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. 2023 Mar 6;11(2):e04150-22. doi: 10.1128/spectrum.04150-22

Molecular Organization and Functional Analysis of a Novel Plasmid-Borne cps Gene Cluster from Lactiplantibacillus plantarum YC41

Jieran An a, Yuchen Zhang a, Zhaoer Zhao a, Ran Huan a, Huaxi Yi c, Hui Wang a, Chunguang Luan d, Shengbao Feng e, Heqiang Huang e, Shanwen Li e, Deliang Wang d, Zhengyuan Zhai a,✉, Yanling Hao b,✉
Editor: Zhenjiang Zech Xuf
PMCID: PMC10100969  PMID: 36877018

ABSTRACT

Capsular polysaccharide (CPS) can tightly attach to bacterial surfaces and plays a critical role in protecting microorganisms from environmental stresses. However, the molecular and functional properties of some plasmid-borne cps gene clusters are poorly understood. In this study, comparative genomics of the draft genomes of 21 Lactiplantibacillus plantarum strains revealed that the specific gene cluster for CPS biosynthesis was observed only in the 8 strains with a ropy phenotype. Furthermore, the complete genomes showed that the specific gene cluster cpsYC41 was located on the novel plasmid pYC41 in L. plantarum YC41. In silico analysis confirmed that the cpsYC41 gene cluster contained the dTDP-rhamnose precursor biosynthesis operon, the repeating-unit biosynthesis operon, and the wzx gene. The insertional inactivation of the rmlA and cpsC genes abolished the ropy phenotype and reduced the CPS yields by 93.79% and 96.62%, respectively, in L. plantarum YC41 mutants. These results revealed that the cpsYC41 gene cluster was responsible for CPS biosynthesis. Moreover, the survival rates of the YC41-rmlA− and YC41-cpsC− mutants under acid, NaCl, and H2O2 stresses were decreased by 56.47 to 93.67% compared to that of the control strain. Furthermore, the specific cps gene cluster was also confirmed to play a vital role in CPS biosynthesis in L. plantarum MC2, PG1, and YD2. These findings enhance our understanding of the genetic organization and gene functions of plasmid-borne cps gene clusters in L. plantarum.

IMPORTANCE Capsular polysaccharide is well known to protect bacteria against various environmental stresses. The gene cluster for CPS biosynthesis is typically organized in the chromosome in bacteria. It is worth noting that complete genome sequencing showed that a novel plasmid pYC41-borne cpsYC41 gene cluster was identified in L. plantarum YC41. The cpsYC41 gene cluster included the dTDP-rhamnose precursor biosynthesis operon, the repeating-unit biosynthesis operon, and the wzx gene, which was verified by the significantly decreased CPS yield and the absent ropy phenotype in the corresponding mutants. The cpsYC41 gene cluster plays an important role in bacterial survival under environmental stress, and the mutants had decreased fitness under stress conditions. The vital role of this specific cps gene cluster in CPS biosynthesis was also confirmed in other CPS-producing L. plantarum strains. These results advanced a better understanding of the molecular mechanisms of plasmid-borne cps gene clusters and the protective functionality of CPS.

KEYWORDS: Lactiplantibacillus plantarum, capsular polysaccharide, cps gene cluster, plasmid, environmental stresses

INTRODUCTION

Lactiplantibacillus plantarum is a facultative heterofermentative lactic acid bacterium (LAB). The larger genome (~3.3 Mb) and the higher proportion of regulatory genes than those of other Lactobacillus species allow this species to survive in a variety of different environmental niches (1). L. plantarum has been widely used in fermented food and feed production, such as dairy, meats, vegetables, and silage (2–4). In addition, it can exhibit various health-promoting effects, including reducing cholesterol levels (5), regulating the immune system (6), alleviating memory impairment (7), and enhancing epithelial barrier function (8). However, during manufacturing or consumption, L. plantarum suffers from unavoidable stress such as dehydration, extreme temperatures, osmotic stress, acid, and bile salt (9). Hence, L. plantarum has developed various adaptive mechanisms to cope with different stresses.

Bacterial polysaccharides represent a large group of carbohydrate polymers, which are either tightly attached to the surface of the microbial cell in the form of capsular polysaccharide (CPS) or loosely attached and secreted into the environment as exopolysaccharide (EPS) (10, 11). They are well known to protect bacteria against environmental stresses by forming a physical barrier on the cell surface (12, 13). When exposed to acidic conditions, an EPS-producing strain showed 20-fold increases in viability compared with a non-EPS-producing strain in Lactobacillus paracasei NFBC 338 (14). Compared with the wild type, the survival rate of an epsE mutant was decreased by 10% under heat shock in Lactobacillus johnsonii FI9785 (15). It has also been reported that CPS resulted in a 20% improvement in bacterial survival in the presence of 1% bile salt in L. plantarum LTC-113 (16). However, in L. plantarum Lp90 and SF2A35B, there were no significant differences in survival rates between the wild-type strains and the corresponding cps deletion mutants under gastrointestinal (GI) tract-mimicking conditions (17). These results indicated that the relationship between CPS and survival strategies remains unclear, even within one species.

The CPS biosynthesis genes are organized as cps gene clusters in L. plantarum (18). The cps gene clusters generally include regulatory genes encoding the regulatory proteins CpsABCD, repeating-unit synthesis genes encoding glycosyltransferases, assembly machinery genes encoding Wzx and Wzy, sugar precursor synthesis genes, and genes for sugar residue modification (19). Up to now, 192 L. plantarum strains with complete genome sequences and 539 L. plantarum strains with draft genome sequences have been deposited in the NCBI database. cps gene clusters located in chromosomes are found in 43 L. plantarum strains, and the genetic organizations of the gene clusters were further characterized in 12 strains, L. plantarum WCFS1, ZJ316, ST-III, NC8, JDM1, P8, 16, SF2A35B, Lp90, LTC-113, SN35N, and K25 (16, 20, 21). Furthermore, plasmid-borne cps gene clusters have been found in 10 L. plantarum strains, L. plantarum ZJ316, 16, C410L1, HFC8, LZ227, TMW1.1623, ZJ102, LTC-113, SN35N, and K25 (16, 20, 21). Only the plasmid-borne cps gene cluster in L. plantarum LTC-113 has been well characterized (16). Thus, it will be necessary to further characterize cps gene clusters on plasmids in L. plantarum.

In this study, L. plantarum YC41 was isolated from sauerkraut, a traditional brine-salted Chinese fermented food. Genetic and physical experiments revealed that the cpsYC41 gene cluster was responsible for CPS biosynthesis. Furthermore, cpsYC41-derived CPS also showed increased tolerance to environmental stresses, including acid, NaCl, and H2O2 stresses. These results provide insight into the molecular genetic mechanism of cps clusters on plasmids in L. plantarum and the beneficial role of CPS in bacterial survival under stress.

RESULTS

Eight L. plantarum strains formed ropy colonies.

One hundred three colonies exhibited clear zones on de Man-Rogosa-Sharpe (MRS) agar supplemented with 2% (wt/vol) CaCO3, and 21 strains were identified as L. plantarum by 16S rRNA gene sequencing (see Table S3 in the supplemental material). Among these 21 L. plantarum strains, 13 strains could not form any strand, while L. plantarum YC41, APC2, MNN1, MC2, PG1, PG2, YD1, and YD2 showed a ropy phenotype (Fig. 1A). The length of the strands was >1.2 cm. Notably, L. plantarum YC41 had the longest strand, which was >80 mm (Fig. 1A1). In addition, these strains with a ropy phenotype remained in suspension during incubation in MRS broth, whereas the nonropy strains were easier to aggregate, as evidenced by clarified medium with cells at the bottom (Fig. 1B). These results suggested that the eight L. plantarum strains could produce CPS.

FIG 1.

FIG 1

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Characteristic features of L. plantarum strains with a ropy phenotype. (A) Detection of ropy L. plantarum strains on MRS agar containing 2% (wt/vol) sucrose. (B) Autoaggregation of L. plantarum incubated at 37°C for 20 h in MRS broth. Nonropy 1, L. plantarum JY; nonropy 2, L. plantarum YC1.2; 1, L. plantarum YC41; 2, L. plantarum APC2; 3, L. plantarum PG1; 4, L. plantarum PG2; 5, L. plantarum MNN1; 6, L. plantarum MC2; 7, L. plantarum YD1; 8, L. plantarum YD2.

The L. plantarum strains with a ropy phenotype had a specific gene cluster for CPS biosynthesis.

To identify the genes responsible for the ropy phenotype, the draft genomes of 21 L. plantarum strains were sequenced in this study. The average nucleotide identities (ANIs) between YC41 and 7 ropy strains were >99.94%. However, the ANIs between YC41 and 13 nonropy strains were 98.98% to 99.20% (Table S5). It is worth noting that five particular regions, R1 to R5, were present only in the 8 ropy strains, which are the eight innermost circles in Fig. 2A and are marked with black stars. According to the gene annotation, R1, with 29,589 bp, contained 33 genes, where most of them encoded acyl carrier protein, 3-oxoacyl–acyl carrier protein reductase, 3-oxoacyl–acyl carrier protein synthase II, an acetyl-CoA carboxylase biotin carboxyl carrier protein subunit, acetyl-CoA carboxylase, biotin carboxylase, and acetyl-coenzyme A carboxylase carboxyl transferase subunit beta 2, etc. R2, with 10,858 bp, harbored 10 genes, which mainly encoded RNA degradosome polyphosphate kinase, K01514 exopolyphosphatase, and RNA polymerase (RNAP)-binding regulatory protein, etc. R3, with 6,573 bp, contained 9 genes, where most of them encoded hypothetical proteins. R4, with 12,571 bp, contained 9 genes, which mainly encoded hypothetical proteins. Notably, only R5, with 11,060 bp, included a series of genes involved in CPS biosynthesis. There were 14 genes in R5, including the cpsA gene encoding a LytR-CpsA-Psr (LCP) family protein, the cpsB gene encoding a tyrosine-protein phosphatase, the cpsC gene encoding a chain length regulator, the cpsD gene encoding a tyrosine-protein kinase, the cpsE gene encoding a priming glycosyltransferase, and 6 genes encoding glycosyltransferases (Fig. 2B). However, genes encoding flippase and polymerase were not found in this gene cluster, suggesting that this cluster might be incomplete. Therefore, complete genomic sequencing of L. plantarum YC41 was carried out in this study.

FIG 2.

FIG 2

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Comparative genomic analysis of 21 L. plantarum strains. (A) Comparative genomic analysis of 21 L. plantarum strains based on BLAST_VERSION+ bin analysis by BLAST Ring Image Generator v0.95. Matches with <50% identity or regions with no BLAST matches appear as blank spaces in each ring. The inner circle represents the reference sequence of L. plantarum YC41. Rings from the outermost to the center are nonropy strains YC1.2, XXS, XJSC, S2.9, S2.6, S2.13, QZSL, PG3, LC.H2.1, JY, JS13, GY3, and FB1 and ropy strains YD2, YD1, PG2, PG1, MC2, MNN1, APC2, and YC41, which are marked with black stars. R1 to R5 are five particular regions that were present only in the 8 ropy strains. R5 containing the putative CPS cluster is marked in red. (B) Physical map of the putative specific cps gene cluster in the 8 ropy strains.

The specific cps gene cluster cpsYC41 is located in a novel plasmid, pYC41.

Complete genomic sequencing indicated that YC41 contained a 3,249,498-bp circular chromosomal DNA, with a G+C content of 44.65%. Notably, the cpsYC41 gene cluster was located in a 53,923-bp plasmid with a G+C content of 39.14%, which was designated pYC41 (Fig. 3A). The nucleotide sequence of pYC41 showed <57% identity with other plasmids in GenBank, suggesting that it might be a novel plasmid. Sequence analysis showed that plasmid pYC41 had 68 open reading frames (ORFs), including the repA gene encoding replication initiator protein A, 26 genes involved in CPS biosynthesis, 14 genes encoding transposases, 3 genes encoding MobA/MobL family proteins, and genes encoding hypothetical proteins (Fig. 3A). In addition, a real-time quantitative PCR (qPCR) assay showed that the copy number of plasmid pYC41 was 2 copies in each cell (Fig. S4). Compared with single-copy genes in the chromosome, the expression levels of plasmid-borne genes can be increased due to the higher copy number (22, 23).

FIG 3.

FIG 3

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Gene organizations of the plasmid pYC41-borne CPS biosynthetic gene cluster. (A) Physical map and gene organization of plasmid pYC41. (B) Physical map of the cpsYC41 gene cluster.

The cpsYC41 gene cluster was 22,074 bp in length and contained 26 putative ORFs (Fig. 3B). Based on amino acid sequence and protein structural similarity, putative functions were assigned to 26 genes within this gene cluster. Among them, the cpsA gene encoding an LCP domain-containing protein was assigned to regulation. The cpsB gene encoding a CpsD/CapB family tyrosine-protein kinase, the cpsC gene encoding a tyrosine-protein kinase transmembrane modulator, and the cpsD gene encoding a tyrosine-protein phosphatase were assigned chain-length control functions in CPS biosynthesis. The priming glycosyltransferase encoded by the P-gtf gene had a domain architecture similar to that of CpsE from Streptococcus agalactiae, which could transfer the first nucleotide sugar to a lipid carrier to initiate repeating-unit synthesis. The central region of the cpsYC41 gene cluster encoded 4 glycosyltransferases, CpsF, CpsG, CpsK, and CpsL; the flippase Wzx; transposases; and several hypothetical proteins. In addition, the rmlA gene encoding glucose-1-phosphate thymidylyltransferase, the rmlB gene encoding dTDP-4-dehydrorhamnose 3,5-epimerase, the rmlC gene encoding dTDP-glucose 4,6-dehydratase, and the rmlD gene encoding dTDP-4-dehydrorhamnose reductase were assigned to dTDP-rhamnose precursor biosynthesis. However, no gene encoding the polymerase Wzy was found in the cpsYC41 gene cluster. In fact, there was a gene encoding the polymerase Wzy in the cps2 gene cluster in the chromosome of L. plantarum YC41 (Fig. S3). The wzy gene encoded a protein with high identity (97.41%) to the polysaccharide polymerase of the cps4 gene cluster located in the chromosome of L. plantarum WCFS1. There may have been collaboration and communication of cps gene clusters in L. plantarum YC41 that contributed to capsular polysaccharide production. Notably, promoter and terminator predictions suggested that there were two operons in the cpsYC41 gene cluster. A reverse transcription-polymerase chain reaction (RT-PCR) assay with cDNA as the template further suggested that genes cpsA to cpsL formed a polycistronic operon (Fig. 4C and D). Moreover, rmlA, rmlC, rmlB, rmlD, and GM_3203 were cotranscribed and formed another operon in this gene cluster (Fig. 4A and B).

FIG 4.

FIG 4

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In silico analysis and RT-PCR assays to verify the cotranscription of the specific cpsYC41 gene cluster. (A) Linear map of the dTDP-rhamnose precursor biosynthesis genes. (B) RT-PCR assays to verify the cotranscription of rmlA to GM_3203. Lane M, DNA marker (2,000 bp); lanes 1, 4, 7, and 10, negative controls omitting the reverse transcriptase in the RT reaction; lanes 2, 5, 8, and 11, positive controls of PCR using genomic DNA as the template; lanes 3, 6, 9, and 12, test groups with the reverse transcriptase in the RT reaction. (C) Linear map of the repeating-unit biosynthesis genes. (D) RT-PCR assays to verify the cotranscription of cpsA to cpsL. Lane M, DNA marker (2,000 bp); lanes 1, 4, 7, 10, 13, 16, 19, and 22, negative controls omitting the reverse transcriptase in the RT reaction; lanes 2, 5, 8, 11, 14, 17, 20, and 23, positive controls of PCR using genomic DNA as the template; lanes 3, 6, 9, 12, 15, 18, 21, and 24, test groups with the reverse transcriptase in the RT reaction.

The cpsYC41 gene cluster was responsible for CPS biosynthesis.

In order to confirm the function of the cpsYC41 gene cluster in CPS biosynthesis, a small internal portion of the rmlA or cpsC gene was cloned into a suicide plasmid, pUC19E. Next, homologous recombination led to insertional mutations and a polar effect on the operons. The resulting mutants were verified by PCR using the specific primers listed in Table S2. Sequencing showed that the PCR product consisted of the expected fragments and the erythromycin (Em) resistance gene, indicating the correct integration of the recombinant plasmid into the chromosome via single-crossover homologous recombination. In addition, the YC41_GM003225-3226 genes were also inactivated to obtain the corresponding mutant as a control. The inactivation of YC41_GM003225-3226 had no significant effects on cell growth and CPS yields (Fig. S6).

During incubation in MRS broth, the YC41-rmlA− and YC41-cpsC− mutants could aggregate at the bottom of the tube, whereas the control strain YC41-control check (YC41-CK) remained in suspension (Fig. 5A). In addition, the colonies of YC41-rmlA− and YC41-cpsC− were not able to produce any strands when extended with an inoculation loop, while the colony of the control mutant had long strands (Fig. 5C). When fresh cultures grown overnight were mixed with India ink, clear halos could be observed around the cells of YC41-CK. However, no clear halos could be observed around the rmlA- and cpsC-deleted mutants, suggesting that the cpsYC41 gene cluster was involved in CPS biosynthesis (Fig. 6A). Moreover, transmission electron microscopy (TEM) images showed that the control mutant had a visible outer layer formed by polysaccharides, whereas this layer was absent in the rmlA- and cpsC-deleted mutants (Fig. 6B). Notably, YC41-CK produced 66.19 mg/L polysaccharides, while the strains with inactivated rmlA and cpsC significantly reduced the polysaccharide yields to 4.11 and 2.90 mg/L, respectively (Fig. 5B). These results indicated that the cpsYC41 gene cluster played a critical role in CPS biosynthesis and the ropy phenotype.

FIG 5.

FIG 5

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Functional analysis of the cpsYC41 gene cluster. (A) Phenotypes of YC41-CK, YC41-rmlA−, and YC41-cpsC− cultured for 20 h in MRS broth. (B) Yields of CPS in YC41-CK, YC41-rmlA−, and YC41-cpsC−. All results were obtained from at least three independent experiments. Data are reported as the means ± SD from three independent experiments (****, P < 0.001). (C) Detection of the ropy phenotype in YC41-CK, YC41-rmlA−, and YC41-cpsC− on MRS agar containing 2% (wt/vol) sucrose.

FIG 6.

FIG 6

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Functional analysis of the cpsYC41 gene cluster. (A) Light microscopy observations of CPS in YC41-CK, YC41-rmlA−, and YC41-cpsC− with India ink (magnification, ×1,000). (B) TEM images of YC41-CK, YC41-rmlA−, and YC41-cpsC−. CM, cell membrane; CW, cell wall.

The cpsYC41 gene cluster was associated with CPS biosynthesis in other L. plantarum strains.

In order to confirm the relationship between the cps gene cluster and CPS biosynthesis, the corresponding mutants were also obtained in other ropy L. plantarum strains such as MC2, PG1, and YD2. Compared with the wild-type strains, the cells of the corresponding rmlA- and cpsC-deleted mutants could aggregate after the stationary phase, and also, a clear halo could not be observed by capsule staining (Fig. 7A and B). In addition, the levels of CPS in rmlA- and cpsC-deleted mutants of other L. plantarum strains were also decreased by over 90% (P < 0.001) (Fig. 7C). These findings indicated that the cpsYC41 gene cluster was responsible for CPS biosynthesis in other ropy L. plantarum strains.

FIG 7.

FIG 7

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Further functional analysis of the cpsYC41 gene cluster. (A) Phenotypes of YC41-CK, YC41-rmlA−, and YC41-cpsC− cultured for 20 h in MRS broth. (B) Light microscopy observations of CPS in YC41-CK, YC41-rmlA−, and YC41-cpsC− stained with India ink (magnification, ×1,000). (C) Yields of CPS in YC41-CK, YC41-rmlA−, and YC41-cpsC−. All results were obtained from at least three independent experiments. Data are reported as the means ± SD from three independent experiments (****, P < 0.001).

CPS improved the resistance of YC41 to environmental stresses.

In order to investigate whether CPS could protect YC41 against environmental stresses, the survival capacity was measured by subjecting stationary-phase cells to low pH, NaCl, and H2O2. The results showed that the survival rate of YC41-CK was maintained at 101.90% at pH 3.0. However, the survival rates of YC41-rmlA− and YC41-cpsC− were only 11.65% and 6.79%, respectively (P < 0.001) (Fig. 8A). The survival rate of the control mutant reached 59.57% in the presence of 5% (wt/vol) NaCl, while the survival rates of the rmlA- and cpsC-deleted mutants were only 11.21 and 3.77%, respectively (P < 0.001) (Fig. 8B). Under 1.5 mM H2O2 stress, the survival rate of YC41-CK was 75.40%, which was ~2-fold higher than those of the rmlA- and cpsC-deleted mutants (P < 0.001) (Fig. 8C). These results showed that CPS could confer to L. plantarum YC41 enhanced tolerance to multiple environmental stresses.

FIG 8.

FIG 8

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Resistance provided by the cpsYC41 gene cluster. (A) Survival rates of YC41-CK, YC41-rmlA−, and YC41-cpsC− in MRS broth at pH 3.0. (B) Survival rates of YC41-CK, YC41-rmlA−, and YC41-cpsC− in MRS broth with 5% NaCl. (C) Survival rates of YC41-CK, YC41-rmlA−, and YC41-cpsC− in MRS broth with 1.5 mM H2O2. All results were obtained from at least three independent experiments. Data are reported as the means ± SD from three independent experiments (***, P < 0.001; ****, P < 0.001).

DISCUSSION

The complete genome sequencing of more than 180 L. plantarum strains enabled a better understanding of the gene clusters related to EPS biosynthesis. Four major EPS biosynthetic gene clusters have been identified in L. plantarum, which are designated cps1, cps2, cps3, and cps4. In the well-characterized L. plantarum strain WCFS1, four cps gene clusters are all present in the chromosomal DNA (18). In addition, some EPS biosynthesis gene clusters were located on mobile genetic elements such as plasmids in L. plantarum C410L1, HFC8, LZ227, ZJ102, and LTC-113 (16, 20, 21). Among these, only the molecular organization of the plasmid pYZ1-borne cpsWc gene cluster has been well characterized in L. plantarum LTC-113 (16). The cpsWc cluster was located in the 68.1-kb plasmid pYZ1 and contained 11 genes in an operon. Furthermore, this plasmid was confirmed to be responsible for CPS biosynthesis by plasmid-curing experiments (16). In this study, insertional inactivation experiments demonstrated that the cpsYC41 gene cluster, which contained the dTDP-rhamnose precursor biosynthesis operon and the repeating-unit biosynthesis operon, was responsible for CPS biosynthesis. Sequence alignment by BLAST Ring Image Generator v0.95 (BRIG) showed that pYC41 had 53% similarity with plasmid unnamed3 (GenBank accession no. CP024061.1) of L. plantarum strain KACC 92189, whereas the coverage was only 53% (see Fig. S1 in the supplemental material). These results indicated that plasmid pYC41 was a novel plasmid harboring CPS biosynthetic gene clusters in L. plantarum.

In the cpsYC41 gene cluster, the rmlACBD operon was highly conserved in some L. plantarum strains, which shared high nucleotide identity with rfbACBD in L. plantarum K25 plas3 (GenBank accession no. CP020096.1) (98.37%), L. plantarum 16 plasmid Lp16H (GenBank accession no. CP006041.1) (97.92%), and the L. plantarum G1 chromosome (GenBank accession no. CP053912.1) (98%), respectively (Fig. S2). Meanwhile, the cpsACBD operon showed 96.64% nucleotide identity with that in L. plantarum SKO-001 plasmid unnamed2 (GenBank accession no. CP040376.1) (Fig. S2). The gene encoding the flippase Wzx shared 99.44% nucleotide identity with the wzx gene in the chromosome of Limosilactobacillus fermentum HFD1 (GenBank accession no. CP050919.1) (Fig. S2). Moreover, the wzx gene was separated by two genes encoding transposases in the cpsYC41 gene cluster. Taken together, there were also some mobile genetic elements, including 14 genes encoding transposases and 3 genes encoding MobA/MobL family proteins, flanking or within the cpsYC41 gene cluster in pYC41. Therefore, the mosaic organization of the cpsYC41 gene cluster probably resulted from horizontal gene transfer and homologous recombination events.

Capsular polysaccharide is known to enhance bacterial tolerance to harsh environmental stresses, maintaining the viability of the bacteria during industrial manufacturing (12, 24). Traditional Chinese sauerkraut is generally harvested in 2% to 10% salt brines at pH 3.0 (25, 26). CPS with the presence of OH groups can hold water around cells and further prevent dehydration to cope with osmotic stress (12, 27). When exposed to 5 mol/L NaCl, Lactobacillus mucosae DPC 6426, which could produce EPS, had a 2-fold increase in survival compared with the non-EPS-producing strain DPC 6420 (28). Under acid stress, CPS carrying negatively charged groups like phosphate groups can prevent proton diffusion into cells by the negative charge on the cell surface (12). A CPS-producing strain, L. plantarum LTC-113, had a 1.4-fold increase in tolerance to an acidic environment compared to its non-CPS-producing mutant (16). Our results also showed that the CPS-producing strain exhibited 15.0- and 15.80-fold increases in tolerance compared to the non-CPS-producing strain in the presence of low acid and 5% NaCl, respectively. In addition, L. plantarum strains suffer from oxidative stress during industrial processes. CPS can also scavenge harmful reactive oxygen species to prevent cell damage (29). When exposed to 20 mM H2O2, the cell counts of an EPS-deficient mutant declined by over 90% (30). Similarly, the survival rate of the CPS-producing strain was >2-fold higher than those of the rmlA- and cpsC-deleted mutants under 1.5 mM H2O2 stress in this study. Taken together, CPS is an important protection mechanism for bacteria to cope with and resist harsh environmental conditions. In addition, the increased gene copy number might also result in greater overall CPS production to protect the YC41 strain from environmental stresses (Fig. S4).

MATERIALS AND METHODS

Isolation and identification of Lactobacillus strains.

A total of 95 samples isolated mainly from fermented food, fruit, and meat were collected and serially diluted 10-fold in sterile 0.85% (wt/vol) NaCl. An aliquot of 0.1 mL of each dilution was plated onto de Man-Rogosa-Sharpe (MRS) agar with 2% CaCO3 and incubated at 37°C for 24 to 48 h. Colonies that produced a clear zone on MRS agar with CaCO3 were subjected to Gram staining. The 16S rRNA genes of Gram-positive Lactobacillus cultures were amplified by colony PCR using the primers listed in Table S2 in the supplemental material. The PCR products were sequenced by Sangon Biotech (Beijing, China) and analyzed by nucleotide BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The strains with high similarity to the 16S rRNA sequence of the L. plantarum reference strain (E value of 0 and maximum identity of ≥99%) were regarded as L. plantarum strains. The L. plantarum strains were maintained in MRS broth with 50% sterile glycerol and stored at −80°C (Table S3).

Screening of CPS-producing L. plantarum strains.

Screening for the ability of L. plantarum isolates to produce CPS was performed according to methods described previously (31). Briefly, L. plantarum isolates were streaked onto MRS agar supplemented with 20 g/L sucrose and incubated at 37°C for 48 h. Colonies were dragged up using a sterile metal loop. If the length of the slime was more than 1.5 mm, the strains were considered CPS producers.

Genome sequencing and comparative analysis.

The total cellular DNA was extracted using an E.Z.N.A. bacterial DNA kit according to the manufacturer’s instructions (Omega Bio-Tek Inc., Doraville, GA, USA). The draft genomes of 21 L. plantarum strains and the complete genome of YC41 were sequenced using the PacBio Sequel platform and the Illumina NovaSeq PE150 platform. Gene functions were predicted by GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), and COG (Clusters of Orthologous Groups). Genome similarity analysis was carried out using OrthoANIu (available at https://www.ezbiocloud.net/tools/ani) (32). Comparative genomic analysis was conducted based on BLAST_VERSION+ bin analysis by BLAST Ring Image Generator v0.95 (BRIG) (33). Matches with <50% identity or regions with no BLAST matches appear as blank spaces in each ring. Accession numbers of the genomes deposited used in the present study are provided in Table S3. Functional annotation of the cluster region was carried out by combining the results of BLASTN, BLASTP, and CD-search of the Conserved Domain Database (CDD). Glycosyltransferases were predicted by the Carbohydrate-Active Enzymes database.

Validation of the dTDP-rhamnose precursor biosynthesis operon and repeating-unit biosynthesis operon by reverse transcription-PCR.

Total RNA was extracted using an RNA extraction kit (Tiangen, Beijing, China) according to the manufacturer’s instructions and digested with RNase-free DNase I to remove DNA. RNA concentrations were quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Subsequently, reverse transcription was carried out using a HiScript III RT SuperMix for qPCR kit (Vazyme Biotech, Nanjing, China), with 1 μg of the total RNA as the template. Standard PCR was carried out using Q5 high-fidelity DNA polymerase (New England BioLabs, Ipswich, MA), with cDNA as the template. To determine whether adjacent genes were cotranscribed, specific primers amplifying the intergenic region between genes were used, as listed in Table S2. PCR products were analyzed by electrophoresis on a 1% (wt/vol) agarose gel and verified by DNA sequencing.

Insertional inactivation of genes involved in CPS biosynthesis.

To study the role of the cps gene cluster in CPS biosynthesis, a series of mutants of L. plantarum YC41 was constructed using single-crossover homologous recombination, as shown in Fig. S5. Internal fragments of the target genes were amplified by PCR with the primers listed in Table S2 and inserted into the XbaI and KpnI sites of suicide plasmid pUC19EM (34). The recombinant plasmid was transformed into L. plantarum strains by electroporation. The insertional mutants were selected on MRS agar plates containing 10 μg/mL erythromycin (Em). To confirm integration at the correct locus, PCR was performed using the specific primers listed in Table S2, and the resulting amplicon was confirmed by sequencing. Meanwhile, the insertional inactivation of the YC41_GM003225-3226 genes encoding hypothetical proteins was carried out using the same procedure to construct a control strain. Bacterial strains and plasmids are listed in Table S1.

Growth assay and capsule staining of L. plantarum mutants.

Cultures of L. plantarum mutants and control strains grown overnight were inoculated (1%, vol/vol) into 50 mL MRS broth. Growth curves were determined by measuring the optical density at 600 nm or performing viable cell counts at 2-h intervals. For capsule staining, 10 μL of the fresh cultures grown overnight was mixed on a microscope slide with 10 μL of India ink and covered with a coverslip. The slide was examined using a Leica DM1000 microscope with an oil immersion lens at a ×1,000 magnification.

Transmission electron microscopy assay.

Cultures grown overnight were fixed in 2.5% glutaraldehyde at 4°C for 14 h as described previously (35). After fixation, the cells were rinsed in 0.1 M phosphate buffer (pH 7.4). The pellets were then postfixed in 2% osmium tetroxide at 4°C for 2 h, dehydrated in a graded alcohol series followed by acetone, and embedded in resin. Then, the resin was transferred into Leica EM UC7 to prepare sections. Sections were stained with uranyl acetate followed by lead citrate for 5 min each and examined using the JEM1200EX transmission electron microscope (JEOL, Japan) at 100 kV.

Isolation and quantification of CPS.

The extraction of CPS was carried out using a method reported previously (36), with minor modifications. Briefly, cultures grown overnight were centrifuged (8,000 × g for 20 min), washed twice, and then resuspended in sterile 1 mol/L NaCl. CPS was dissociated from the cells by sonication at 60 W for 5 min at 4°C. The proteins in the supernatant were precipitated by using 4% (vol/vol) trichloroacetic acid (TCA). The obtained supernatant was collected and precipitated with 75% ethanol at 4°C for 12 h, followed by centrifugation under the same conditions as the ones described above. The crude CPS was dialyzed with a membrane with a molecular weight cutoff of 14 kDa. The yield of CPS was estimated by the phenol-sulfuric acid method using glucose as a carbohydrate standard and expressed as milligrams per liter (37).

Survival assay of L. plantarum mutants under acid, oxidative, and osmotic stresses.

Five milliliters of cultures grown overnight was collected and centrifuged at 6,000 × g for 3 min. Cell pellets were collected and washed twice with sterile phosphate-buffered saline (PBS) buffer. For acid stress assays, cell pellets were resuspended in 5 mL MRS broth adjusted to pH 3.0 and incubated at 37°C for 3 h. For osmotic stress assays, cell pellets were resuspended in 5 mL MRS broth supplemented with 5% (wt/vol) NaCl and incubated at 37°C for 24 h. For oxidative stress assays, cell pellets were resuspended in 5 mL MRS broth with 1.5 mM H2O2 and incubated at 37°C for 30 min. Survival for each treatment was determined by 10-fold serial dilutions and enumeration on MRS plates with erythromycin. The survival rate was calculated by dividing the viable cell counts after treatment by the value obtained before treatment. Three independent biological replicates were performed in this study.

Determination of the relative plasmid copy number (PCN) by real-time PCR.

Amplification and detection were carried out in an ABI Step One Plus real-time PCR system (Applied Biosystems Inc.) using SYBR green qPCR master mix +genomic DNA (+gDNA) wiper (Vazyme Biotech, Nanjing, China). The alanine racemase gene (alr), a single-copy gene in the chromosome of L. plantarum WCFS1, was used as the reference gene (GenBank accession no. LP_RS02190 (Gene ID: 1061815)). A 219-bp fragment of the alr gene and a 147-bp fragment of the repA gene were separately amplified using the primers indicated in Table S2. The relative copy number of plasmid pYC41 was calculated as Nrelative = (1 + E)−ΔCT (38), where E and ΔCT represent the PCR amplification efficiency and the difference between the threshold cycle number (CT) of the alr reaction and that of the repA reaction, respectively.

Statistical analysis.

Data were analyzed using GraphPad Prism 6 software for Windows (GraphPad Software Inc., La Jolla, CA, USA). All data are reported as means ± standard deviations (SD). When two groups were compared, unpaired Student’s t test with Welch’s correction was used to calculate P values.

Data availability.

The complete genome of YC41 was deposited in GenBank under accession no. CP101640. The accession numbers of the 21 newly assembled draft genomes were deposited at GenBank under the following accession numbers: JANEZN000000000, JANEYV000000000, JANEYX000000000, JANEZL000000000, JANEZI000000000, JANEZK000000000, JANEZG000000000, JANEYY000000000, JANEZH000000000, JANEZJ000000000, JANEYZ000000000, JANEZC000000000, JANEZD000000000, JANEZE000000000, JANEZF000000000, JANEZB000000000, JANEZM000000000, JANEZA000000000, JANFNB000000000, JANFNC000000000, JANEYW000000000. The detail genomic features of the 21 draft genomes are listed in Table S3.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (no. 31972055).

Jieran An designed the study and wrote the manuscript. Jieran An, Yuchen Zhang, and Zhaoer Zhao performed the experiments. Jieran An analyzed and evaluated the data. Jieran An, Zhengyuan Zhai, and Yanling Hao revised the manuscript. All authors critically revised the manuscript and approved the final version to be published.

We 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.

Footnotes

Supplemental material is available online only.

Supplemental file 1
Supplemental material. Download spectrum.04150-22-s0001.pdf, PDF file, 0.8 MB (865.3KB, pdf)

Contributor Information

Zhengyuan Zhai, Email: zhaizy@cau.edu.cn.

Yanling Hao, Email: haoyl@cau.edu.cn.

Zhenjiang Zech Xu, 南昌大学.

REFERENCES

  • 1.Duar RM, Lin XB, Zheng J, Martino ME, Grenier T, Pérez-Muñoz ME, Leulier F, Gänzle M, Walter J. 2017. Lifestyles in transition: evolution and natural history of the genus Lactobacillus. FEMS Microbiol Rev 41:S27–S48. doi: 10.1093/femsre/fux030. [DOI] [PubMed] [Google Scholar]
  • 2.Kwaw E, Ma Y, Tchabo W, Apaliya MT, Wu M, Sackey AS, Xiao L, Tahir HE. 2018. Effect of Lactobacillus strains on phenolic profile, color attributes and antioxidant activities of lactic-acid-fermented mulberry juice. Food Chem 250:148–154. doi: 10.1016/j.foodchem.2018.01.009. [DOI] [PubMed] [Google Scholar]
  • 3.Mu L, Xie Z, Hu L, Chen G, Zhang Z. 2020. Cellulase interacts with Lactobacillus plantarum to affect chemical composition, bacterial communities, and aerobic stability in mixed silage of high-moisture amaranth and rice straw. Bioresour Technol 315:123772. doi: 10.1016/j.biortech.2020.123772. [DOI] [PubMed] [Google Scholar]
  • 4.Luan X, Feng M, Sun J. 2021. Effect of Lactobacillus plantarum on antioxidant activity in fermented sausage. Food Res Int 144:110351. doi: 10.1016/j.foodres.2021.110351. [DOI] [PubMed] [Google Scholar]
  • 5.Ye X, Huang D, Dong Z, Wang X, Ning M, Xia J, Shen S, Wu S, Shi Y, Wang J, Wan X. 2022. Fxr signaling-mediated bile acid metabolism is critical for alleviation of cholesterol gallstones by Lactobacillus strains. Microbiol Spectr 10:e00518-22. doi: 10.1128/spectrum.00518-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Liu Y, Xiao W, Yu L, Tian F, Wang G, Lu W, Narbad A, Chen W, Zhai Q. 2021. Evidence from comparative genomic analyses indicating that Lactobacillus-mediated irritable bowel syndrome alleviation is mediated by conjugated linoleic acid synthesis. Food Funct 12:1121–1134. doi: 10.1039/d0fo02616f. [DOI] [PubMed] [Google Scholar]
  • 7.Lin S-W, Tsai Y-S, Chen Y-L, Wang M-F, Chen C-C, Lin W-H, Fang TJ. 2021. Lactobacillus plantarum GKM3 promotes longevity, memory retention, and reduces brain oxidation stress in SAMP8 mice. Nutrients 13:2860. doi: 10.3390/nu13082860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Fast D, Duggal A, Foley E. 2018. Monoassociation with Lactobacillus plantarum disrupts intestinal homeostasis in adult Drosophila melanogaster. mBio 9:e01114-18. doi: 10.1128/mBio.01114-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Garcia-Gonzalez N, Battista N, Prete R, Corsetti A. 2021. Health-promoting role of Lactiplantibacillus plantarum isolated from fermented foods. Microorganisms 9:349. doi: 10.3390/microorganisms9020349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Whitfield C, Wear SS, Sande C. 2020. Assembly of bacterial capsular polysaccharides and exopolysaccharides. Annu Rev Microbiol 74:521–543. doi: 10.1146/annurev-micro-011420-075607. [DOI] [PubMed] [Google Scholar]
  • 11.Daba GM, Elnahas MO, Elkhateeb WA. 2021. Contributions of exopolysaccharides from lactic acid bacteria as biotechnological tools in food, pharmaceutical, and medical applications. Int J Biol Macromol 173:79–89. doi: 10.1016/j.ijbiomac.2021.01.110. [DOI] [PubMed] [Google Scholar]
  • 12.Nguyen P-T, Nguyen T-T, Bui D-C, Hong P-T, Hoang Q-K, Nguyen H-T. 2020. Exopolysaccharide production by lactic acid bacteria: the manipulation of environmental stresses for industrial applications. AIMS Microbiol 6:451–469. doi: 10.3934/microbiol.2020027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Nguyen P-T, Nguyen T-T, Vo T-N-T, Nguyen T-T-X, Hoang Q-K, Nguyen H-T. 2021. Response of Lactobacillus plantarum VAL6 to challenges of pH and sodium chloride stresses. Sci Rep 11:1301. doi: 10.1038/s41598-020-80634-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Stack HM, Kearney N, Stanton C, Fitzgerald GF, Ross RP. 2010. Association of beta-glucan endogenous production with increased stress tolerance of intestinal lactobacilli. Appl Environ Microbiol 76:500–507. doi: 10.1128/AEM.01524-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Dertli E, Mayer MJ, Narbad A. 2015. Impact of the exopolysaccharide layer on biofilms, adhesion and resistance to stress in Lactobacillus johnsonii FI9785. BMC Microbiol 15:8. doi: 10.1186/s12866-015-0347-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Gao Y, Niu M, Yu X, Bao T, Wu Z, Zhao X. 2021. Horizontally acquired polysaccharide-synthetic gene cluster from Weissella cibaria boosts the probiotic property of Lactiplantibacillus plantarum. Front Microbiol 12:692957. doi: 10.3389/fmicb.2021.692957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Lee IC, Caggianiello G, van Swam II, Taverne N, Meijerink M, Bron PA, Spano G, Kleerebezem M. 2016. Strain-specific features of extracellular polysaccharides and their impact on Lactobacillus plantarum-host interactions. Appl Environ Microbiol 82:3959–3970. doi: 10.1128/AEM.00306-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Remus DM, van Kranenburg R, van Swam II, Taverne N, Bongers RS, Wels M, Wells JM, Bron PA, Kleerebezem M. 2012. Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling. Microb Cell Fact 11:149. doi: 10.1186/1475-2859-11-149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Zeidan AA, Poulsen VK, Janzen T, Buldo P, Derkx PMF, Øregaard G, Neves AR. 2017. Polysaccharide production by lactic acid bacteria: from genes to industrial applications. FEMS Microbiol Rev 41:S168–S200. doi: 10.1093/femsre/fux017. [DOI] [PubMed] [Google Scholar]
  • 20.Jiang Y, Zhang J, Zhao X, Zhao W, Yu Z, Chen C, Yang Z. 2018. Complete genome sequencing of exopolysaccharide-producing Lactobacillus plantarum K25 provides genetic evidence for the probiotic functionality and cold endurance capacity of the strain. Biosci Biotechnol Biochem 82:1225–1233. doi: 10.1080/09168451.2018.1453293. [DOI] [PubMed] [Google Scholar]
  • 21.Jiang Y, Yang Z. 2018. A functional and genetic overview of exopolysaccharides produced by Lactobacillus plantarum. J Funct Foods 47:229–240. doi: 10.1016/j.jff.2018.05.060. [DOI] [Google Scholar]
  • 22.Nicoloff H, Hjort K, Levin BR, Andersson DI. 2019. The high prevalence of antibiotic heteroresistance in pathogenic bacteria is mainly caused by gene amplification. Nat Microbiol 4:504–514. doi: 10.1038/s41564-018-0342-0. [DOI] [PubMed] [Google Scholar]
  • 23.Rodríguez-Beltrán J, DelaFuente J, León-Sampedro R, MacLean RC, San Millán Á. 2021. Beyond horizontal gene transfer: the role of plasmids in bacterial evolution. Nat Rev Microbiol 19:347–359. doi: 10.1038/s41579-020-00497-1. [DOI] [PubMed] [Google Scholar]
  • 24.Caggianiello G, Kleerebezem M, Spano G. 2016. Exopolysaccharides produced by lactic acid bacteria: from health-promoting benefits to stress tolerance mechanisms. Appl Microbiol Biotechnol 100:3877–3886. doi: 10.1007/s00253-016-7471-2. [DOI] [PubMed] [Google Scholar]
  • 25.Wu R, Yu M, Liu X, Meng L, Wang Q, Xue Y, Wu J, Yue X. 2015. Changes in flavour and microbial diversity during natural fermentation of suan-cai, a traditional food made in Northeast China. Int J Food Microbiol 211:23–31. doi: 10.1016/j.ijfoodmicro.2015.06.028. [DOI] [PubMed] [Google Scholar]
  • 26.Xiong T, Li J, Liang F, Wang Y, Guan Q, Xie M. 2016. Effects of salt concentration on Chinese sauerkraut fermentation. LWT Food Sci Technol 69:169–174. doi: 10.1016/j.lwt.2015.12.057. [DOI] [Google Scholar]
  • 27.Ortega-Morales BO, López-Cortés A, Hernández-Duque G, Crassous P, Guezennec J. 2001. Extracellular polymers of microbial communities colonizing ancient limestone monuments. Methods Enzymol 336:331–339. doi: 10.1016/s0076-6879(01)36599-0. [DOI] [PubMed] [Google Scholar]
  • 28.London LE, Price NP, Ryan P, Wang L, Auty MA, Fitzgerald GF, Stanton C, Ross RP. 2014. Characterization of a bovine isolate Lactobacillus mucosae DPC 6426 which produces an exopolysaccharide composed predominantly of mannose residues. J Appl Microbiol 117:509–517. doi: 10.1111/jam.12542. [DOI] [PubMed] [Google Scholar]
  • 29.Zhang L, Liu C, Li D, Zhao Y, Zhang X, Zeng X, Yang Z, Li S. 2013. Antioxidant activity of an exopolysaccharide isolated from Lactobacillus plantarum C88. Int J Biol Macromol 54:270–275. doi: 10.1016/j.ijbiomac.2012.12.037. [DOI] [PubMed] [Google Scholar]
  • 30.Chen J, Lee SM, Mao Y. 2004. Protective effect of exopolysaccharide colanic acid of Escherichia coli O157:H7 to osmotic and oxidative stress. Int J Food Microbiol 93:281–286. doi: 10.1016/j.ijfoodmicro.2003.12.004. [DOI] [PubMed] [Google Scholar]
  • 31.Fguiri I, Ziadi M, Atigui M, Ayeb N, Arroum S, Assadi M, Khorchani T. 2016. Isolation and characterisation of lactic acid bacteria strains from raw camel milk for potential use in the production of fermented Tunisian dairy products. Int J Dairy Technol 69:103–113. doi: 10.1111/1471-0307.12226. [DOI] [Google Scholar]
  • 32.Gosselin S, Fullmer MS, Feng Y, Gogarten JP. 2022. Improving phylogenies based on average nucleotide identity, incorporating saturation correction and nonparametric bootstrap support. Syst Biol 71:396–409. doi: 10.1093/sysbio/syab060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ranjan A, Shaik S, Nandanwar N, Hussain A, Tiwari SK, Semmler T, Jadhav S, Wieler LH, Alam M, Colwell RR, Ahmed N. 2017. Comparative genomics of Escherichia coli isolated from skin and soft tissue and other extraintestinal infections. mBio 8:e01070-17. doi: 10.1128/mBio.01070-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ma X, Wang G, Zhai Z, Zhou P, Hao Y. 2018. Global transcriptomic analysis and function identification of malolactic enzyme pathway of Lactobacillus paracasei L9 in response to bile stress. Front Microbiol 9:1978. doi: 10.3389/fmicb.2018.01978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Álvarez B, Krogh-Andersen K, Tellgren-Roth C, Martínez N, Günaydın G, Lin Y, Martín MC, Álvarez MA, Hammarström L, Marcotte H. 2015. An exopolysaccharide-deficient mutant of Lactobacillus rhamnosus GG efficiently displays a protective llama antibody fragment against rotavirus on its surface. Appl Environ Microbiol 81:5784–5793. doi: 10.1128/AEM.00945-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Yang Z, Li S, Zhang X, Zeng X, Li D, Zhao Y, Zhang J. 2010. Capsular and slime-polysaccharide production by Lactobacillus rhamnosus JAAS8 isolated from Chinese sauerkraut: potential application in fermented milk products. J Biosci Bioeng 110:53–57. doi: 10.1016/j.jbiosc.2009.12.010. [DOI] [PubMed] [Google Scholar]
  • 37.Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. 1956. Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356. doi: 10.1021/ac60111a017. [DOI] [Google Scholar]
  • 38.Lee CL, Ow DSW, Oh SKW. 2006. Quantitative real-time polymerase chain reaction for determination of plasmid copy number in bacteria. J Microbiol Methods 65:258–267. doi: 10.1016/j.mimet.2005.07.019. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

Supplemental material. Download spectrum.04150-22-s0001.pdf, PDF file, 0.8 MB (865.3KB, pdf)

Data Availability Statement

The complete genome of YC41 was deposited in GenBank under accession no. CP101640. The accession numbers of the 21 newly assembled draft genomes were deposited at GenBank under the following accession numbers: JANEZN000000000, JANEYV000000000, JANEYX000000000, JANEZL000000000, JANEZI000000000, JANEZK000000000, JANEZG000000000, JANEYY000000000, JANEZH000000000, JANEZJ000000000, JANEYZ000000000, JANEZC000000000, JANEZD000000000, JANEZE000000000, JANEZF000000000, JANEZB000000000, JANEZM000000000, JANEZA000000000, JANFNB000000000, JANFNC000000000, JANEYW000000000. The detail genomic features of the 21 draft genomes are listed in Table S3.

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