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

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.

Unraveling the mechanism of raffinose utilization in Ligilactobacillus salivarius Ren by transcriptomic analysis.

In the gastrointestinal tract, some dietary carbohydrates, such as xylose, raffinose and arabinose, are able to stimulate the growth of Lactobacillus and Bifidobacterium. In this study, the growth rate of Ligilactobacillus salivarius Ren in raffinose was 0.91 ± 0.03 h-1, which was higher than that in glucose (0.83 ± 0.01 h-1). However, limited information is available on specific transporters and glycoside hydrolases responsible for raffinose uptake and catabolism in L. salivarius. Transcriptomic analysis revealed the differential expression of 236 genes (∣log2FoldChange∣ > 0.8) in response to raffinose, which were mainly associated with raffinose transport, raffinose hydrolysis, galactose metabolism and pyruvate metabolism. Notably, gene rafP encoding lactose/raffinose permease was 101.86-fold up-regulated. Two α-galactosidase gene galA1 and galA2 were 117.82-fold and 2.66-fold up-regulated, respectively. To further investigate the role of these genes in raffinose utilization, insertional inactivation was performed using the pORI28-pTRK669 system. The growth assay of these mutants in modified MRS containing 2% (w/v) raffinose indicated that RafP played an important role in raffinose transport and GalA1 was the primary enzyme involved in raffinose hydrolysis. To our knowledge, this is the first report on the molecular mechanism of raffinose utilization in L. salivarius. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03280-6.

L-Malic Acid Protects Lacticaseibacillus paracasei L9 from Glycodeoxycholic Acid Stress via the Malolactic Enzyme Pathway.

Bile stress tolerance is a crucial characteristic of probiotics for surviving in the human gastrointestinal tract. The mechanism underlying the effect of l-malic acid on enhancing the glycodeoxycholic acid (GDCA) tolerance of Lacticaseibacillus paracasei L9 was investigated herein. Bile tolerance specificity assays revealed that Lc. paracasei L9 was more sensitive to GDCA than to taurocholic acid, glycocholic acid, and taurodeoxycholic acid. Notably, l-malic acid significantly enhanced the GDCA tolerance of Lc. paracasei L9 by increasing the pH of the medium. The role of the malolactic enzyme pathway in enhancing GDCA resistance was investigated using molecular techniques. Confocal laser scanning and scanning electron microscopy revealed that l-malic acid preserved membrane permeability and cellular morphology, thereby protecting bacterial cells from GDCA stress-induced damage. The study also demonstrated that l-malic acid enhanced bile tolerance in different species of lactobacilli. These findings provide a novel protective mechanism for coping with bile stress in lactobacilli.

Transcriptome analysis revealed growth phase-associated changes of a centenarian-originated probiotic Bifidobacterium animalis subsp. lactis A6.

BACKGROUND: The physiology and application characteristics of probiotics are closely associated with the growth phase. Bifidobacterium animalis subsp. lactis A6 is a promising probiotic strain isolated from the feces of a healthy centenarian in China. In this study, RNA-seq was carried out to investigate the metabolic mechanism between the exponential and the stationary phase in B. lactis A6. RESULTS: Differential expression analysis showed that a total of 815 genes were significantly changed in the stationary phase compared to the exponential phase, which consisted of 399 up-regulated and 416 down-regulated genes. The results showed that the transport and metabolism of cellobiose, xylooligosaccharides and raffinose were enhanced at the stationary phase, which expanded carbon source utilizing profile to confront with glucose consumption. Meanwhile, genes involved in cysteine-cystathionine-cycle (CCC) pathway, glutamate dehydrogenase, branched-chain amino acids (BCAAs) biosynthesis, and Clp protease were all up-regulated in the stationary phase, which may enhance the acid tolerance of B. lactis A6 during stationary phase. Acid tolerance assay indicated that the survival rate of stationary phase cells was 51.07% after treatment by pH 3.0 for 2h, which was 730-fold higher than that of 0.07% with log phase cells. In addition, peptidoglycan biosynthesis was significantly repressed, which is comparable with the decreased growth rate during the stationary phase. Remarkably, a putative gene cluster encoding Tad pili was up-regulated by 6.5 to 12.1-fold, which is consistent with the significantly increased adhesion rate to mucin from 2.38% to 4.90% during the transition from the exponential phase to the stationary phase. CONCLUSIONS: This study reported growth phase-associated changes of B. lactis A6 during fermentation, including expanded carbon source utilizing profile, enhanced acid tolerance, and up-regulated Tad pili gene cluster responsible for bacterial adhesion in the stationary phase. These findings provide a novel insight into the growth phase associated characteristics in B. lactis A6 and provide valuable information for further application in the food industry.

Low concentrations of FA exhibits the Hormesis effect by affecting cell division and the Warburg effect.

Formaldehyde (FA), a ubiquitous indoor environmental pollutant, has been classified as a carcinogen. There are many studies showed that low levels of FA could promote cell proliferation, however, little is known about the signal pathways. To determine the potential molecular mechanisms, human chronic myeloid leukemia cells (K562 cells) and human bronchial epithelial cells (16HBE cells) were exposed to different concentrations of FA. The data showed that FA at 0-125 µM or 0-60 µM promoted the proliferation of K562 cells or 16HBE cells respectively, indicating that FA did have the Hormesis effect. FA at 75 µM (K562 cells) and 40 µM (16HBE cells) significantly promoted cell proliferation, increased intracellular reactive oxygen species (ROS) levels, and decreased glutathione (GSH) content. At the same time, FA treatment induced a marked increase in the key molecules of cell division like CyclinD-cdk4 and E2F1. In addition, pyruvate kinase isozyme M2 (PKM2), glucose, glucose transporter 1 (GLUT1), lactic acid and lactate dehydrogenase A (LDHA) content in the Warburg effect were increased. Administering Vitamin E (VE), significantly disrupted cell division and disturbed the Warburg effect, effectively indicating the decrease of cell activity. Conclusively, these findings suggested that low concentrations of FA could promote cell proliferation by accelerating cell division process or enhancing the Warburg effect to embody the Hormesis effect.

Illumination with 630 nm Red Light Reduces Oxidative Stress and Restores Memory by Photo-Activating Catalase and Formaldehyde Dehydrogenase in SAMP8 Mice.

AIMS: Pharmacological treatments for Alzheimer's disease (AD) have not resulted in desirable clinical efficacy over 100 years. Hydrogen peroxide (H2O2), a reactive and the most stable compound of reactive oxygen species, contributes to oxidative stress in AD patients. In this study, we designed a medical device to emit red light at 630 ± 15 nm from a light-emitting diode (LED-RL) and investigated whether the LED-RL reduces brain H2O2 levels and improves memory in senescence-accelerated prone 8 mouse (SAMP8) model of age-related dementia. RESULTS: We found that age-associated H2O2 directly inhibited formaldehyde dehydrogenase (FDH). FDH inactivity and semicarbazide-sensitive amine oxidase (SSAO) disorder resulted in endogenous formaldehyde (FA) accumulation. Unexpectedly, excess FA, in turn, caused acetylcholine (Ach) deficiency by inhibiting choline acetyltransferase (ChAT) activity in vitro and in vivo. Interestingly, the 630 nm red light can penetrate the skull and the abdomen with light penetration rates of ∼49% and ∼43%, respectively. Illumination with LED-RL markedly activated both catalase and FDH in the brains, cultured cells, and purified protein solutions, all reduced brain H2O2 and FA levels and restored brain Ach contents. Consequently, LED-RL not only prevented early-stage memory decline but also rescued late-stage memory deficits in SAMP8 mice. INNOVATION: We developed a phototherapeutic device with 630 nm red light, and this LED-RL reduced brain H2O2 levels and reversed age-related memory disorders. CONCLUSIONS: The phototherapy of LED-RL has low photo toxicity and high rate of tissue penetration and noninvasively reverses aging-associated cognitive decline. This finding opens a promising opportunity to translate LED-RL into clinical treatment for patients with dementia. Antioxid. Redox Signal. 00, 000-000.