Loss of a Cardiolipin Synthase in Helicobacter pylori G27 Blocks Flagellum Assembly

Bacterial strains and growth conditions.E. coli DH5α was used for cloning and plasmid construction. E. coli strains were grown in Luria-Bertani broth or agar medium. Medium was supplemented with ampicillin (100 μg/ml), chloramphenicol (30 μg/ml) or kanamycin (30 μg/ml) when appropriate. For routine growth of H. pylori strains, the cultures were grown microaerobically under an atmosphere consisting of 10% CO₂, 4% O₂, and 86% N₂ at 37°C on tryptic soy agar (TSA) supplemented with 5% horse serum or in tryptic soy broth (TSB) supplemented with 5% heat-inactivated horse serum with shaking under an atmosphere consisting of 5% CO₂, 10% H₂, 10% O₂, and 75% N₂. Growth of H. pylori strains for glycerophospholipid extraction was done at 37°C in Mueller-Hinton broth (MHB) with 10% horse serum and 20 mM 2-(4-morpholino)-ethanesulfonic acid (MES; pH 6.0) under an atmosphere consisting of 10% CO₂, 5% O₂ and 85% N₂. Kanamycin (30 μg/ml) or chloramphenicol (30 μg/ml) was added to the medium used to culture H. pylori when appropriate.

Strain construction.All primers used for PCR in the construction of H. pylori mutants are listed in Table S1 in the supplemental material. Genomic DNA from H. pylori B128 was purified using the Wizard genomic DNA purification kit (Promega) and used as the PCR template to construct the clsC knockout. DNA was amplified from H. pylori B128 gDNA using Phusion polymerase (New England Biolabs), and the resulting amplicons were incubated with Taq polymerase (Promega) at 72°C for 10 min to add 3′-A overhangs for T/A cloning with pGEM-T Easy plasmid (Promega). To disrupt clsC in H. pylori G27 and B128, a 634-bp upstream DNA fragment was amplified that included 493 bp of frdB (the gene immediately upstream of clsC; it encodes succinate dehydrogenase subunit B), an intergenic region of 21 bp, and 99 bp of the 5′ end of clsC that includes a 21-bp sequence complementary to a chloramphenicol resistance (cat) cassette using the primers M54 and M55. A 604- bp downstream DNA fragment that included 508 bp downstream of clsC and 75 bp of the 3′ end of clsC, together with a 21-bp sequence complementary to the cat cassette, was amplified using the primers M56 and M57. A cat cassette was PCR amplified from pUC20 cat using the primers cat5 and cat6, generating a 742-bp fragment (55). PCR-based gene splicing by overlap extension (PCR SOEing) was used to join the upstream and downstream regions to the cat cassette, and the resulting amplicon was cloned into pGEM-T Easy to generate the suicide plasmid pJC030, which was introduced into H. pylori strains G27 and B128 by natural transformation. Chloramphenicol-resistant transformants were selected on TSA supplemented with horse serum and chloramphenicol. Replacement of clsC with the cat cassette in the H. pylori G27 and B128 chromosome was confirmed by PCR using primers that flanked the clsC locus. The resulting amplicons were sequenced (Eton Bioscience) to verify that clsC had been replaced with the cat cassette.

Complementation of the clsC mutation.Plasmid pJC032 is a derivative of the shuttle vector pHel3 (56) that carries H. pylori G27 clsC under the control of its native promoter located upstream of frdA. To construct pJC032, the frdA promoter and clsC coding regions were amplified using the primer pairs P069/P070 and P071/P072, respectively, and H. pylori G27 gDNA as the template. The resulting amplicons were joined by PCR SOEing. Primers that contained 25 bp of homologous sequence with pHel3 were used to amplify the fusion product, and the resulting amplicon was cloned into pHel3 cut with SphI using the sequence- and ligation-independent cloning (SLIC) method (57) to generate pJC032. For complementation assays, pJC032 was introduced into the H. pylori clsC mutants by natural transformation, which were then examined for CL, motility, and the presence of flagella, as described below.

Motility assay.Motility was assessed using a soft-agar medium comprised of MHB, 10% heat-inactivated horse serum, 20 mM MES (pH 6.0), 5 μM FeSO₄, and 0.4% Noble agar. Plates were stab inoculated with cells from TSA plates grown for 5 days using wooden sticks and incubated at 37°C under an atmospheric condition consisting of 10% CO₂, 4% O₂, and 86% N₂. Diameters of swim halos were measured after 7 days of incubation. The two-sample t test was used to determine statistical significance.

Transmission electron microscopy.H. pylori strains were grown to late log phase in TSB supplemented with 10% heat-inactivated horse serum to an optical density at 600 nm (OD₆₀₀) of ∼1.0. One milliliter of culture was collected by centrifugation (550 × g) and then resuspended in 125 μl of 0.1 M phosphate-buffered saline (PBS). Cells were fixed by adding 50 μl of 16% EM-grade formaldehyde and 25 μl of 8% EM-grade glutaraldehyde to the cell resuspension. After incubation at room temperature for 5 min, 10 μl of the cell suspension was added to 300-mesh, Formvar-coated copper grids, followed by incubation at room temperature for 5 min. The cell suspension was wicked off the grids using a filter paper, and the grids were washed three times with 10 μl of water. Cells were stained by applying 10 μl of 1% uranyl acetate to the grids for 30 s. After the stain was removed with filter paper, the grids were washed three times with 10 μl of water and then air dried. Cells were visualized using a JEOL JEM 1011 transmission electron microscope. Flagellum counts were determined for at least 100 cells for each strain. The Mann-Whitney U test was used to determine whether there were statistically significant differences in number of flagella per cell for the various H. pylori strains.

RNA extraction and cDNA synthesis.H. pylori cultures were grown in TSB supplemented with 5% horse serum for 24 h, diluted in fresh medium to a cell density of 5 Klett units, and then allowed to grow to a cell density of 90 Klett units. RNA was harvested from the H. pylori cultures using the Zymo Direct-zol RNA MiniPrep Plus kit and quantified using a NanoDrop Lite instrument (Thermo Scientific). RNA quality was assessed using a 1% bleach gel (58). RNA preparations were treated with Turbo DNA-free kit (Ambion) according to the manufacturer’s protocol to eliminate contaminating gDNA, and the absence of gDNA was confirmed by PCR. RNA samples were analyzed using a Bioanalyzer 2100 instrument (Agilent), and each sample had a minimum RNA integrity number of 9.5. Single-strand cDNA was synthesized from 200 ng of RNA using an iScript cDNA synthesis kit (Bio-Rad).

RNA-seq assay.Ribosomal RNAs were depleted from RNA preparations and libraries were prepared using 5 μg of total RNA. Illumina sequencing was performed using a NextSeq500 instrument at the University of Georgia Genomics Facility. Quality of the reads were assessed using FastQC and trimmed using Trimmomatic, and the resulting reads were assembled to the H. pylori G27 genome (accession no. NC_011333.1) using Bowtie2 and visualized using Geneious (59, 60). The DESeq2 package in RStudio was used to analyze raw fragment counts and differentially regulated genes were identified with a log₂ fold cutoff of 1.5 (61).

RT-qPCR.The transcript levels of flhA, fliF, flgE, flaB, and flaA were monitored by RT-qPCR as previously described (25, 62). The primers used for this experiment are listed in Table S1 in the supplemental material. RT-qPCR was performed on serially diluted cDNA samples, and concentrations for all samples were determined using a standard curve of serially diluted gDNA. Each RT-qPCR mixture totaled 20 μl, consisting of 10 μl of Luna universal qPCR mix (New England BioLabs), 5 μl of 100-fold diluted cDNA, and 5 μl of the primers (final concentrations of 250 nM final for each primer). Evaluation of single products in all reactions was performed after each experiment by a melt curve analysis. The Applied Biosystems StepOnePlus real-time PCR system was used to perform all experiments in technical triplicates for three biological replicates of each condition. The 2^–ΔΔCq method (63) was used to quantify gene expression levels, and statistical significance was determined using a two-sample t test.

Allelic exchange mutagenesis.gDNA was isolated from the H. pylori B128 clsC mutant using a Wizard genomic DNA purification kit (Promega) and used to transform the H. pylori G27 clsC mutant as follows. The recipient strain was patched onto fresh TSA plates and incubated for 4 days. The cells were collected, transferred to a fresh plate, and then transformed with 3 μg of gDNA. After 24 h of incubation, the cells were collected and resuspended in 1 ml of TSB and spot plated onto the soft agar medium used for motility assays. After 7 days of incubation, cells from the edge of the swim halo were used to inoculate fresh soft agar medium, and the inoculated plates were incubated for 7 days. Cells from the edge of the swim halo were streaked onto TSA supplemented with 5% horse serum and chloramphenicol, and the resulting isolated colonies were screened for motility in the soft agar medium.

DNA sequencing and analysis.Following the instructions supplied with the Illumina iTruSeq adaptor kit, genomic libraries were prepared using 500 ng of gDNA from various H. pylori strains. The resulting genomic libraries were sequenced at the University of Georgia Genomics Facility by Illumina sequencing. The quality of the reads was assessed using FastQC, and the reads were trimmed using Trimmomatic (59, 60). The reads for H. pylori G27 gDNA sequences were mapped using Bowtie2 using the published NCBI genome for H. pylori G27 (accession no. NC_011333.1) and visualized using Geneious. The alignment generated was used to align the gDNA sequence of the H. pylori G27 clsC mutant, which served as the backbone for aligning the gDNA sequences of the suppressor mutant strains. SNPs were identified in the genome sequences of the motile recipient strains using Geneious. Candidate genes were identified by using RStudio to cross-reference SNPs that were common to all the motile G27 clsC recipients.

Replacement of specific alleles in the H. pylori G27 clsC mutant.For the following PCR and cloning steps, target DNA was amplified using Phusion polymerase, and the resulting amplicons were incubated with Taq polymerase at 72°C for 10 min to add 3′-A overhangs to facilitate T/A cloning into plasmid pGEM-T Easy. Primer pair P079 and P080 were used to amplify 558 bp of the ureA promoter from H. pylori G27. Using the pKSF plasmid (64) as a template, a 2,978-bp DNA fragment was amplified that included the kanamycin cassette and a promoterless sacB gene. The two amplicons were joined by PCR SOEing, which generated a 3,536-bp product and was cloned into pGEM-T Easy that resulted in the plasmid pJC038. Regions upstream (568 bp) and downstream (393 bp) of H. pylori B128 icfA were amplified using the primer pairs P165/P166 and P167/P168, respectively. The resulting amplicons were joined by PCR SOEing and cloned into pGEM-T Easy to generate plasmid pJC068. The kan-sacB cassette was introduced into unique NheI and XhoI sites in plasmid pJC068 to generate a suicide vector (pJC069) that was used to replace the icfA and kdsA alleles in the H. pylori G27 clsC mutant with the corresponding alleles from H. pylori B128. The primers P165 and P189 were used to amplify 495 bp of DNA upstream of H. pylori G27 icfA. The primers P190 and P168 were used to amplify a region of H. pylori B128 gDNA corresponding to the entire coding region of icfA and the first 164 bp of the kdsA coding region. The resulting amplicons were joined using PCR SOEing and cloned into pGEM-T Easy to generate plasmid pJC075. Plasmid pJC075 was introduced by natural transformation into the H. pylori G27 clsC strain that carried the kan-sacB cassette in the icfA locus. Transformants in which the kan-sacB cassette was replaced with the icfA-kdsA alleles carried on pJC075 were isolated following a sucrose-based counterselection as described (64). The icfA-kdsA regions for several kanamycin-sensitive, sucrose-resistant isolates were amplified by PCR, and the resulting amplicons were sequenced to confirm that the H. pylori G27 clsC mutant contained the B128 ifcA-kdsA alleles.

The primer pairs P169/P170 and P187/P188 were used to amplify 616 and 503 bp, respectively, of DNA sequence upstream and downstream of H. pylori G27 flgI. The two amplicons were joined using PCR SOEing, and the resulting amplicon was cloned into pGEM-T Easy to generate plasmid pJC074. A kan-sacB cassette was inserted in unique NheI and XhoI sites in pJC074 to generate the suicide vector pJC077 that was used to replace flgI coding sequence in the H. pylori G27 clsC mutant with the kan-sacB cassette. The primer pair P169/P191 was used to amplify 552 bp of DNA upstream of H. pylori G27 flgI, the primer pair P192/P172 was used to amplify 552 bp of DNA downstream of H. pylori G27 flgI, and the primer pair P193/P194 was used to amplify H. pylori B128 flgI (1,029 bp of DNA sequence). The three amplicons were joined using PCR SOEing, and the resulting amplicon was cloned into pGEM-T Easy to generate plasmid pJC078. Plasmid pJC078 was transformed into the H. pylori G27 clsC strain where flgI had been replaced with the kan-sacB cassette to introduce the H. pylori B128 flgI allele into the strain using a sucrose-based counterselection.

Glycerophospholipid extraction.Cells were harvested, and pellets were washed once with 5 ml of PBS. Isolation of glycerophospholipids was carried out using a modified method of Bligh and Dyer, as described previously (65). Briefly, cell pellets were resuspended in 5 ml of single-phase Bligh-Dyer mixture consisting of chloroform-methanol-water (1:2:0.8, vol/vol/vol) and then incubated for 20 min at room temperature. Samples were centrifuged at 1,800 × g for 10 min. The resulting supernatants were transferred to clean glass tubes and converted into a two-phase Bligh-Dyer mixture consisting of chloroform-methanol-water (2:2:1.8, vol/vol/vol). This mixture was vortexed and centrifuged to separate the organic and aqueous phases. The lower organic phase was removed and placed in a clean glass tube. A second extraction was performed by adding 2.6 ml of chloroform back to the tube containing the upper phase. The mixture was vortexed and centrifuged. The resulting lower phase was pooled with the previous lower phase and converted again into a two-phase Bligh-Dyer mixture by adding 5.2 ml of methanol and 4.7 ml of water. The resulting mixture was vortexed and centrifuged. Finally, the lower phase containing glycerophospholipids was removed and placed into a new tube, which was subsequently dried under a stream of nitrogen.

Analysis of ³²P-labeled glycerophospholipids.Bacterial glycerophospholipids were uniformly radiolabeled with ³²P (20 μCi/ml) following a dilution to an OD₆₀₀ of ∼0.1 using an overnight liquid culture and then incubated for 24 h. Bacteria were transferred from growth bottles into Teflon screw-cap glass tubes (16 by 125 mm) and pelleted by centrifugation at 1,800 × g for 15 min. The cells were resuspended in PBS and then repelleted by centrifugation at 1,800 × g for 15 min. ³²P-labeled glycerophospholipids were extracted, spotted on a Silica Gel 60 TLC plate (20,000 cpm), and separated in a glass solvent chamber using a mobile phase consisting of chloroform, methanol, and acetic acid (65:25:5, vol/vol/vol). Upon migration of the solvent front to the top of the TLC plate (∼2 h), the plates were dried, covered in plastic wrap, and exposed overnight to a phosphor screen. Visualization of the ³²P-labeled glycerophospholipids was performed using an Amersham Typhoon imager.

Mass spectrometry.All solvents were high-pressure liquid chromatography grade. Glycerophospholipid samples from unlabeled cells were resuspended in 100 μl of chloroform-methanol (4:1, vol/vol). A solution of 9-aminoacridine matrix (Sigma) was prepared at a concentration of 15 mg/ml in chloroform-methanol (2:1, vol/vol). Sample solutions were mixed with matrix solution (1:1, vol/vol), and 1 μl of the mixture was loaded onto the target plate. A peptide standard mixture (s6104-10ug; ProteoChem, Inc.) was prepared according to manufacturer’s recommendations and used as an external calibrant. MALDI-TOF mass spectra of glycerophospholipid extracts were acquired in reflector mode on Autoflex speed mass spectrometer (Bruker Daltonics). A total of 500 single laser shots were averaged from each mass spectrum. Data were acquired in negative ion mode and processed using FlexControl 3.4 and FlexAnalysis 3.4 software (Bruker Daltonics).

Sample preparation for cryo-EM observation.H. pylori strains were cultured on plates for 4 days. The cells on the agar surface were collected and then washed with PBS buffer. Colloidal gold solution (10 nm in diameter) was added to the diluted H. pylori samples to yield a 10-fold dilution and then deposited on a freshly glow-discharged, holey carbon grid for 30 s. The grid was blotted with filter paper and rapidly plunge-frozen in liquid ethane in a homemade plunger apparatus, as described previously (66).

Cryo-ET data collection and image processing.The frozen-hydrated specimens of H. pylori G27 clsC mutant were transferred to Titan Krios electron microscope (FEI) equipped with a 300-kV field emission gun and a direct electron detector (Gatan K2 Summit). The images were collected at a defocus near to 0 μm using Volta Phase Plate and the energy filter with 20 eV slit. The data were acquired automatically with SerialEM software (67). During the data collection, the phase shift is monitored at the range of pi/3 to ∼pi2/3; when the phase shift is out of the above range, next spot of phase plate will be switched to be charged for the use. A total dose of 50 e⁻/Ų is distributed among 35 tilt images covering angles from −51° to +51° at tilt steps of 3°. The starting tilting angle is +36° instead of 0°. For every single tilt series collection, the dose-fractionated mode was used to generate 8 to 10 frames per projection image. Collected dose-fractionated data were first subjected to the motion correction program to generate drift-corrected stack files (68–70). The stack files were aligned using gold fiducial markers and volumes reconstructed by the weighted back-projection method using IMOD and Tomo3d software to generate tomograms (71, 72).

Subtomogram analysis with i3 packages.Bacterial flagellar motors were detected manually using the i3 program (73, 74). The orientation and geographic coordinates of selected particles were then estimated. In total, six subtomograms of H. pylori G27 clsC flagellar motors were used to perform subtomogram averaging analysis. The i3 tomographic package was used on the basis of the “alignment by classification” method with missing wedge compensation for generating the averaged structure of the motor, as described previously (66).

Data availability.The raw data used for analysis is available in NCBI Short Read Archive (SRA database) under accession numbers PRJNA545123 (DNA sequencing; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA545123) and PRJNA545152 (RNA-seq; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA545152).