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The coordination of single or multiple microorganisms are required for the manufacture of traditional fermented foods, improving the flavour and nutrition of the food materials. However, both the additional economic benefits and safety concerns have been raised by microbiotas in fermented products. Among the fermented products, Lactobacillus and Saccharomyces cerevisiae are one of the stable microbiotas, suggesting their interaction is mediated by coexistence-relevant mechanisms and prevent to be excluded by other microbial species. Thus, aiming to guide the manufacture of fermented foods, this review will focus on interactions of coexistence-relevant mechanisms between Lactobacillus and S. cerevisiae, including metabolites communications, aggregation, and polymicrobial biofilm. Also, the molecular regulatory network of the coexistence-relevant mechanisms is discussed according to omics researches.

Pediococcus acidilactici may significantly reduce the pH-value, and thus has different influence, including serving as a probiotic in human microbiota but a spoilage in human food as it could change the flavor. Pediococcus acidilactici is also capable of entering into the viable but non-culturable (VBNC) state causing false negative results of standard culture-based detection method. Thus, development of detection method for VBNC state P. acidilactici is of great significance. In this study, propidium monoazide (PMA) combined with cross priming amplification (CPA) was developed to detect the VBNC cells of P. acidilactici and applied on the detection in different systems. With detection limit of 104 cells/ml, high sensitivity, and 100% specificity, PMA-CPA can successfully detect VBNC cells of P. acidilactici and be applied in with high robustness.

Food safety and foodborne infections and diseases have been a leading hotspot in public health, and methicillin-resistant Staphylococcus aureus (MRSA) has been recently documented to be an important foodborne pathogen, in addition to its recognition to be a leading clinical pathogen for some decades. Standard identification for MRSA has been commonly performed in both clinical settings and food routine detection; however, most of such so-called “standards,” “guidelines,” or “gold standards” are incapable of detecting viable but non-culturable (VBNC) cells. In this study, two major types of staphylococcal food poisoning (SFP), staphylococcal enterotoxins A (sea) and staphylococcal enterotoxins B (seb), as well as the panton-valentine leucocidin (pvl) genes, were selected to develop a cross-priming amplification (CPA) method. Limit of detection (LOD) of CPA for sea, seb, and pvl was 75, 107.5, and 85 ng/μl, indicating that the analytical sensitivity of CPA is significantly higher than that of conventional PCR. In addition, a rapid VBNC cells detection method, designated as PMA-CPA, was developed and further applied. PMA-CPA showed significant advantages when compared with PCR assays, in terms of rapidity, sensitivity, specificity, and accuracy. Compared with conventional VBNC confirmation methods, the PMA-CPA showed 100% accordance, which had demonstrated that the PMA-CPA assays were capable of detecting different toxins in MRSA in VBNC state. In conclusion, three CPA assays were developed on three important toxins for MRSA, and in combination with PMA, the PMA-CPA assay was capable of detecting virulent gene expression in MRSA in the VBNC state. Also, the above assays were further applied to real samples. As concluded, the PMA-CPA assay developed in this study was capable of detecting MRSA toxins in the VBNC state, representing first time the detection of toxins in the VBNC state.

Salmonella enterica is a typical foodborne pathogen with multiple toxic effects, including invasiveness, endotoxins, and enterotoxins. Viable but nonculturable (VBNC) is a type of dormant form preserving the vitality of microorganisms, but it cannot be cultured by traditional laboratory techniques. The aim of this study is to develop a propidium monoazide-crossing priming amplification (PMA-CPA) method that can successfully detect S. enterica rapidly with high sensitivity and can identify VBNC cells in food samples. Five primers (4s, 5a, 2a/1s, 2a, and 3a) were specially designed for recognizing the specific invA gene. The specificity of the CPA assay was tested by 20 different bacterial strains, including 2 standard S. enterica and 18 non-S. enterica bacteria strains covering Gram-negative and Gram-positive isolates. Except for the two standard S. enterica ATCC14028 and ATCC29629, all strains showed negative results. Moreover, PMA-CPA can detect the VBNC cells both in pure culture and three types of food samples with significant color change. In conclusion, the PMA-CPA assay was successfully applied on detecting S. enterica in VBNC state from food samples.

Formation of viable but non-culturable (VBNC) status in methicillin-resistant Staphylococcus aureus (MRSA) has never been reported, and it poses a significant concern for food safety. Thus, this study aimed to firstly develop a rapid, cost-effective, and efficient testing method to detect and differentiate MRSA strains in the VBNC state and further apply this in real food samples. Two targets were selected for detection of MRSA and toxin, and rapid isothermal amplification detection assays were developed based on cross-priming amplification methodology. VBNC formation was performed for MRSA strain in both pure culture and in artificially contaminated samples, then propidium monoazide (PMA) treatment was further conducted. Development, optimization, and evaluation of PMA-crossing priming amplification (CPA) were further performed on detection of MRSA in the VBNC state. Finally, application of PMA-CPA was further applied for detection on MRSA in the VBNC state in contaminated food samples. As concluded in this study, formation of the VBNC state in MRSA strains has been verified, then two PMA-CPA assays have been developed and applied to detect MRSA in the VBNC state from pure culture and food samples.

Site-directed protein immobilization allows the homogeneous orientation of proteins with high retention of activity, which is advantageous for many applications. Here, we report a facile, specific, and efficient strategy based on the SpyTag-SpyCatcher chemistry. Two SpyTag-fused model proteins, that is, the monomeric red fluorescent protein (RFP) and the oligomeric glutaryl-7-aminocephalosporanic acid acylase, were easily immobilized onto a SpyCatcher-modified resin directly from cell lysates, with activity recoveries in the range of 85-91%. This strategy was further adapted to protein purification, which proceeded through the selective capture of the SpyCatcher-fused target proteins by a SpyTag-modified resin, with the aid of an intein to generate authentic N-termini. For two model proteins, that is, RFP and a variable domain of a heavy chain antibody, the yields were ∼3-7 mg/L culture with >90% purities. This approach could provide a versatile tool for producing high-performance immobilized protein devices and proteins for industrial and therapeutic uses.

Cronobacter sakazakii is an emerging opportunistic foodborne pathogen causing rare but severe infections in neonates. Furthermore, the formation of biofilm allows C. sakazakii to persist in different environments. We have demonstrated that the mutator phenotype ascribed to deficiency of the pmrA gene results in more biomass in the first 24 h but less during the post maturation stage (7–14 d) compared with BAA 894. The present study aimed to investigate the regulatory mechanism modulating biofilm formation due to pmrA mutation. The transcriptomic analyses of BAA 894 and s-3 were performed by RNA-sequencing on planktonic and biofilm cells collected at different time points. According to the results, when comparing biofilm to planktonic cells, expression of genes encoding outer membrane proteins, lysozyme, etc. were up-regulated, with LysR family transcriptional regulators, periplasmic proteins, etc. down-regulated. During biofilm formation, cellulose synthase operon genes, flagella-related genes, etc. played essential roles in different stages. Remarkably, pmrA varies the expression of a number of genes related to motility, biofilm formation, and antimicrobial resistance, including srfB, virK, mviM encoding virulence factor, flgF, fliN, etc. encoding flagellar assembly, and marA, ramA, etc. encoding AraC family transcriptional regulators in C. sakazakii. This study provides valuable insights into transcriptional regulation of C. sakazakii pmrA mutant during biofilm formation.

The CRISPR-Cas12a system has been demonstrated as an attractive tool for bacterial genome engineering. In particular, FnCas12a recognizes protospacer-adjacent motif (PAM) sites with medium or low GC content, which complements the Cas9-based systems. Here we explored Francisella novicida Cas12a (FnCas12a) for genome editing in Pseudomonas aeruginosa. By using a two-plasmid system expressing the constitutive FnCas12a nuclease, the inducible λRed recombinase, a CRISPR RNA (crRNA), we achieved gene deletion, insertion and replacement with high efficiency (in most cases > 75%), including the deletion of large DNA fragments up to 15 kb and the serial deletion of duplicate gene clusters. This work should provide a useful and complementary addition to the genome engineering toolbox for the study of P. aeruginosa biology and physiology.

To deregulate the purine operon of the purine biosynthetic pathway and optimize energy generation of the respiratory chain to improve the yield of guanosine in Bacillus amyloliquefaciens XH7. The 5′-untranslated region of the purine operon, which contains the guanine-sensing riboswitch, was disrupted. The native promoter Pw in B. amyloliquefaciens XH7 was replaced by different strong promoters. Among the promoter replacement mutants, XH7purE::P41 gave the highest guanosine yield (16.3 g/l), with an increase of 23% compared with B. amyloliquefaciens XH7. The relative expression levels of the purine operon genes (purE, purF, and purD) in the XH7purE::P41 mutant were upregulated. The concentration of inosine monophosphate (IMP), the primary intermediate in the purine pathway, was also significantly increased in the XH7purE::P41 mutant. Combined modification of the low-coupling branched respiratory chains (cytochrome bd oxidase) improved guanosine production synergistically. The final guanosine yield in the XH7purE::P41△cyd mutant increased by 41% to 19 g/l compared with B. amyloliquefaciens XH7. The combined modification strategy used in this study is a novel approach to improve the production of guanosine in industrial bacterial strains.

To expand the repertoire of strong promoters for high level expression of proteins based on the transcriptome of Bacillus licheniformis. The transcriptome of B. licheniformis ATCC14580 grown to the early stationary phase was analyzed and the top 10 highly expressed genes/operons out of the 3959 genes and 1249 operons identified were chosen for study promoter activity. Using beta-galactosidase gene as a reporter, the candidate promoter pBL9 exhibited the strongest activity which was comparable to that of the widely used strong promoter p43. Furthermore, the pro-transglutaminase from Streptomyces mobaraensis (pro-MTG) was expressed under the control of promoter pBL9 and the activity of pro-MTG reached 82 U/ml after 36 h, which is 23% higher than that of promoter p43 (66.8 U/ml). In our analyses of the transcriptome of B. licheniformis, we have identified a strong promoter pBL9, which could be adapted for high level expression of proteins in the host Bacillus subtilis.

This study investigated the effect of the β2 receptor agonist terbutaline on the single channel activity of BKCa channel. The effects of racemate and two isomers of terbutaline were all assessed. β2 adrenoceptors were stably overexpressed on HEK293 cells by lentiviral transduction method and chicken BKCa channels were transiently expressed on normal HEK293 cell line or HEK293 cells overexpressing β2 receptors. Data showed that terbutaline significantly increased the single channel open probability of BKCa channel within 10min. The channel activating effects of terbutaline are stereoselective and mainly stay with the R-enantiomers. The opening probability of BKCa channel at 10min after drug application normalized to that just before drug application (Po10/Po0s) for R- and S-terbutaline were 7.85±3.20 and 1.06±0.45 respectively at 1μM concentration, corresponding to 28.37±9.96 and 2.68±1.09 at the higher concentration of 10μM. ICI 118551 blocked the effect of R- but not S-terbutaline (10μM), whereas atropine blocked the channel activating effects of S-terbutaline of higher concentration. In addition, the muscarinic receptor agonist carbachol increased the BKCa channel activity in an atropine-sensitive manner as an positive control experiment, which indicate the involvement of M receptor in the channel activating effect of S-terbutaline.

Tannin acyl hydrolase (tannase, EC3.1.1.20) catalyzes the hydrolysis of hydrolyzable tannins. It is used in the manufacture of instant tea and in the production of gallic acid. In this study, we reported that the overexpression, purification and characterization of an Aspergillus niger tannase. The tannase gene was cloned from A. niger SH-2 and expressed in the A. niger strain Bdel4 which is low-background of secreted proteins. The recombinant tannase was purified by desalting, followed by gel filtration for characterization. The tannase activity achieved 111.5 U/mL at 168 h, and the purity of the enzyme in the broth supernatant was estimated to be over 70%. The optimum temperature and pH of the recombinant tannase was ∼40 °C and 7.0, respectively. The tannase activity was inhibited by Mg2+, Ca2+, Cu2+, Ba2+, Ni2+ and EDTA, and was enhanced by Mn2+ and Co2+. Since A. niger is a GRAS microorganism, the recombinant tannase could be purification-free due to its high purity. The results of this study suggested that this recombinant strain could be subjected to large-scale production of A. niger tannase.

To construct a promoter probe vector, pBE-bgaB, to screen strong promoters from Bacillus amyloliquefaciens. 266 colonies containing active promoter elements from the genomic DNA of B. amyloliquefaciens were identified. Among these, promoter P41 exhibited the strongest β-Gal activity in Escherichia coli and B. amyloliquefaciens. Sequence analysis showed that promoter P41 contained P ykuN , a ykuN gene encoding flavodoxin. Optimization of the ribosome-binding site from P41 to P382 improved β-Gal activity by ~ 200%. A new strong promoter for protein expression and genetic engineering of Bacillus species.

Compared to classical antibodies, camel heavy chain antibodies (HCAbs) are smaller in size due to lack of the light chain and the first constant domain of the heavy chain (CH1 region). The variable regions of HCAbs (VHHs) are more soluble and stable than that of conventional antibodies (VHs). Even with such simple structure, they are still functional in antigen binding. Although HCAbs have been extensively investigated over the past two decades, most efforts have been based upon low throughput sequence analysis, and there are only limited reports trying to analyze and describe the complete immune repertoire (IR) of camel HCAbs. Here we leveraged the high-throughput data generated by Next Generation Sequencing (NGS) of the variable domains of the antibody heavy chains from three Bactrian camels to conduct in-depth comparative analyses of the immunoglobulin repertoire. These include analyses of the complementary determining region 3 (CDR3) length and distribution, mutation rate, antibody characteristic amino acids, the distribution of the cysteine (Cys) codons, and the non-classical VHHs. We found that there is higher diversity in the CDR2 than in the other sub-regions, and there is a higher mutation rate in the VHHs than in the VHs (P < 0.05). In addition to substitutions at amino acid (AA) residue positions NO.49/50/52 between VH and VHH clones, we also observed other substitutions at the positions NO.40/54/57/96/101 that could lead to additional structural alterations. We also found that VH-derived VHH clones, referred to as non-classical VHH clones in this study, accounted for about 8% of all clones. Further, only 5%-10% clones had the Trp > Arg AA substitution at the first position of framework 4 for all types of clones. We present, for the first time, a relatively complete picture of the Bactrian camel antibody immune repertoire, including conventional antibody (Ab) and HCAbs, using PCR and in silico analysis based on high-throughput NGS data.