SIRT3 Regulates Clearance of Apoptotic Cardiomyocytes by Deacetylating Frataxin.

BACKGROUND: Efferocytosis is an activity of macrophages that is pivotal for the resolution of inflammation in hypertension. The precise mechanism by which macrophages coordinate efferocytosis and internalize apoptotic cardiomyocytes remains unknown. The aim of this study was to determine whether SIRT3 (sirtuin-3) is required for both apoptotic cardiomyocyte engulfment and anti-inflammatory responses during efferocytosis. METHODS: We generated myeloid SIRT3 knockout mice and FXN (frataxin) knock-in mice carrying an acetylation-defective lysine to arginine K189R mutation (FXNK189R). The mice were given Ang II (angiotensin II) infusion for 7 days. We analyzed cardiac macrophages' mitochondrial iron levels, efferocytosis activity, and phenotype both in vivo and in vitro. RESULTS: We showed that SIRT3 deficiency exacerbated Ang II-induced downregulation of the efferocytosis receptor MerTK (c-Mer tyrosine kinase) and proinflammatory cytokine production, accompanied by disrupted mitochondrial iron homeostasis in cardiac macrophages. Quantitative acetylome analysis revealed that SIRT3 deacetylated FXN at lysine 189. Ang II attenuated SIRT3 activity and enhanced the acetylation level of FXNK189. Acetylated FXN further reduced the synthesis of ISCs (iron-sulfur clusters), resulting in mitochondrial iron accumulation. Phagocytic internalization of apoptotic cardiomyocytes increased myoglobin content, and derived iron ions promoted mitochondrial iron overload and lipid peroxidation. An iron chelator deferoxamine improved the levels of MerTK and efferocytosis, thereby attenuating proinflammatory macrophage activation. FXNK189R mice showed improved macrophage efferocytosis, reduced cardiac inflammation, and suppressed cardiac fibrosis. CONCLUSIONS: The SIRT3-FXN axis has the potential to resolve cardiac inflammation by increasing macrophage efferocytosis and anti-inflammatory activities.

Effect of composite organic amendment on Cd(II) ions stabilization and microbial activity under various ammonium sulfate levels.

To attenuate the risk of Cadmium(Cd) contamination and the deterioration of soil quality caused by excessive nitrogen fertilizer application in greenhouse, a composite organic amendment (spend mushroom substrate and its biochar) was prepared to remedy Cd(II) ions contaminated soil (0.6 mg/kg) under different N fertilizer levels. The results showed that in the absence of a composite organic amendment, the soil pH decreased by 0.15 when the N level increased from 0.1 to 0.8 g N⋅kg-1. However, the pH increased by 0.86-0.91, the exchangeable Cd(II) ions content decreased by 26.0%-26.7%, the microbial biomass increased by 34.34%-164.46%, and the number of copies of the AOB gene increased by 13-20 times with the application of composite organic amendment and the increase of N level. Both Pearson correlation analysis and Mantel test demonstrated the reduction in Cd(II) ions availability, the restoration of soil properties and the increase in microbial biomass all contributed to the composite organic amendment, which is of importance for soil remediation under excessive N fertilizer.

Mutations in VWA8 cause autosomal-dominant retinitis pigmentosa via aberrant mitophagy activation.

BACKGROUND: Although retinitis pigmentosa (RP) is the most common type of hereditary retinal dystrophy, approximately 25%-45% of cases remain without a molecular diagnosis. von Willebrand factor A domain containing 8 (VWA8) encodes a mitochondrial matrix-targeted protein; its molecular function and pathogenic mechanism in RP remain unexplained. METHODS: Family members of patients with RP underwent ophthalmic examinations, and peripheral blood samples were collected for exome sequencing, ophthalmic targeted sequencing panel and Sanger sequencing. The importance of VWA8 in retinal development was demonstrated by a zebrafish knockdown model and cellular and molecular analysis. RESULTS: This study recruited a Chinese family of 24 individuals with autosomal-dominant RP and conducted detailed ophthalmic examinations. Exome sequencing analysis of six patients revealed heterozygous variants in VWA8, namely, the missense variant c.3070G>A (p.Gly1024Arg) and nonsense c.4558C>T (p.Arg1520Ter). Furthermore, VWA8 expression was significantly decreased both at the mRNA and protein levels. The phenotypes of zebrafish with VWA8 knockdown are similar to those of clinical individuals harbouring VWA8 variants. Moreover, VWA8 defects led to severe mitochondrial damage, resulting in excessive mitophagy and the activation of apoptosis. CONCLUSIONS: VWA8 plays a significant role in retinal development and visual function. This finding may provide new insights into RP pathogenesis and potential genes for molecular diagnosis and targeted therapy.

CRISPR/Cas12a trans-cleavage mediated photoelectrochemical biosensor based on zeolitic imidazolate framework-67 for ATP determination.

An efficient PEC biosensor is proposed for ATP detection based on exciton energy transfer from CdTe quantum dots (CdTe QDs) to Au nanoparticles (AuNPs), integrating CRISPR/Cas12a trans-cleavage activity and specific recognition of ZIF-67 to ATP. Exciton energy transfer between CdTe QDs and AuNPs system is firstly constructed as photoelectrochemical (PEC) sensing substrate. Then, the activator DNAs, used to activate CRISPR/Cas12a, are absorbed on the surface of ZIF-67. In the presence of ATP, the activator DNAs are released due to more efficient adsorption of ZIF-67 to ATP. The released activator DNA activates trans-cleavage activity of CRISPR/Cas12a to degrade ssDNA on the electrode, leading to the recovery of photocurrent due to the interrupted energy transfer. Benefiting from the specific recognition of ZIF-67 to ATP and CRISPR/Cas12a-modulated amplification strategy, the sensor is endowed with excellent specificity and high sensitivity.

Light-harvesting iridium (III) complex-sensitized NiO photocathode for photoelectrochemical bioanalysis.

A novel iridium (III) complex bearing boron dipyrromethene (Bodipy) as the light-harvesting antenna has been synthesized and is firstly employed as photosensitizer to assemble a dye-sensitized NiO photocathode. The assembled photocathode exhibits significantly improved photoelectrochemical (PEC) performance. Integrating the prepared photocathode with hybridization chain reaction (HCR)--based signal amplification strategy, a cathodic PEC biosensor is proposed for the detection of microRNA-133a (miRNA-133a). In the presence of the target, HCR is triggered to form long duplex concatamers on the photocathode, which allows numerous manganese porphyrins (MnPP) to bind in the dsDNA groove. With the help of H2O2, MnPP with peroxidase-like activity catalyzes 4--chloro-1-naphthol (4-CN) to produce benzo--4--chlorohexadienone (4-CD) precipitate on the electrode, leading to a significant decrease of photocurrent signal. The decreased photocurrent correlates linearly with the target concentration from 0.1 fM to 1 nM with a detection limit of 66.2 aM (S/N = 3). The proposed PEC strategy exhibits delightful selectivity, reproducibility and stability.

Promoting Surface Reconstruction of Low-Cost Stainless Steel Catalyst for Efficient Oxygen Evolution Reaction.

The development of cost-effective transition metal catalysts for oxygen evolution reaction (OER) is critical for the production of hydrogen fuel from water splitting. Low-cost and efficient stainless steel-based catalysts are expected to replace the scarce platinum group metals for large-scale energy applications. Here in this work, we report the conversion of commonly available inexpensive and easily accessible 434-L stainless steel (SS) into highly active and stable electrodes by corrosion and sulfuration strategies. The Nix Fe1-x S layer as a pre-catalyst and S-doped Nix Fe oxyhydroxides in situ formed on the catalyst surface are the true active species for OER. The optimized 434-L stainless steel-based electrocatalyst exhibits a low overpotential of 298 mV at 10 mA cm-2 in 1.0 M KOH with a small OER kinetics (the Tafel slope of 54.8 mV dec-1 ) and good stability. This work reveals the 434-L alloy stainless steel with Fe and Cr as the main elements can be used as qualified OER catalysts by surface modification, along with a new mentality to solve the energy and resource waste problems.

Constructing Hierarchical Porous MoO2 @Mo2 N@C Composite via a Confined Pyrolysis Synthetic Strategy Towards Lithium-Ion Battery Anodes.

Molybdenum dioxide (MoO2 ) demonstrates a big potential toward lithium-ion storage due to its high theoretical capacity. The sluggish reaction kinetics and large volume change during cycling process, however, unavoidably lead to inferior electrochemical performance, thus failing to satisfy the requirements of practical applications. Herein, we developed a molybdenum-based oxyacid salt confined pyrolysis strategy to achieve a novel hierarchical porous MoO2 @Mo2 N@C composite. A two-step successive annealing process was proposed to obtain a hybrid phase of MoO2 and Mo2 N, which was used to further improve the electrochemical performance of MoO2 -based anode. We demonstrate that the well-dispersed MoO2 nanoparticles can ensure ample active sites exposure to the electrolyte, while conductive Mo2 N quantum dots afford pseudo-capacitive response, which conduces to the migration of ions and electrons. Additionally, the interior voids could provide buffer spaces to surmount the effect of volume change, thereby avoiding the fracture of MoO2 nanoparticles. Benefiting from the aforesaid synergies, the as-obtained MoO2 @Mo2 N@C electrode demonstrates a striking initial discharge capacity (1760.0 mAh g-1 at 0.1 A g-1 ) and decent long-term cycling stability (652.5 mAh g-1 at 1.0 A g-1 ). This work provides a new way for the construction of advanced anode materials for lithium-ion batteries.

Psychometric evaluation of the Chinese version of the Elderly-Constipation Impact Scale: a translation and validation study.

OBJECTIVE: The objective of this study was to translate the Elderly-Constipation Impact Scale into Chinese and to examine its reliability and validity in a population of older people suffering from chronic constipation. METHODS: In this study, the scale was paraphrased, back-translated, cross-culturally adapted and pre-experimented using the Brislin double translation-back-translation method to create the initial Chinese version of the Elderly-Constipation Impact Scale. A convenience sampling method was used to select 564 study participants who met the inclusion and exclusion criteria in Liaoning and Shanxi, China, to evaluate the reliability and validity of the scale. General information about the study population was using descriptive statistics; item analysis was used to screen the items of the scale. Content validity, exploratory factor analysis, and validation factor analysis were chosen to validate the scales; internal consistency, spilt-half reliability and retest reliability were used determine the reliability of the measurement scales. RESULTS: The Chinese version of the Elderly-Constipation Impact Scale contains 7 dimensions and 21 items. The Cronbach's alpha coefficient for the total scale was 0.901 and the range of Cronbach's alpha values for each dimension was 0.707 to 0.918. The split-half reliability of the scale was 0.736 and the retest reliability was 0.763. The exploratory factor analysis showed a KMO value of 0.873 and a Bartlett's spherical test X2 value of 3499.978 (p < 0.001). A total of seven common factors were extracted, namely daily activities, treatment satisfaction, lack of control of bodily function, diet restriction, symptom intensity, anxiety and preventive actions, with a cumulative variance contribution of 77.813%. Each item had a loading value > 0.4 on its common factor. In the validation factor analysis, the model fit results were X2 / df = 1.886, GFI = 0.910, AGFI = 0.874, PGFI = 0.654, IFI = 0.955, TLI = 0.942, CFI = 0.954, RMSEA = 0.056 and PNFI = 0.718. The model fit indicators were all within acceptable limits. CONCLUSION: The Chinese version of the E-CIS has good reliability and validity in the chronic constipation population of elderly individuals. The results of the questionnaire can effectively and comprehensively reflect the impact of chronic constipation on the quality of life of elderly individuals. It provides a meaningful reference for identifying targets for intervention.

Enhancing the biosynthesis of nicotinamide mononucleotide in Lactococcus lactis by heterologous expression of FtnadE.

BACKGROUND: Nicotinamide mononucleotide (NMN), a key intermediate of nicotinamide adenine dinucleotide, plays an important in anti-aging and disease. Lactococcus lactis, an important probiotic lactic acid bacteria (LAB), has shown great potential for the biosynthesis of NMN, which will significantly affect the probiotic effects of the dairy products. RESULTS: We used the CRISPR/nCas9 technique to knockout nadR gene of L. lactis NZ9000 to enhance the accumulation of NMN by 61%. The nadE* gene from Francisella tularensis with codon optimization was heterologous in L. lactis NZ9000ΔnadR and has a positive effect on NMN production. Combined with optimization of the concentration of substrate nicotinamide, a final intracellular NMN titer was 2289 µmol L-1  mg-1 with 10 g L-1 nicotinamide supplement, which was 5.7-fold higher than that of the control. The transcription levels of key genes (pncA, nadD and prs1) involved in NMN biosynthesis were up-regulated by more than two-fold, indicating that the increase of NMN titer was attributed to FtnadE* heterologous expression. CONCLUSION: Our study provides a better understanding of the NMN biosynthesis pathway in L. lactis, and can facilitate NMN production in LAB via synthetic biology approaches. © 2022 Society of Chemical Industry.

Psychometric evaluation of the Chinese version of the Positive Health Behaviours Scale for clinical nurses: a cross-sectional translation.

BACKGROUND: Occupational health is essential for nurses in clinical nursing practice. However, there is no specific tool for measuring the health behaviour of clinical nurses in China. This study aimed to translate the Positive Health Behaviours Scale into Chinese and validate its psychometric properties among clinical nurses. DESIGN: A cross-sectional design with repeated measures. METHODS: A total of 633 clinical nurses were recruited by convenience sampling from hospitals in Liaoning Province, China. After obtaining the authorization of the original author, the PHBS was translated into Chinese by the Brislin back-translation method. Item analysis was completed to evaluate item discrimination, and the Delphi method was adopted to analyse content validity. Exploratory factor analysis and confirmatory factor analysis were conducted to explore and validate the underlying factor structure. Internal consistency and test-retest reliability were calculated to evaluate reliability. RESULTS: A total of 29 items were retained in the item analysis, and the content validity index of the translated scale was 0.956. In the EFA, four common factors were extracted (nutrition, physical activity, relaxation and behaviours related to mental health and preventive behaviours), explaining 60.81% of the total variance. The results of the CFA were as follows: χ2/df = 1.363, GFI = 0.902, NFI = 0.909, IFI = 0.974, TLI = 0.971, CFI = 0.974, RMSEA = 0.034, and RMR = 0.023. The results of the EFA and CFA showed that the translated scale had good structural validity. Cronbach's α coefficient, the split-half reliability and the test-retest reliability of the Chinese version of the PHBS were 0.928, 0.953 and 0.891, respectively. At the same time, the translated scale had good reliability. CONCLUSIONS: The Chinese version of the PHBS for clinical nurses had good psychometric properties. The results of the questionnaire survey effectively and comprehensively reflect the level of health behaviours in clinical nurses, which provides a scientific reference for determining the intervention target.

Tuning C1 /C2 Selectivity of CO2 Electrochemical Reduction over in-Situ Evolved CuO/SnO2 Heterostructure.

Heterostructured oxides with versatile active sites, as a class of efficient catalysts for CO2 electrochemical reduction (CO2 ER), are prone to undergo structure reconstruction under working conditions, thus bringing challenges to understanding the reaction mechanism and rationally designing catalysts. Herein, we for the first time elucidate the structural reconstruction of CuO/SnO2 under electrochemical potentials and reveal the intrinsic relationship between CO2 ER product selectivity and the in situ evolved heterostructures. At -0.85 VRHE , the CuO/SnO2 evolves to Cu2 O/SnO2 with high selectivity to HCOOH (Faradaic efficiency of 54.81 %). Mostly interestingly, it is reconstructed to Cu/SnO2-x at -1.05 VRHE with significantly improved Faradaic efficiency to ethanol of 39.8 %. In situ Raman spectra and density functional theory (DFT) calculations reveal that the synergetic absorption of *COOH and *CHOCO intermediates at the interface of Cu/SnO2-x favors the formation of *CO and decreases the energy barrier of C-C coupling, leading to high selectivity to ethanol.

Transcriptomic and metabolomic analyses for providing insights into the influence of polylysine synthetase on the metabolism of Streptomyces albulus.

ε-poly-L-lysine (ε-PL) is the main secondary metabolite of Streptomyces albulus, and it is widely used in the food industry. Polylysine synthetase (Pls) is the last enzyme in the ε-PL biosynthetic pathway. Our previous study revealed that Pls overexpressed in S. albulus CICC11022 result in the efficient production of ε-PL. In this study, a Pls gene knockout strain was initially constructed. Then, genomic, transcriptomic and metabolomic approaches were integrated to study the effects of the high expression and knockout of Pls on the gene expression and metabolite synthesis of S. albulus. The high expression of Pls resulted in 598 significantly differentially expressed genes (DEGs) and 425 known differential metabolites, whereas the inactivation of Pls resulted in 868 significant DEGs and 374 known differential metabolites. The expressions of 8 and 35 genes were negatively and positively associated with the Pls expression, respectively. Subsequently, the influence mechanism of the high expression and inactivation of Pls on the ε-PL biosynthetic pathway was elucidated. Twelve metabolites with 30% decreased yield in the high-expression strain of Pls but 30% increased production in the Pls knockout strain were identified. These results demonstrate the influence of Pls on the metabolism of S. albulus. The present work can provide the theoretical basis for improving the production capacity of ε-PL by means of metabolic engineering or developing bioactive substances derived from S. albulus.

Novel selective κ agonists SLL-039 and SLL-1206 produce potent antinociception with fewer sedation and aversion.

SLL-039 (N-cyclopropylmethyl-7α-4'-(N'-benzoyl) amino-phenyl-6,14-endoethano-tetrahydronorthebaine) and SLL-1206 (N-cyclopropylmethyl-7α-3'-(p-methoxybenzyl) amino-phenyl-6,14-endoethano-tetrahydronorthebaine) are two 4,5-epoxymorphinan-based high selective κ receptor agonists that we recently discovered. In the present study we characterized their pharmacological properties in comparison with arylacetamide-based typical κ agonist U50,488H. We showed that both SLL-039 and SLL-1206 produced potent and long-lasting antinociceptive actions in three different rodent models of pain via activation of κ opioid receptor. In hot-plate assay, the antinociceptive potency of SLL-039 and SLL-1206 increased about 11-and 17.3-fold compared to U50,488H and morphine, respectively, with ED50 values of 0.4 mg/kg. Following repeated administration, SLL-1206, SLL-039, and U50,488H all developed analgesic tolerance tested in hot-plate assay. U50,488H and SLL-039 produced antipruritic effects in a dose-dependent manner, whereas SLL-1206 displayed some antipruritic effects only at very low doses. In addition, SLL-1206 was capable of decreasing morphine-induced physical dependence. More importantly, SLL-039 and SLL-1206 at effective analgesic doses did not cause sedation and conditioned place aversion (CPA), whereas U50,488H did. In comparison with SLL-039, SLL-1206 caused similar antinociceptive responses, but fewer sedation and CPA. In conclusion, our results suggest that SLL-039 and SLL-1206 have potential to be developed as novel analgesic agents, and 4,5-expoxymorphinan scaffold is an attractive structure for the development of selective κ agonists with fewer side effects.

Large scale application of pulse oximeter and auscultation in screening of neonatal congenital heart disease.

PURPOSE: To conduct a retrospective evaluation of a large clinical implementation of combined pulse oximeter (POX) and cardiac auscultation as a fast-screening device for congenital heart disease (CHD). METHODS: Every newborn in a large maternity healthcare center received auscultation and POX screening within 24 hours after delivery. When an abnormal heart murmur or SpO2 level was detected, an echocardiogram was ordered to confirm the diagnosis of CHD. RESULTS: From January 1, 2018 to December 31, 2019, there were 44,147 livebirths at the studied hospital where 498 suspected CHD were identified: 27 newborns by POX screening and 471 by cardiac auscultation. The diagnosis was further confirmed in 458 neonates through echocardiogram. This result put forth an overall diagnosis rate of 92.0%. Cardiac auscultation detected the majority of CHD cases 438 (95.6%) while POX only screened 20 (4.4%) cases. Interestingly, no CHD case was detected by both auscultation examination and POX screening. Auscultation detected most of the common types of CHD, but POX excelled in identifying rare and critical cases. POX screening alone had a very low accuracy of 74.07% in positive predict value (PPV). On the other hand, auscultation functioned well in terms of PPV and negative predict value (NPV) (92.99 and 99.95%, respectively), but the addition of POX improved the overall screening performance resulting in 100% NPV. We also validate the finding with the data 6 months after the study period. CONCLUSION: Our study demonstrated that addition of pulse oximetry to routine cardiac auscultation could be used as an accurate and feasible screening for early screening of CHD in newborns in large-scale clinical practice.

CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus.

Streptococcus thermophilus is used extensively in the dairy industry and has shown great promise as a chassis cell for the biosynthesis of high-value metabolites. However, metabolic engineering in S. thermophilus lacks effective genetic modification tools to modulate gene expression to relieve metabolic burden and maximize the production of desired compounds. Here, we developed a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system for efficient gene transcriptional modulation in S. thermophilus. Our CRISPRi system typically achieved 66 to 98% knockdown of single or multiple gene expression. We used CRISPRi for the biosynthesis of a new exopolysaccharide (EPS) as a paradigm model. Repression of galK at module of uridine diphosphate glucose sugar metabolism and overexpression of epsA and epsE at EPS synthesis module resulted in an approximately 2-fold increase in EPS titer (277 mg/L) when compared with a control strain. This study demonstrated the effectiveness of CRISPRi as a powerful metabolic engineering tool and synthetic biology strategy for S. thermophilus.

High-efficiency transformation of Streptococcus thermophilus using electroporation.

BACKGROUND: Streptococcus thermophilus, one of the most important lactic acid bacteria, is widely used in food fermentation, which is beneficial to improve food quality. However, the current genetic transformation systems are inefficient for S. thermophilus S-3, which hinders its further study. RESULTS: We developed three electroporation transformation methods for S. thermophilus S-3, and optimized various parameters to enhance the transformation efficiency up to 1.3 × 106 CFU/µg DNA, which was 32-fold higher than that of unoptimized. Additionally, transcriptional analysis showed that a series of competence genes in S. thermophilus S-3 were remarkedly up-regulated after optimization, indicating that improvement of transformation efficiency was attributed to the expression level of competence genes. Furthermore, to prove their potential, expression of competence genes (comEA, cbpD and comX) were employed to increase transformation efficiency. The maximum transformation efficiency was obtained by overexpression of comEA, which was 14-fold higher than that of control. CONCLUSION: This is the first report of competence gene expression for enhancing transformability in S. thermophilus, which exerts a positive effect on the development of desirable characteristics strains. © 2021 Society of Chemical Industry.

Integrative analysis and validation of dysregulated long non-coding RNAs in colon cancer.

It is an increasing evidence that long non-coding RNAs (lncRNAs) are involved in tumour initiation and progression. Here, we analysed RNA-sequencing data from the Cancer Genome Atlas (TCGA) datasets. Totally, 1176lncRNAs, 245miRNAs and 2081mRNAs were identified to be differentially expressed (DE) in colon cancer tissues compared with normal tissues. CASC21, a novel lncRNA located in 8q24.21 locus, was significantly overexpressed in 30 colon cancer tissues compared with matched normal tissues by qRT-PCR assay. CASC21 tended to higher expression as the increase of the tumour-node-metastasis (TNM) classification. Functionally, CASC21 promoted cell proliferation by regulating cell cycle and enhanced tumour metastasis by epithelial-mesenchymal transition (EMT) in colon cancer. Mechanism study indicated that CASC21 might be involved in activating WNT/ß-catenin pathway in colon cancer. In addition, we also built a competing endogenous RNA (ceRNNA) network by bioinformatic analysis using TCGA datasets. Together, our results not only provide novel lncRNAs as potential candidates for further study but also prove that CASC21 is an oncogenic regulator through activating WNT/ß-catenin signalling in colon cancer.

Short communication: An inducible CRISPR/dCas9 gene repression system in Lactococcus lactis.

Lactococcus lactis, one of the most important probiotic lactic acid bacteria (LAB), is widely used in the dairy industry as a cell factory for recombinant protein production. Currently, a nisin-controlled inducible expression system is used for this purpose and represents the only commercial expression system in LAB. However, the available genetic modification methods are rather limited for modulating gene expression in L. lactis. Here, we developed a 2-plasmid system for gene transcription repression in L. lactis NZ9000 that uses inducible clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9. An inducible promoter Pnisin was used to drive the expression of dCas9 from Streptococcus pyogenes, whereas a strong constitutive promoter P44 drove single guide RNA expression for single or multiple target genes. dCas9 enabled CRISPR interference-mediated silencing of single or multiple target genes with significant reduction of gene expression, up to 99%. In addition, LLNZ_07335, a putative penicillin acylase, was identified as bile salt hydrolase for bile salt resistance in NZ9000 using this system. To our knowledge, this report is the first for a functional gene for bile salt tolerance in L. lactis. Overall, our work introduces a new gene repression tool for various applications in L. lactis or other LAB.

Comparison of gal-lac operons in wild-type galactose-positive and -negative Streptococcus thermophilus by genomics and transcription analysis.

Streptococcus thermophilus is one of the most important homo-fermentative thermophilic bacteria, which is widely used as a starter culture in dairy industry. Both wild-type galactose-negative (Gal-) S. thermophilus AR333 and galactose-positive (Gal+) S. thermophilus S-3 in this study were isolated from Chinese traditional dairy products. Here, to access the mechanism of the difference of galactose utilization between strains AR333 and S-3, the expression of gal-lac operons was examined using real-time qPCR in the presence of different sugars, and the gene organization of gal-lac operons was characterized using comparative genomics analysis. As compared with medium containing glucose, the expression of gal-lac operons in AR333 and S-3 was significantly activated (> 5-fold) in the presence of galactose or lactose in the medium. More importantly, the expression of gal operon in S-3 was higher than that of AR333, suggesting that the strength of gal promoter in AR333 and S-3 may be different. The genomes of AR333 and S-3 were the first time sequenced to provide insight into the difference of gal-lac operons in these two strains. Comparative genomics analysis showed that gene order and individual gene size of gal-lac operons are conserved in AR333 and S-3. The DNA sequence of gal operon responsible for galactose utilization between AR333 and S-3 is almost identical except that galK promoter of S-3 possesses single base pair mutation (G to A substitution) at -9 box galK region. Moreover, the expression of red fluorescent protein can be activated by galK promoter of S-3, but cannot by galK promoter of AR333 in galactose medium, suggesting that gal operon is silent in AR333 and active in S-3 under galactose-containing medium. Overall, our results indicated that single point mutation at -9 box in the galK promoter can significantly affect the expression of gal operon and is largely responsible for the Gal+ phenotype of S. thermophilus.

Genomic and phenotypic analyses of exopolysaccharide biosynthesis in Streptococcus thermophilus S-3.

Streptococcus thermophilus, one of the most important industrial lactic acid bacteria, is widely used as a starter culture in the dairy industry. Streptococcus thermophilus S-3 isolated from Chinese traditional dairy products has shown great potential for the production of larger amounts of exopolysaccharides (EPS), which significantly affect the organoleptic properties of fermented milk products. To understand the relationship between the genotype and phenotype of S. thermophilus S-3 in terms of EPS biosynthesis, its genome of strain S-3 was sequenced and the genes related to carbohydrate utilization, nucleotide sugars synthesis, and EPS biosynthesis were investigated. The genomic analysis revealed that S. thermophilus S-3 can use sucrose, mannose, glucose, galactose, and lactose. Phenotypic analysis showed that S-3 prefers fermenting lactose to fermenting glucose or galactose. The genetic analysis of nucleotide sugars and EPS biosynthesis revealed that S-3 can synthesize uridine diphosphate (UDP)-glucose, deoxythymidine diphosphate-glucose, deoxythymidine diphosphate-rhamnose, UDP-galactose, UDP-N-acetylgalactosamine, and UDP-N-acetylglucosamine. A high yield of EPS from S-3 cultivated with lactose rather than glucose as the carbon source was correlated with high transcriptional levels of the genes associated with metabolism of these nucleotide sugars and EPS biosynthesis. Our results provide a better understanding of EPS biosynthesis in S. thermophilus and can facilitate enhanced EPS production by lactic acid bacteria fermentation via genetic and metabolic engineering approaches.