Lactobacillus casei Zhang-G-1200 was isolated from a long-term laboratory-based evolution experiment performed in our laboratory (Wang et al., 2017). The strain Zhang-G-1200 exhibited a higher resistance to gentamycin compared with the parental cells (minimum inhibitory concentration, MIC, of 32 μg/mL for Zhang-G-1200 vs. 2 μg/mL for the parental strain). The acquisition of resistance was a result of multigenerational and stepwise increase during the prolonged cultivation under gentamycin stress. Growth curves of the 2 strains were constructed based on optical density (OD) measurement every 2 h along 30-h fermentation in LSM supplemented with 1 μg/mL gentamycin. Meanwhile, changes in pH and viable counts were determined. All analyses were performed in triplicate.

The bacterial strain, Escherichia (E. coli) DH5α, was used as a host for standard cloning procedures. It was propagated aerobically in Luria Bertani broth at 37°C. Chloramphenicol (10 μg/ml for both E. coli and L. casei Zhang-G-1200) and erythromycin (250 and 5 μg/ml for E. coli and L. casei Zhang-G-1200, respectively) were used for selecting genetically modified bacterial clones.

Both the parental and L. casei Zhang-G-1200 cells were collected after 24 h of cultivation in LSM supplemented with 1 μg/mL gentamycin. Four biological replicates were prepared for each of the L. casei Zhang and the Zhang-G-1200 strains. Cells were harvested by centrifugation, followed by washing with phosphate buffered saline for 4 times. One milliliter of lysis buffer was added to each sample, followed by sonication on ice and centrifugation at 13,000 rpm for 10 min at 4°C. The protein concentration of sample supernatants was determined by using the bicinchoninic acid protein assay.

Hundred microgram protein was transferred to a new tube and adjusted to a protein concentration of 1 μg/μL with 100 mM triethylammonium bicarbonate (TEAB). Five microliters of 200 mM DTT were added and incubated at 55°C for 1 h, then 5 μL of 375 mM iodoacetamide was added to the sample and incubated for 30 min at room temperature in the dark. For each sample, proteins were precipitated with ice-cold acetone before re-dissolving in 20 μL TEAB. Proteins were then tryptically digested with sequencing grade modified trypsin (Promega, Madison, WI), and the resultant peptide mixture was labeled using reagents from the iTRAQ reagents kit.

The peptide mixture was redissolved in buffer A (buffer A: 10 mM ammonium formate in water, pH 10, adjusted with ammonium hydroxide) and then fractionated by high pH separation using an Aquity UPLC system (Waters Corporation, Milford, MA) connected to a reverse phase column (BEH C18 column, 2.1 × 150 mm, 1.7 μm, 300 Å, Waters Corporation, Milford, MA). High pH separation was done using a linear gradient, starting from 0% B to 45% B in 35 min (B: 10 mM ammonium formate in 90% acetonitrile, pH 10.0, adjusted with ammonium hydroxide). The column flow rate and temperature were maintained at 250 μL/min and 45°C, respectively. Sixteen fractions were collected, and each fraction was dried in a vacuum concentrator prior to the next step.

The dried fractions were re-suspended in a solution made of solvent C and D (C: water with 0.1% formic acid; D: acetonitrile with 0.1% formic acid), separated by nano LC, and analyzed by on-line electrospray tandem mass spectrometry. The experiments were performed on a Nano Aquity UPLC system (Waters Corporation, Milford, MA) connected to a quadrupole-Orbitrap mass spectrometer (Q-Exactive) (Thermo Fisher Scientific, Bremen, Germany) equipped with an online nano-electrospray ion source. Eight microliters of peptide sample were loaded onto the trap column (Thermo Scientific Acclaim PepMap C18, 100 μm × 2 cm), with a flow of 10 μl/min for 3 min, and subsequently separated on an analytical column (Acclaim PepMap C18, 75 μm × 25 cm) with a linear gradient, from 5% D to 30% D in 95 min. The column was re-equilibrated to the initial conditions for 15 min. The column flow rate and temperature were maintained at 300 nL/min and 45°C, respectively. An electrospray voltage of 2.0 kV was used against the inlet of the mass spectrometer.

The Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra (m/z 350–1,600) were acquired with a mass resolution of 70 K, followed by 15 sequential high-energy-collisional-dissociation (HCD) MS/MS scans with a resolution of 17.5 K. In all cases, one micro-scan was recorded using dynamic exclusion of 30 s, with an MS/MS fixed first mass of 100.

Tandem mass spectra were extracted by the Proteome Discoverer software (Thermo Fisher Scientific, version 1.4.0.288). The mass profiles generated from all samples were searched with Mascot (Matrix Science, London, UK; version 2.3) against the NCBI database (Taxonomy: L. casei Zhang). We used the percolator algorithm <1% to control peptide level false discovery rates. Only unique peptides were used for protein quantification and the normalization on protein median was applied to correct any experimental bias. The minimum number of proteins that must be observed was set to 1,000. Students't-tests were performed with the software package R, with p < 0.05 considered statistically significant. A 2.0-fold change was used as the threshold for selection of differentially regulated proteins. All regulated proteins were distributed over clusters of orthologous genes (COGs) and searched against the Kyoto Encyclopedia of Genes and Genomes database.

Based on the results of proteomic analysis, 1 protein (the alkaline shock protein, coded by the gene LCAZH_0227) was selected for PRM analysis. This protein was most up-regulated among all differentially expressed proteins.

Experiments were performed on a Q Exactive mass spectrometer coupled with Easy-nLC1200. Peptide mixtures were separated by C18-reversed phase chromatography on an Easy column (75 μm × 25 cm), and the analytical separation was run for 90 min using a linear gradient of ACN/FA 2%/0.1% (Solvent A) and ACN/FA 80%/0.1% (Solvent B) at a flow rate of 300 nL/min. The gradient programme was run as follows: 5% B at 1 min, ramping to 23% B at 41 min, 29% B at 51 min, rapid ramping to 100% B over 8 min and holding 100% B for 6 min before returning to initial condition of 5% B. The column was re-equilibrated to 5% B for 30 min after each run. All samples were analyzed using a multiplexed PRM method based on a scheduled inclusion list containing the target precursor ions representing the standard peptides. The full scan event was collected at m/z 300–1300, an Orbitrap resolution of 70,000, the automatic gain control target at 3e6, and the maximum fill time at 20 ms. Every full scan was followed by 10 PRM scans at a resolution of 17,500 with an isolation window: 2.0 m/z, an AGC value of 5e5, the maximum fill time of 100 ms, and a normalized collision energy of 28 in a higher-energy c-trap dissociation (HCD) cell.

PRM data analysis and data integration were performed with the Skyline Software. Routine assessment of instrument and chromatographic performance was done with a quality control (QC) standard consisting of all synthetic peptides, which was prepared at a concentration of 20 fmol/μl in 0.1% formic acid. Every sample was injected for three times, the peak areas of the target peptides were extracted using Skyline, peak peaking was manually checked and corrected in accordance to the retention time, transitions, mass accuracy, and MS/MS spectra. At least 3 transitions for each peptide were extracted from the PRM data. Peptides were quantified by summing the peak areas under curve (AUC) of each transition. Peptide abundance was normalized based on the total ion current (TIC) extracted from the full scan acquisition for each run. Proteins were quantified by summing the abundances of the selected peptides, and the accurate protein quantities had to match those of the synthetic standard peptides.

The gene LCAZH_0227 (asp23) was selected for target disruption. Plasmids and primers used for gene disruption are listed in Table 1. The mutant line was constructed by using a cre-lox-based system originally developed by Lambert et al. (2007). Briefly, the upstream (amplified with the primers 0227upF and 0227upR,) and downstream (amplified with 0227downF and 0227downR) fragments of the LCAZH_0227 gene were PCR amplified from the genomic DNA of L. casei Zhang-G-1200. The fragments were cloned between the SalI-HF or PmeI and Ecol 53KI or BglII restriction sites of the suicide vector pNZ5319 to form the recombinant mutagenesis vector, pNZ5319-0227 Up-Down. To inactivate the LCAZH_0227 gene, pNZ5319-0227 Up-Down was introduced into L. casei Zhang-G-1200 by electroporation. Chloramphenicol-resistant transformants were selected and replica plated to check for an erythromycin-sensitive phenotype. Candidate double-crossover mutant clones were identified by PCR, and correct integration of the lox66-P32-cat-lox71 cassette into the genome was further verified by PCR using the primers 0227upF or catR and catF or 0227downR. To excise the P32-cat selectable marker cassette, the cre expression plasmid pMSPcre was transformed into the 0227::lox66-P32-cat-lox71 gene replacement mutant. The Cre-mediated recombination and correct excision of the P32-cat cassette were checked by PCR using primers spanning the recombination loci (0227upF and 0227downR). The pMSPcre vector was cured from L. casei Zhang-G-1200 Δ0227 colonies by growth without erythromycin selection pressure. Additionally, PCR products were confirmed by sequencing when necessary.

The growth performance and gentamycin-resistant phenotype of the mutant strain was evaluated by viable counts, OD measurements, and MIC of gentamycin (Guo et al., 2017). Phenotypic differences between the wild-type and mutant strains were determined statistically by Student's t-test.

The growth of the two bacterial lines in the presence of gentamycin was monitored. Growth curves were plotted based on viable counts, pH values, and OD values. As shown in Figures 1A–C, the growth performance of L. casei Zhang-G-1200 was completely different from that of the parental line. The viable counts of L. casei Zhang-G-1200 increased more rapidly than that of the parental line, reaching a maximum cell density of 9.05 × 10⁷ cfu/mL after 20 h (Figure 1A). Meanwhile, the pH of the LSM medium inoculated with L. casei Zhang-G-1200 dropped much faster than that of the parental line, indicating a higher fermentation rate (Figure 1B).

A total of 72 proteins were significantly up-regulated (>2.0-folds, P < 0.05) in L. casei Zhang-G-1200 compared with the parental strain (Table 2). Most of these proteins could be assigned to one of the functional COGs categories (Figure 2), with 26.3% of these proteins involved in carbohydrate transport and metabolism [G] and another 25.3% of them involved in amino acid transport and metabolism [E].

Over half (6 out of 10) of the up-regulated proteins were associated with the COGs category of carbohydrate transport and metabolism [G], which were the phosphotransferase system (PTS)-related components located within 3 different operons. According to the Transporter Classification Database (TCDB), 4 of these differentially regulated proteins belonged to the PTS Galactitol family, including the galacitol transporter subunits EIIA (LCAZH_2649), EIIB (LCAZH_2648), IIB (LCAZH_0604), and EIIC (LCAZH_0605), while the other 2 belonged to the PTS Lactose-N, N'-Diacetylchitobiose-β-glucoside family, namely the cellobiose-specific transporter subunits IIA (LCAZH_1771) and IIB (LCAZH_1772).

Another group of significantly up-regulated proteins was responsible for the uptake of oligopeptides (LCAZH_2023-LCAZH_2026). This genomic cluster coded for the ATPase and 2 permease components of a dipeptide/oligopeptide/nickel ABC transporter, as well as an oligopeptide ABC transporter periplasmic protein. Similar to the Opp system identified in other LAB genomes (Chen et al., 2015), another gene that coded for the ATP-binding subunit of the oligopeptide ABC transporter was found immediately downstream of the oligopeptide ABC transporter periplasmic protein of the genome of L. casei Zhang (Zhang et al., 2010). Besides, several other oligopeptide (LCAZH_0201, LCAZH_1596, LCAZH_1886, and LCAZH_2851) and amino acid (LCAZH_0500 and LCAZH_0418) transporter components, as well as subunits for de novo syntheses of tryptophan (LCAZH_0084) and glutamate (LCAZH_2518 and LCAZH_2519), were identified.

Other up-regulated proteins included those relating to purine biosynthesis (LCAZH_1739, LCAZH_1740, and LCAZH_1743) and stress response (LCAZH_1344 and LCAZH_0227). Notably, the alkaline shock protein (LCAZH_0227) was the most significantly up-regulated protein with a 21.9-fold increase in its expression compared with the parental line, suggesting its important role in gentamycin adaptation.

A total of 36 proteins were significantly down-regulated (<−2.0-folds, P < 0.05) in L. casei Zhang-G-1200 compared with the parental strain (Table 3). According to the COGs functional classification, 33.3% of these proteins were involved in posttranslational modification, protein turnover, chaperones metabolism [G] (Figure 2). Indeed, most of them were stress-responsive proteins, including the protease subunit of ATP-dependent Clp protease (LCAZH_0907), the molecular chaperones GrpE, GroEL, and HSP20-2 (LCAZH_1553, LCAZH_2207, and LCAZH_2811), the co-chaperonin GroES (LCAZH_2208), and the Clp protease/DnaK/DnaJ chaperone ATP-binding subunit (LCAZH_1753), while the others were mainly hypothetical proteins of unknown functions.

The expression of the alkaline shock protein (LCAZH_0227) was confirmed by PRM analysis. Fortunately, 1 unique peptide (FDDAVIAK) corresponding to this protein was found, which enabled the downstream quantification. As revealed by the analysis, the quantity of this alkaline shock protein was significantly higher in L. casei Zhang-G-1200 than the parental cells (2.938 ± 0.144 fmol/μg vs. 0.789 ± 0.057 fmol/μg). This result was consistent with the findings of the proteomic analysis.

The asp23 gene (LCAZH_0227) was selected as a target for mutational analysis because it was the most up-regulated protein in L. casei Zhang-G-1200 when the cells were grown in the presence of gentamycin. As shown in Figure 3, asp23 inactivation did not affect the growth of L. casei Zhang-G-1200 in LSM without antibiotics. However, the L. casei Zhang-G-1200 asp23 disruption mutant was significantly more sensitive to gentamycin with a relatively low MIC value of 8 μg/mL though still higher than that of the parental strain (vs. 2 μg/mL and 32 μg/mL for the parental strain and L. casei Zhang-G-1200, respectively). When the concentration of gentamycin increased to 16 μg/mL, the wild type L. casei Zhang-G-1200 cells survived significantly better than the asp23 disruption mutant.

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.