Table 1 lists all strains and plasmids utilized. E. coli and L. bulgaricus were cultured in LB and Man-Rogosa-Sharpe (MRS) (Beijing Land Bridge Technology Co., Ltd. CM187), respectively, at 37°C with no shaking. Saccharomyces cerevisiae Y₂HGOLD cells, carrying four reporter genes (HIS3, ADE2, AUR1-C, and MEL1) controlled by the GAL4 promoter (Xu et al., 2015), were cultured in Yeast Peptone Dextrose (10 g yeast extract, 20 g peptone, 20 g dextrose per liter) or synthetic defined (SD) medium (BD Difco Ltd., USA) at 28°C. Ampicillin (Amp, Sigma Chemical Co, USA), was used for E. coli at 100 μg/mL. Erythromycin (Em, Sigma) and Amp were used for L. bulgaricus at 50 μg/mL each. Chloramphenicol was used for L. bulgaricus at 10 μg/mL.

The whole genome sequence of the L. bulgaricus ATCC BAA-365 strain was downloaded from NCBI (https://www.ncbi.nlm.nih.gov/nuccore/NC_008529.1), and used for the prediction of TCS. HisKA (PF00512), HATPase-c (PF02518), Response reg (PF00072) from the Protein families database were used in a HMMER search. HisKA.hmm and HATPase-c.hmm were employed for scanning the highly conserved phosphate group binding- and HATPase regions in HPK. Response-reg.hmm was utilized to screen conserved phosphate groups in the response regulator protein.

To construct a LBUL_RS04160 mutant, a 570 bp fragment intermediate region of the LBUL_RS04160 gene was first amplified by PCR using HPK4160-SphI-F (5′-ACGCGCATGCCGCATGAACTTAAGACGCCC-3′) (SphI site underlined) and HPK4160-EcoRI-R (5′-ACATGAATTCTTGCGGCTGTGGCTCTTATC-3′) (EcoRI site underlined) primers, and inserted into pUC19 to generate pUC19-HPK4160. The erythromycin resistance gene was amplified by PCR from pMG76e using the EryB-BfaI-F (5′-CCGCTAGATGACCACCGACGCCGCGACG-3′) and EryB-BstEII-R (5′-CGGGTAACCTCACTGCAACCAGGCTTCCGG-3′) primers, and inserted into pUC19-HPK4160 to create plasmid pUC19-HPK4160::EryBII (Figure 1).

To construct a LBUL_RS00115 mutant, a 819 bp fragment intermediate region of the LBUL_RS00115 gene was first amplified by PCR with the HPK0115-SphI-F (5′-ACGCGCATGCCGCGGGCAGGCAAAAAG-3′) (SphI site underlined) and HPK0115-EcoRI-R (5′-ACATGAATTCAACGCAGCGGATGATGCTTA-3′) (EcoRI site underlined) primers, and inserted into pUC19 to generate pUC19-HPK0115. The erythromycin resistance gene was amplified by PCR from pMG76e with the EryB-BstEII-F (5′-CGGGTAACCATGACCACCGACGCCGCGACG-3′) and EryB-BstYI-R (5′-CGAGATCCTCACTGCAACCAGGCTTCCGG-3′) primers, and inserted into pUC19-HPK0115 to create the pUC19-HPK0115::EryBII plasmid (Figure 2).

All other gene manipulation experiments were carried out as described previously (Pang et al., 2014).

The pUC19-HPK4160::EryBII and pUC19-HPK0115::EryBII plasmids were transformed into L. bulgaricus ATCC BAA-365 as proposed previously (Holo and Nes, 1989; Kim et al., 2009), with minor modifications described in a previous study (Pang et al., 2014).

A 1.3 kb fragment, encompassing the complete LBUL_RS00115 coding region was amplified by PCR using the HPK0115-SalI-F (5′-ACGCGTCGACATGATCAACAGCCTGTTCA-3′) and HPK0115-SphI-R (5′-ACATGCATGCCTATCCCTTCTGAATAACT-3′) primers from the L. bulgaricus BAA-365 genome, and cloned into pMG56e to generate pMG56e-HPK0115. The latter plasmid (pMG56e-HPK0115) was then introduced into the mutant L. bulgaricus H0115-K7, yielding complemented strain L. bulgaricus H0115-K7-com1 (Table 1). Transformants with successful complementation were selected by culture on 10 μg/mL chloromycetin containing plates.

Bacterial suspension (OD600 = 0.4~0.6) was centrifuged to remove the bacterial cells, the supernatant was measured OD260/280 nm, the reading was recorded as A₀. Take the appropriate amount of the above bacterial suspension in the incubator for t hours. Half of the sample was centrifuged to remove the bacterial cells. Measure the OD260/280 nm of the supernatant and the reading was recorded as A_t. The remaining bacterial suspension sonicated (400 w, work 3 s, interval 3 s) until the solution became clear (the cells are completely broken) under ice-cooling, bacterial cells were removed by centrifugation, measure the OD260/280 nm of the supernatant and the reading was recorded as A_s. The autolysis rate is calculated according to the formula:

Groups were compared by One-Way ANOVA and LSD test.

It is difficult to construct a two hybrid system by using full length WP_011677872.1 and WP_011677871.1 genes, because WP_011677872.1 contain six transmembrane regions. Therefore, these transmembrane regions of WP_011677872.1 were removed, and the HATPase-c domain of WP_011677872.1 was selected as the bait protein. The WP_011677872.1 HATPase-c domain gene was PCR amplified with the HATPase-NdeI-F (5′-GGAATTCCATATGATGGTAAATATCGTAAGCATCA-3′) and HATPase-BamHI-R (5′-CGGGATCCCTATCCCTTCTGAATAACT-3′) primers, and inserted into the pGBKT7 vector (Clontech), to create the two-hybrid plasmid pGBK-HPK0115 (Figure 3); the complete LBUL_RS00110 sequence was amplified by PCR using the RR0110- NdeI-F (5′-GGAATTCCATATGATGCTAGCCATCATCATTT-3′) and RR0110-BamHI-R (5′-CGGGATCCTTAAACAAGGTCATTTT-3′) primers, and inserted into the pGADT7 vector (Clontech), to create the two-hybrid plasmid pGAD-RR0110 (Figure 4).

Co-transformation of S. cerevisiae Y₂HGOLD was carried out using various two-hybrid plasmid pairs: pGBK-HPK0115/pGAD-RR0110 (0.15 μg/each), pGBKT7-53/pGADT7-T (positive control), pGBKT7-Lam/ pGADT7-T (negative control). Y₂HGold cell amounts were determined with (His) or without (-His) histidine, to assess HIS3 reporter activation, which in combination with AUR1-C reporter activation (growth on 0.1 mg/mL aureobasidin A, Clontech), reflects protein interaction. Strain S. cerevisiae Y₂HGold, and various vectors were obtained from Clontech Laboratories, Inc.

The whole genome sequence of L. bulgaricus BAA-365 was scanned by the Hmmer software for HisKA, HATPase-c and Response-reg domains. A total of 7 HPKs and 7 RRs were identified, as shown in Table 2. NCBI BLASTP was used for HPK and RR function prediction: the functions of five RRs have been reported, while those of the two remaining RRs remain unknown. The structural domains of WP_011543855.1, WP_003620064.1, WP_011677872.1, and WP_011677871.1 were assessed by utilizing Simple Modular Architecture Research Tool (SMART) (Figure 5).

Two histidine protein kinases, WP_011543855.1 and WP_011677872.1, had transmembrane regions (blue block), which can directly sense changes in the external environment. The PAS domain (PER-ARNTSIM domain) can sense changes of light, oxygen, redox potential, small molecule ligand and total cell energy, and is an important signal receptor domain (Furukawa-Hibi et al., 2015; Guo et al., 2015; Kasai et al., 2015). Compared with the structural regions of WP_011543855.1 and WP_011677872.1, those of WP_003620064.1 and WP_011677871.1 were relatively simple; both genes contained typical signal receiving domains (REC), and WP_011677871.1 contained a LytTR type signal output domain. In order to verify whether there is a correlation between the two TCS and cell autolysis, LBUL_RS04160 (coding gene of WP_011543855.1) and LBUL_RS00115 (coding gene of WP_011677872.1) were knocked out, respectively.

The recombinant plasmids pUC19-HPK4160::EryBII obtained from transformed E. coli DH5α were submitted to digestion by SphI and EcoRI. Electrophoresis data indicated the presence of both the 4.4 kbp pUC19-HPK4160::EryBII and 1.7 kbp HPK4160::EryBII fragments, suggesting successful insertion of EryBII into pUC19-HPK4160. In agreement, PCR reactions using Primers 1 and 2 (Table 3) showed concordant data (Figure 6). Next, pUC19-HPK0115::EryBII identification was carried out in a similar way to pUC19-HPK4160::EryBII. After digestion, the 4.7 kb pUC19-HPK0115::EryBII and 2.0 kb HPK0115::EryBII fragments were all present, also suggesting successful HPK0115::EryBII insertion into the pUC19 vector. These findings were confirmed by PCR using Primers 5 and 6 (Figure 6).

The recombinant plasmids pUC19-HPK4160::EryBII and pUC19-HPK0115::EryBII were transformed into L. bulgaricus ATCC BAA-365, respectively, by electroporation. After culture for 2 h at 37°C, the transformed bacteria were transferred on solid MRS medium with 0.5 M sucrose and erythromycin (50 μg/mL) for selection. Next, candidate colonies were plated onto MRS agar with ampicillin (50 μg/mL). The double-crossover mutant bacteria could not grow in the latter conditions. Finally, three mutants L. bulgaricus ΔH4160 1-3 with pUC19-HPK4160::EryBII and one double-crossover mutant L. bulgaricus ΔH0115 1 with pUC19-HPK0115:: EryBII were chosen in the second round.

DNA was obtained from the L. bulgaricus ΔH4160-1 and wildtype BAA-365 genomes, after 24 h of culture in MRS broth, and amplified by PCR with Primers 1 and 2. This yielded 0.5 and 1.7 kb amplicons from wildtype and mutant genomic DNAs, respectively (Figure 7); the ~1.2 kb difference reflected the inserted erythromycin resistance gene. These data confirmed successful LBUL_RS04160 knockout by inserting the erythromycin resistance gene. The identification of LBUL_RS00115 mutant L. bulgaricus ΔH0115-1 was carried out as described above (Figure 7).

For complementation, pMG56e-HPK0115 was transformed into L. bulgaricus ΔH0115-1 by electroporation. The HPK0115-complemented strain was named rΔH0115-1. As shown in Figure 8D, rΔH0115-1 autolysis was markedly increased in comparison with that of ΔH0115-1, with no significant difference compared with that of wild type. These findings suggested that HPK0115 complementation restituted the autolytic capacity.

In BAA-365, ΔH4160-1, ΔH0115-1, and rΔH0115-1, autolysis monitoring results revealed no significant differences between the LBUL_RS04160 gene knockout strain ΔH4160-1 and wide type strain BAA-365; this indicated that LBUL_RS04160 gene was not associated with cell autolysis. However, autolysis rate of the LBUL_RS00115 gene knockout strain ΔH0115-1 was starkly reduced compared with the value obtained for the wild type strain at the 16 h time point. In addition, a markedly enhanced maximum OD value was obtained in the knockout mutant compared with wild type; this indicated that the density of L. bulgaricus population was significantly increased when the LBUL_RS00115 gene was knocked out (Figure 8). In order to further demonstrate that the autolysis of ΔH0115-1 changed significantly, we monitored the colony counts of the four strains at different time points, the results shows that when the bacteria grown to stationary phase, the viable count of ΔH0115-1 is significantly higher than that of other bacteria (Figure 9). The four bacteria grown to 24 h were observed by electron microscopy, it can be seen only ΔH0115-1 bacterial cell wall is still relatively complete, but the other three strains of cell wall can be seen obvious damage (Figure 10). These findings indicated a significant function for LBUL_RS00115 in L. bulgaricus autolysis. Meanwhile, a reduced autolysis remained in LBUL_RS00115 knockout organisms, implying the contribution of additional genes the autolytic process in L. bulgaricus.

The yeast two-hybrid system represents a well-known method in identifying protein interactions (Chini, 2014; Ferro et al., 2014). To determine whether the WP_011677872.1 (encoded by LBUL_RS00115) and WP_011677871.1 (encoded by LBUL_RS00110) proteins interact, two-hybrid system plasmids were generated with the HATPase-c domain gene of LBUL_RS00115 and full LBUL_RS00110 gene. The two-hybrid plasmids pGBK-HPK0115 and pGAD-RR0110 were cotransformed into S. cerevisiae Y₂HGOLD. The cotransformants could grow on SD/-Trp-Leu-His plates with 0.1 mg·mL⁻¹ aureobasidin A (Clontech) (Figure 11). These data demonstrated that these cotransformants activated both HIS3 and AUR1-C reporters, confirming interaction occurrence between WP_011677872.1 and WP_011677871.1.

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.