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. 2016 Dec 7:6:38122.
doi: 10.1038/srep38122.

Gsy, a novel glucansucrase from Leuconostoc mesenteroides, mediates the formation of cell aggregates in response to oxidative stress

Minghui Yan  1   2 , Jin Han  1 , Xiaofen Xu  1 , Lianliang Liu  3 , Caixia Gao  1 , Huajun Zheng  4 , Yunxia Chen  5 , Yimin Tao  6 , Hu Zhou  6 , Yunfei Li  2 , Zhengjun Wu  1
Affiliations

Gsy, a novel glucansucrase from Leuconostoc mesenteroides, mediates the formation of cell aggregates in response to oxidative stress

Minghui Yan et al. Sci Rep. .

Abstract

Leuconostoc mesenteroides is a member of lactic acid bacteria (LAB) with wide applications in the food and medical industries. Species in the genus Leuconostoc are catalase-negative and generally regarded as facultative anaerobic or aerotolerant organisms. Despite their extensive use in industry, certain issues concerning the aerobic life of L. mesenteroides, e.g., the mechanism involved in the tolerance to oxygen, remain to be addressed. In this manuscript, a survival strategy employed by L. mesenteroides BD3749 in response to oxidative stress was elucidated. BD3749 cells cultivated in medium with sucrose available synthesized large amounts of exopolysaccharides, mostly consisting of insoluble EPS. When BD3749 cells were challenged with oxidative stress, the amount of insoluble EPS was greatly enhanced. The synthesized EPSs reduced the accumulation of reactive oxygen species (ROS) in bacterial cells and improved their survival during chronic oxidative stress. Another study showed that Gsy, a novel glucansucrase in the GH70 family that is induced by sucrose and up-regulated following exposure to oxygen, was responsible for the synthesis of insoluble EPS. Gsy was subsequently demonstrated to play pivotal roles in the formation of aggregates to alleviate the detrimental effects on BD3749 cells exerted by oxygen.

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Figures

Figure 1
Figure 1. Sucrose-dependent aggregation of BD3749 in response to oxygen.
(A,B,C) An overnight culture of BD3749 was diluted 1:100 in fresh medium containing 2% glucose or sucrose and incubated aerobically at 180 rpm and 30 °C. The aggregation characteristics of logarithmic-phase culture in glucose or sucrose was investigated and photographed. Shown are the microscopic images of BD3749 in aggregated (A) and planktonic (B) cells grown in sucrose medium. Cell morphology of BD3749 grown in glucose medium is shown in (C). All microscopic images were recorded with an AxioPlan 2 Imaging system by Carl Zeiss. The boxed areas were enlarged on the right.
Figure 2
Figure 2. ROS accumulation in BD3749 exposed to oxygen.
(A) Relative ROS accumulation in BD3749 cells grown aerobically in medium containing glucose or sucrose as a sole carbon source, as described in Materials and Methods. The amount of ROS was calculated by the intensity of the DCF fluorescence per mg total protein of the cell lysate. The relative ROS level of the BD3749 culture in glucose (Circle) and sucrose (Triangle) are shown. Experiments were performed in triplicate, and the average intensity and standard deviations are shown. (B) Relative ROS level in aggregated and planktonic cells of BD3749. A logarithmic culture was set on the table for 2 min for natural sedimentation of the aggregates. Aggregated and planktonic cells were collected independently, and the ROS level was monitored as described in Materials and Methods. (C) The percent aggregation was monitored for 24 hours of aerobic growth. Aggregated cells were collected as described above, and planktonic cells were harvested by centrifugation at 8000 rpm for 5 minutes. The collected cells were then resuspended in PBS to a volume equivalent to that of the culture after 3 washes with 0.5 M NaOH before sonication. The protein content of sonicated cell lysate was then quantified using the Bradford method. The percent of aggregation was calculated by protein content of aggregated cells divided by that of aggregated cells plus planktonic cells. All the experiments were performed in triplicate, and the standard deviations are shown in the graphs.
Figure 3
Figure 3. Identification of glucansucrase Gsy in BD3749.
(A) The total proteins from the supernatant were prepared as described and equivalent amount of total protein was separated on a 16 × 20 cm 6% SDS-PAGE and stained with Coomassie brilliant blue G250. The lines on the right indicate the differentially expressed protein bands. (B) In situ detection of dextransucrase activity. After electrophoresis, the SDS-PAGE gel was incubated in 50 mM sodium acetate buffer containing 50 g/L sucrose buffered at pH 5.4. The active bands were detected by the appearance of opaque polymer.
Figure 4
Figure 4. Expression analysis of glucansucrase-encoding genes.
(A) Equal amount of total protein from the supernant was separated by 6% SDS-PAGE and immunoblotted with the indicated antibodies after transfer onto PVDF membranes. The gel was run in triplicate and analysed with anti-1322, anti-1323, and anti-1322 plus anti-1323, respectively. (B) Effect of oxygen on the protein level of glucansucrases. Equal amount of total protein from cell pellets from oxic and anoxic cultures were separated and detected with the indicated antibody. (C) Transcription analysis of the putative glucansucrase genes in response to oxygen. The transcription level under oxic conditions was normalized to that under anoxic conditions. Asterisk indicates low transcription level that is close to the limit of detection.
Figure 5
Figure 5. Sequence-based analysis of Gsy.
(A) Domain structure analysis of Gsy. The deduced active sites are marked with asterisks, and the peptide used for antibody production is indicated below the GH70 domain. Cell wall binding repeats were analyzed on Scanprosite (http://prosite.expasy.org/scanprosite). Shown below Gsy is a typical Gtf from S.mutans (AAN58706.1)(http://www.cazy.org/GH70_characterized.html, http://prosite.expasy.org/scanprosite/). (B) Phylogenetic analysis of putative glucansucrases in BD3749 and the characterized GH70 enzymes. The tree was constructed with the maximum-likelihood method based on the JTT matrix-based model using MEGA6. Each protein is labelled with its GenBank accession number. GH70 enzymes of BD3749 are labelled in red. The value on each branch is the estimated confidence limit for the position of the branches, as determined by bootstrap analysis. The scale bar represents a 7% difference in amino acid sequence. It should be noted that CDX66820.1 and CAB65910.2, GH70 enzymes from NRRL B-1299 and NRRL B-1355, respectively, were assigned to Leuconostoc citreum as suggested by Bounaix et al..
Figure 6
Figure 6. Functional blockade of Gsy inhibits aggregation.
(A) The Gsy antibody inhibited the production of insoluble EPS. The antibody was supplemented as mentioned above, and EPS were collected as described in Materials and Methods. The average amount from three experiments and standard deviations are shown. (B) Gsy antibody blocked oxygen-induced aggregate formation. Antibody was added to a final concentration 20 μg/mL, and normalized IgG was used as a negative control. PMSF (100 μM) was added to prevent antibody degradation. The primitive culture was photographed to demonstrate the formation of aggregates.
Figure 7
Figure 7. Increase in EPS in response to oxidative stress.
(A) EPS synthesized by BD3749 under oxic and anoxic conditions. The insoluble and soluble EPS were collected and detected, respectively. (B) EPS synthesized by BD3749 in broth with H2O2 log phase culture of BD3749 was diluted 1:50 into fresh medium supplemented with H2O2 at the indicated concentration. After 48 h of cultivation, insoluble and soluble EPS in the culture were obtained as described in Materials and Methods. Excluding the oxic group, incubation was carried out in the Whitley A35 Anaerobic Workstation. The average amount of three experiments and standard deviations are shown.
Figure 8
Figure 8. Exopolysaccharides improve the survival of BD3749 cells under chronic oxygen stress.
BD3749 cells were inoculated into 30 mL TYC-Suc medium or TYC-Glc medium (in which sucrose was substituted with an equal amount of glucose) and incubated anaerobically at 37 °C for 2 days. The cultures were then shifted to an aerobic incubator at 30 °C and maintained for 5 days, during which the pH value was monitored and viable cells were enumerated by plate counting. The average amount of three experiments and standard deviations are shown in the graph.
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