach, with intermittent cooling on ice. Tubes were spun at 12,000 rpm for 5 min and the aqueous layer was transferred to a new 1.5 ml microcentrifuge tube. To this, 500 μl of PCI solution was addedand mixed gently by inverting the tubes.Tubes were centrifuged again at 12,000 rpm for 5 min and aqueous layer was transferred to another 1.5 ml microcentrifuge tube. Next, 2.5 volume of absolute ethanol was added to the aqueous layer, mixed well and centrifuged at 13,000 rpm for 10 min. Supernatant was decanted and the DNA pellet was washed once with 70% ethanol and centrifuged at 13,000 rpm for 10min. Washed DNA pellet was air-dried and dissolved in 100-200 μl of 1X TE buffer by gently tapping the tubes

Glass bead lysis method

Total protein extraction and immunoblotting

For complementation studies, C. glabrataORFs, CgFTR1(1.22 kb), CgFET3(1.91 kb), CgYFH1(0.53 kb), CgCCW14(0.64 kb), CgMAM3(1.91 kb)andCgHOG1(1.34 kb) were PCR amplified from wild-type genomic DNA using Phusion high-fidelity DNA polymerase and cloned down-stream of the PGK1promoter intothe XmaIand XhoI, XbaIand XmaI, XmaIand XhoI, BamHIand SalI, XmaIand XhoI, and BamHIand XmaIsites, respectively, in the CEN-ARS containing plasmid pGRB2.2 (pRK74). For over-expression studies, C. glabrataORFs, CgCCC1(0.95 kb), CgYAP5(1.05 kb)andCgMRS4(0.92 kb) were PCR amplified from wild-type genomic DNA using Phusion high-fidelity DNA polymerase and cloned down-stream of the constitutive promoter PDC1into the BamHI and SalI, XbaIand XmaI, and XmaI and XhoIsites, respectively, in the CEN-ARS containing plasmid obtained from Addgene (Addgene-ID 45323). All clones were verified by PCR and sequencing analysis

Cloning of C. glabrataORFs

PCR-positive transformants were inoculated in 10 ml YPD medium, allowedto grow for 12 handgenomic DNAwas isolated.Another round of PCR was performed using genomic DNA asatemplate toconfirm the gene deletion

A homologous recombination-based strategy was used to disrupt C. glabraraORFs witha cassette containing the nat1gene, which codes for nourseothricin acetyltransferase and imparts resistance to nourseothricin. Briefly, 5’-and 3’-UTR region (nearly 500-700 bp) of the gene to be deleted were amplified by PCR using wild-type genomic DNA as template. Both 5’-and 3’-UTR amplified products were fused to one half each ofthenat1gene amplified from theplasmid(pRK625). The two nat1-amplified fragments share about 300-350 bp complimentary region. To obtain fusion products, primers were designed insucha way thatthereverse primer for 5’-UTR and the forward primer for 3’-UTRof the gene of interestshare 20 bp complimentary region with the forwardprimer for 5’nat1fragment andthereverse primer for 3’nat1fragment amplification, respectively. The fused PCR products were co-transformed in to the wild-type strain and transformants were plated on YPD-agar plates. Plates were incubated at 30°C for 16 h to allow the homologous recombination between nat1fragments,and 5’-and 3’-UTR atthegenomic loci. Post incubation,cells were replica plated ontoYPD-agar plate supplemented with 200 μg/ml nourseothricin and incubated for another 24 h. Nourseothricin-resistant colonies were purified and verified for gene disruption viahomologous recombination by PCR using appropriate set of primers

Generation of C. glabratadeletion strains

For Yeast colony PCR, yeast cells were subjected to zymolyase (MP Biomedicals, 0832092) digestion to obtain thespheroplast. To perform zymolyase digestion, a digestion cocktail was prepared in 1XPBS consisting of zymolyase (2.5mg/ml) and sorbitol (1.2 M). The cocktail was dispensed in 0.2ml PCR tubes in 10 μl aliquots anda tip-full of yeast cells wasadded to these tubes. Tubes were incubated at 37°C for 2-3 h and 1 μl of digested mixture was used as a template in a PCR reaction

Yeast colony PCR

To perform restriction digestion of plasmid DNA and PCR-amplified DNA products, restriction enzymes were procured from NEW ENGLAND Biolabs(NEB). Restriction digestion was set in 50 μl reaction volume with appropriate buffer and 1X BSA. For ligation of DNA fragments obtained after restriction digestion, T4 DNA Ligase enzyme (NEB, M0202M) was used. All ligation reactions were set in 20 μl reaction volume containing 1X ligase buffer, 3-10 units of DNA ligase enzyme and vector to insert molar ratio of 1:3. The ligation mixture waseitherincubated at 16°C for 16h or at room temperature for 2-3 h. Post incubation, ligation reaction was inhibited by heatingtubes at 65°C for 15-20 min.2-5 μl of ligation mixture was used to transform ultra-competent E. coliDH5αcells

To extract DNA from agarose gels,the QIAquick® gel extraction kit (QIAGEN, 28706) was used. For purification of PCR amplified DNA products,the QIAquick® PCR purification kit (QIAGEN, 28106) was used. Clean-up of enzymatic reactions was performedusing the MinElute® Reaction Cleanup kit (QIAGEN, 28204). Allprotocolswere followed as per manufacturer’s instructions

Restriction digestion and ligation

Gel extraction, PCR purification and Reaction clean-up

Bacterial plasmid DNA was isolatedusing the QIAprep® spin Miniprep kit (QIAGEN, 27106). 10 ml of LB medium supplemented with appropriate antibioticswas inoculated withasingle bacterial colony and incubated at 37°C for 12-16 h. Cultures were spun down at 8,000 rpm for 5 min, supernatant was discarded and the cell pellet was processed to isolate plasmid DNA as per instructions given in the kit. DNA was eluted either in nuclease-free water or in elution buffer provided in the kit and stored at -20°C until use

Plasmid isolation

were chosen for qPCR. For all qPCR reactions,0.4 μl of cDNA template was used in a 20 μl reaction volume. Reactionswere performed anddata wereanalysed in ABI7500 real-time qPCR machine. Amplified products were run on 2% agarose gel to confirm amplification ofthecorrect size product. CTvalues of respective products were normalized with corresponding CTvalue of the housekeeping gene CgACT1. Relative change in expression was determined by comparative CTmethod,also referred as 2-∆∆CTmethod, utilizing following equation.Fold change upon treatment=2-∆∆CT∆∆CT=∆CT Treated-∆CT Untreated∆CTTreated= CTvalue for gene of interest upon treatment-CTvalue of internal control (CgACT1)upon treatment∆CTUntreated= CTvalue for gene of interest without treatment-CTvalue of internal control (CgACT1)without treatmentThe reaction cycling conditions were as follows1)95°C for 10 min (initial activation)2)95°C for 15 sec (denaturation)3)55°C for 30 sec (annealing)4)72°C for40 sec (extension)5)Go to step 2 (40 cycles)6)72°C for 10 min (final extension)

MESA GREEN qPCR mastermix (RT-SY2X-03+WOULR) supplied by Eurogentech was used in all qPCR experiments. Primers for real-time qPCR experiments were designed by using the Primer3 plus software to obtain 120-200 bp amplification products. Standardization of optimaltemplate and primer concentrationconditionswas done in a PCR reaction and concentrations resulting in good amplification withoutprimer dimers

Quantitative Real-time PCR(qPCR)

Reverse transcriptase “Superscript III” (Invitrogen, 18080-051) was used to perform cDNA synthesis. Briefly, 500 ng of DNase I-digested RNA was incubated with 1 μl of 10 mM dNTP and 50 μM oligo(dT) at 65°C for 5 min in a 10 μl reaction mixture followed by cooling on ice for 5 min. Post incubation, 10 μl of cDNA synthesis mixture was added which contained 2 μl of 10XRT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, 1 μl of RNase out (40 units) and 1μl of Superscript III (200 units). Tubes were incubated at 50°C for 1 h and thereaction was terminated at 85°C for 5 min. The quality of synthesized cDNA was checked by using it as a template in a PCR reaction to amplify the housekeeping gene CgACT1. Amplification of CgACT1was indicative of proper cDNA synthesis

Complementary DNA (cDNA) synthesis

Deoxyribonuclease I (DNase I) enzyme (Invitrogen) was used to remove the DNA contamination from RNA samples,if any. Briefly,1 μg of RNA was subjected to DNase I digestion by using 1 U of DNase I in a 10 μl reaction mixture which contained 1X DNase I buffer and appropriate volume of water. The reaction mixture was incubated at room temperature for 15 min. Post incubation,to inhibit DNase I enzyme activity,1 μl of 25 mM EDTA was added to the reaction mixture and tubes were heated at 65°C for 10 min. DNase I-digested RNA samples were used as template to perform PCR for the amplification of CgACT1geneandabsence of amplification product was used as criterion toconfirmproper DNase I digestion and lackof DNAcontaminationin the RNA sample

DNase I digestion

microcentrifuge tube. For precipitation of RNA, 1/10thvolume of 3 M sodium acetate (pH 5.3) and 2.5 volume of 100% ice-coldethanol was added. In order to facilitate precipitation, tubes werekept at -20°C for 20 min. Tubes were centrifuged at 13,000 rpm for 10 min in a refrigerated centrifuge. The RNA pellet was washed with 70% ethanol,resuspendedin 100-200 μl of nuclease-free water and stored at -20°C untiluse.Care was taken to keep allreagents and tubes on ice to maintain the cold temperature throughout theRNA extractionprocess

All reagents required for RNA extraction were preparedin DEPC-treated water. RNasecontamination from non-autoclavable items wasremoved by wiping them with RNaseZap® (Ambion). Total RNA from yeast cells was extractedusing acid phenolextractionmethod. Briefly, yeast cells were grown underappropriate conditions and at suitabletime points,cells were harvested by centrifugation at 4,000 rpm for 5 min. The cell pellet was washed twice with ice-cold DEPC-treated water, resuspended in 350 μl of AE buffer and transferred toa1.5 ml microcentrifuge tube. To this,40 μl of 10% SDS and 400 μl of acid phenol (pH 4.3) was added. The cell suspension was mixed well by vortexing thrice, short pulsesof10 seconds each,and incubated at 65°C for 15 min with continuous agitation at 800 rpm. Post incubation, cells were kepton ice for 5 min and centrifuged at 13,000 rpm in a refrigerated centrifuge set at 4°C for 10 min. After centrifugation, aqueous layer was transferred to a new1.5 ml microcentrifuge tube and 400 μl of chloroform was added. Tubes were mixed well by gentlyinverting them 4-5 times and centrifuged at 13,000 rpm for 10 min. The aqueous layer was separated and transferred to a new1.5 ml

Total RNA isolation

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Spheroplast lysis method

Yeast genomic DNA was isolated by mechanically lysing the yeast cells. Briefly, 10 ml of overnight grown yeast culture was transferred toa 15 ml centrifuge tube andcells were spun down at 4,000 rpm for 5 min. Media was decanted and cells were washed with 10 ml sterile water. Washedcells were resuspended in 500 μl of Buffer A and transferred to a 1.5 ml microcentrifuge tube. Tubes were incubated at 65°C for 15 min. Post incubation,500 μl of PCI (25:24:1) solution was added. To this, 0.5 g of 0.5 mm glass beads were added and cells were lysed mechanically in a bead-beatinghomogenizer (MP Biomedicals,FastPrep®-24) thrice, 45 seceach, with intermittent cooling on ice. Tubes were spun at 12,000 rpm for 5 min and the aqueous layer was transferred to a new 1.5 ml microcentrifuge tube. To this, 500 μl of PCI solution was addedand mixed gently by inverting the tubes.Tubes were centrifuged again at 12,000 rpm for 5 min and aqueous layer was transferred to another 1.5 ml microcentrifuge tube. Next, 2.5 volume of absolute ethanol was added to the aqueous layer, mixed well and centrifuged at 13,000 rpm for 10 min. Supernatant was decanted and the DNA pellet was washed once with 70% ethanol and centrifuged at 13,000 rpm for 10min. Washed DNA pellet was air-dried and dissolved in 100-200 μl of 1X TE buffer by gently tapping the tubes

Glass bead lysis method

Yeast genomic DNA isolation

Molecular biology methods

To phenotypically characterize C. glabratamutants,serial dilution spot growth assays were performed. Briefly, the optical density of overnight-grown C. glabratacultures wasnormalized to OD600of 1.0andnormalized cultures were further diluted 10-fold in 1X sterile PBS five times. 3 μl of serially diluted culture were spotted on test plates. Plates were incubated at 30°C (unless mentioned otherwise) for 24-48hand growth was recorded by capturing plate images. For experiments involvingchecking theability of mutants to utilize non-fermentable carbon sources,growth was scoredafter 6-7 days of incubation

Serial dilution spot growth assay

phorbol myristateacetate (PMA) (Tsuchiya et al., 1982). For PMA treatment, THP-1 cells were allowed to grow till 70-80% confluence and were collectedin a centrifuge tube by centrifugationat 1,000 rpm for 3 min. THP-1 cell pelletswere resuspended in 4-5 ml of pre-warmed complete RPMI-1640 medium, 100 μl of this cell suspension was appropriately diluted in PBS (1X) and viability was determined by counting trypan blue stained cellsusing hemocytometer. THP-1 cell suspension was diluted appropriately to obtainafinal cell density of 106cells/ml with pre-warmed complete RPMI-1640 medium. PMA was added totheTHP-1 cell suspension at a final concentration of 16 nM and mixed well by gently inverting the tubes. PMA-treated cells were seeded either in 24-well cell culture plates or in cell culture dishes and allowed to grow for 12 h under tissue culture conditions i.e. at 37°C and 5% CO2.After 12 h incubation, spent medium was replaced withfresh pre-warmed complete RPMI-1640 medium and cells were allowed to recover for another 12 h

THP-1 monocytes getdifferentiated intophagocytic macrophages upon treatment with

PMA (Phorbol myristateacetate) treatment of THP-1 monocytic cells

Cells were grown in 35 mm dishes at 20% initial confluence. At 40-50% confluence, cells were treated with different genotoxic agents as described in Section 2.2.2. Post treatment, cells were washed twice with PBS and replaced with fresh media to allow recovery for different lengths of time. At each time point, cells were harvested and fixed with 70% ethanol at -20°C overnight. Fixation was carried out by adding 70% ethanol drop by drop, while the cells were being vortexed at a low speed.The fixed samples were brought to room temperature, pelleted down at 2000 rpm for 3 min and washed twice with PBS. Cells were stained with PI solution containing 0.1% Triton X-100, 0.2 mg RNase and 20μg propidium iodide and incubated at 37ºC for 30 min in the dark. Samples were analyzed by flow cytometry (FACS ARIA, BD). Data was analyzed usingFACS DIVA (BD) and FlowJo (FLOWJO, LLC) softwares to identify different stages of the cell cycle

Cell cycle analysis by PI staining