Expression and Characterization of an Efficient Alginate Lyase from Psychromonas sp. SP041 through Metagenomics Analysis of Rotten Kelp.

Alginate is derived from brown algae, which can be cultivated in large quantities. It can be broken down by alginate lyase into alginate oligosaccharides (AOSs), which exhibit a higher added value and better bioactivity than alginate. In this study, metagenomic technology was used to screen for genes that code for high-efficiency alginate lyases. The candidate alginate lyase gene alg169 was detected from Psychromonas sp. SP041, the most abundant species among alginate lyase bacteria on selected rotten kelps. The alginate lyase Alg169 was heterologously expressed in Escherichia coli BL21 (DE3), Ni-IDA-purified, and characterized. The optimum temperature and pH of Alg169 were 25 °C and 7.0, respectively. Metal ions including Mn2+, Co2+, Ca2+, Mg2+, Ni2+, and Ba2+ led to significantly increased enzyme activity. Alg169 exhibited a pronounced dependence on Na+, and upon treatment with Mn2+, its activity surged by 687.57%, resulting in the highest observed enzyme activity of 117,081 U/mg. Bioinformatic analysis predicted that Alg169 would be a double-domain lyase with a molecular weight of 65.58 kDa. It is a bifunctional enzyme with substrate specificity to polyguluronic acid (polyG) and polymannuronic acid (polyM). These results suggest that Alg169 is a promising candidate for the efficient manufacturing of AOSs from brown seaweed.

CRISPR RNA-Guided Transposases Facilitate Dispensable Gene Study in Phage.

Phages provide a potential therapy for multi-drug-resistant (MDR) bacteria. However, a significant portion of viral genes often remains unknown, posing potential dangers. The identification of non-essential genes helps dissect and simplify phage genomes, but current methods have various limitations. In this study, we present an in vivo two-plasmid transposon insertion system to assess the importance of phage genes, which is based on the V. cholerae transposon Tn6677, encoding a nuclease-deficient type I-F CRISPR-Cas system. We first validated the system in Pseudomonas aeruginosa PAO1 and its phage S1. We then used the selection marker AcrVA1 to protect transposon-inserted phages from CRISPR-Cas12a and enriched the transposon-inserted phages. For a pool of selected 10 open-reading frames (2 known functional protein genes and 8 hypothetical protein genes) of phage S1, we identified 5 (2 known functional protein genes and 3 hypothetical protein genes) as indispensable genes and the remaining 5 (all hypothetical protein genes) as dispensable genes. This approach offers a convenient, site-specific method that does not depend on homologous arms and double-strand breaks (DSBs), holding promise for future applications across a broader range of phages and facilitating the identification of the importance of phage genes and the insertion of genetic cargos.

Fatty acid esters of hydroxy fatty acids: A potential treatment for obesity-related diseases.

Obesity, a burgeoning worldwide health system challenge, is associated with multiple chronic diseases, including diabetes and chronic inflammation. Fatty acid esters of hydroxy fatty acids (FAHFAs) are newly identified lipids with mitigating and anti-inflammatory effects in diabetes. Increasing work has shown that FAHFAs exert antioxidant activity and enhance autophagy in neuronal cells and cardiomyocytes. We systematically summarized the biological activities of FAHFAs, including their regulatory effects on diabetes and inflammation, antioxidant activity, and autophagy augmentation. Notably, the structure-activity relationships and potential biosynthesis of FAHFAs are thoroughly discussed. FAHFAs also showed potential roles as diagnostic biomarkers. FAHFAs are a class of resources with promising applications in the biomedical field that require in-depth research and hotspot development, as their structure has not been fully resolved and their biological activity has not been fully revealed.

Corrigendum: First report on the rapid detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) in viable but non-culturable (VBNC) under food storage conditions.

[This corrects the article DOI: 10.3389/fmicb.2020.615875.].

Microbial Interaction between Lactiplantibacillus plantarum and Saccharomyces cerevisiae: Transcriptome Level Mechanism of Cell-Cell Antagonism.

Lactiplantibacillus plantarum and Saccharomyces cerevisiae are frequently co-isolated in food, although playing different roles. This study aimed at investigating the microbial interaction between L. plantarum and S. cerevisiae, especially cell-cell direct interaction and their mechanism. Cell-cell and supernatant-cell coculture models were set up, with CFU counting, OD600 measurement, optical and atomic force microscopy performed to examine the growth and morphology of L. plantarum and S. cerevisiae cells. In cell-cell coculture model, L. plantarum cells inhibited S. cerevisiae growth (inhibition rate ~80%) with its own growth pattern unaffected. Cell-cell aggregation happened during coculture with surface roughness changed and partial S. cerevisiae cell lysis. Mature (24 h) L. plantarum cell-free culture supernatant showed inhibition (35%-75%) on S. cerevisiae growth independent of pH level, while supernatant from L. plantarum-S. cerevisiae coculture showed relatively stronger inhibition. Upon transcriptomics analysis, hypothesis on the mechanism of microbial interaction between L. plantarum and S. cerevisiae was demonstrated. When L. plantarum cell density reached threshold at 24 h, all genes in lamBDCA quorum sensing (QS) system was upregulated to potentially increase adhesion capability, leading to the aggregation to S. cerevisiae cell. The downregulation of whole basic physiological activity from DNA to RNA to protein, cell cycle, meiosis, and mitogen-activated protein kinase (MAPK) signaling pathways, as well as growth maintenance essential genes ari1, skg6, and kex2/gas1 might induce the decreased growth and proliferation rate and partial death of S. cerevisiae cells in coculture. IMPORTANCE L. plantarum and S. cerevisiae are frequently co-isolated in food, although playing different roles. The co-existence of L. plantarum and S. cerevisiae could result in variable effects, raising economic benefits and safety concerns in food industry. Previous research has reported the microbial interaction between L. plantarum and S. cerevisiae mainly rely on the signaling through extracellular metabolites. However, cell-cell aggregation has been observed with mechanism remain unknown. In the current study, the microbial interaction between L. plantarum and S. cerevisiae was investigated with emphasis on cell-cell direct interaction and further in-depth transcriptome level study showed the key role of lamBDCA quorum sensing system in L. plantarum. The results yield from this study demonstrated the antagonistic effect between L. plantarum and S. cerevisiae.

Genomic Iterative Replacements of Large Synthetic DNA Fragments in Corynebacterium glutamicum.

Synthetic genomics will advance our understanding of life and allow us to rebuild the genomes of industrial microorganisms for enhancing performances. Corynebacterium glutamicum, a Gram-positive bacterium, is an important industrial workhorse. However, its genome synthesis is impeded by the low efficiencies in DNA delivery and in genomic recombination/replacement. In the present study, we describe a genomic iterative replacement system based on RecET recombination for C. glutamicum, involving the successive integration of up to 10 kb DNA fragments obtained in vitro, and the transformants are selected by the alternative use of kanR and speR selectable markers. As a proof of concept, we systematically redesigned and replaced a 54.3 kb wild-type sequence of C. glutamicumATCC13032 with its 55.1 kb synthetic counterpart with several novel features, including decoupled genes, the standard PCRTags, and 20 loxPsym sites, which was for the first time incorporated into a bacterial genome. The resulting strain semi-synCG-A1 had a phenotype and fitness similar to the wild-type strain under various stress conditions. The stability of the synthetic genome region faithfully maintained over 100 generations of nonselective growth. Genomic deletions, inversions, and translocations occurred in the synthetic genome region upon induction of synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE), revealing potential genetic flexibility for C. glutamicum. This strategy can be used for the synthesis of a larger region of the genome and facilitate the endeavors for metabolic engineering and synthetic biology of C. glutamicum.

A strategy design based on antibiotic­resistance and plasmid replicons genes of clinical Escherichia coli strains.

Since antimicrobial resistance, especially ß-lactam resistance genes were common in clinical Escherichia coli strains, this study had designed and developed multiplex amplification platform for rapid and accurate detection of such resistance genes in 542 clinical E. coli isolates. The obtained specimens were subjected to bacteriological examination, antimicrobial susceptibility testing, and detection of ß-lactamase genes and plasmid replicons. The major virulence genes were detected by 7 groups of multiplex PCR and eight groups of multiplex PCR were designed to detect 8 different plasmid replicons including parA-parB, iteron, repA, and RNAI. It was found that most MDR isolates were co-resistant to penicillins (AMP) and fluoroquindones (LVX, CIP) and distribution of LVX and CIP resistance was significantly higher among female than male gender. RNAI (AY234375) showed the highest detection rate, followed by the iteron (J01724) and repA (M26308), indicating the relatively higher carriage rate of corresponding plasmids. BlaOXA acquired the highest carriage rate, followed by group 2 blaCTX-M and blaSHV-1, indicating their prevalence among clinical E. coli. Among the ß-lactamase genes, blaOXA acquired the highest carriage rate, followed by group 2 blaCTX-M and blaSHV-1, indicating their prevalence among clinical E. coli. The RNAI (AY234375) showed the highest detection rate, followed by the iteron (J01724) and repA (M26308), indicating the relatively higher carriage rate of the corresponding plasmids by clinical E. coli isolates. It is shown that the developed multiplex amplification methodology is applicable to AMR detection, and such identification of plasmid replicons and ß-lactamase genes may aid in the understanding of clinical E. coli isolate epidemiology.

Intense pulsed light for inactivating planktonic and biofilm molds in food.

It has been reported that about a quarter of the world's agriculture products is unable to be consumed each year because of mold contamination, resulting in incalculable economic losses. Despite modern food technology and the various preservation techniques available, the problem of mold contamination of food is still not adequately controlled. In this study, we simulated the biofilm formed by Aspergillus niger and Penicillium glaucum in liquid and solid food in 96 well cell culture plates and polycarbonate membrane models, respectively, and investigated the fungicidal effect of IPL on planktonic and biofilm molds at three different capacitance parameters at room and refrigerator temperatures. The results show that IPL can achieve fungicidal rates of over 99% for planktonic molds and over 90% for biofilm molds, and that the smaller the capacitance, the more frequent the irradiation required to achieve the same fungicidal rate. In addition, temperature, A. niger or Penicillium glaucum have no effect on the fungicidal effect of IPL. We believe that IPL is a promising non-thermal physical sterilization technique for fungal inhibition on food surfaces.

Significant downtrend of antimicrobial resistance rate and rare ß-lactamase genes and plasmid replicons carriage in clinical Pseudomonas aeruginosa in Southern China.

OBJECTIVES: Pseudomonas aeruginosa is a medically important pathogen showing intrinsic low permeability to various antimicrobial agents and its potential to acquire multiple resistance mechanism. A longitudinal surveillance aimed to investigate the antimicrobial resistance and its determinants of Pseudomonas aeruginosa in Southern China. A total of 2163 P. aeruginosa isolates were obtained from patients in Southern China during 2004-2016. METHODS: The antimicrobial susceptibility of the isolates was performed by disk diffusion and Vitek 2 automated system and interpreted according to the Clinical and Laboratory Standard Institute (CLSI) 2015. RESULTS: A significant downtrend of resistant rate (>10.0%) was observed for tested antibiotic agents including ciprofloxacin (>30.0%), gentamicin (29.0%), tobramycin (24.2%) and ceftazidime (24.0%) except for aztreonam and amikacin. A total of 269 randomly selected isolates were further studied on the carriage of ß-lactam resistance genes by using 7 groups of multiplex PCRs targeting on 20 genes. ß-lactam resistance genes were rarely detected with a rate lower than 8%. Among all ß-lactam resistance genes, blaSHV acquired the highest identification rate (18/269, 6.7%), followed by blaOXA-1-like (6/269, 2.2%) and blaPER (6/269, 2.2%). In addition, 8 different plasmid replicons were amplified using 8 groups of multiplex PCRs including 18 sets of primers. Only five plasmid replicons were identified in 5 different P. aeruginosa isolates. Insignificant clonal relatedness among the positive strains identified by regular PCR were further verified by randomly amplified polymorphic DNA (RAPD)-PCR. CONCLUSION: This study has provided comprehensive knowledge on current antimicrobial resistance, ß-lactam resistance genes and plasmid replicons carriage in a large scale of clinical P. aeruginosa isolates.

Development of a Direct and Rapid Detection Method for Viable but Non-culturable State of Pediococcus acidilactici.

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.

Efficient genome editing for Pseudomonas aeruginosa using CRISPR-Cas12a.

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.

Pathogenic and Virulence Factor Detection on Viable but Non-culturable Methicillin-Resistant Staphylococcus aureus.

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.

Impact of pmrA on Cronobacter sakazakii planktonic and biofilm cells: A comprehensive transcriptomic study.

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.

Direct Detection of Viable but Non-culturable (VBNC) Salmonella in Real Food System by a Rapid and Accurate PMA-CPA Technique.

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.

Polymicrobial interaction between Lactobacillus and Saccharomyces cerevisiae: coexistence-relevant mechanisms.

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.

Spy chemistry-enabled protein directional immobilization and protein purification.

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.

First Report on the Rapid Detection and Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) in Viable but Non-culturable (VBNC) Under Food Storage Conditions.

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.

Determination and optimization of a strong promoter element from Bacillus amyloliquefaciens by using a promoter probe vector.

OBJECTIVE: To construct a promoter probe vector, pBE-bgaB, to screen strong promoters from Bacillus amyloliquefaciens. RESULTS: 266 colonies containing active promoter elements from the genomic DNA of B. amyloliquefaciens were identified. Among these, promoter P41 exhibited the strongest ß-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 ß-Gal activity by ~ 200%. CONCLUSION: A new strong promoter for protein expression and genetic engineering of Bacillus species.

High level expression and characterization of tannase tan7 using Aspergillus niger SH-2 with low-background endogenous secretory proteins as the host.

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.

Optimization of the purine operon and energy generation in Bacillus amyloliquefaciens for guanosine production.

OBJECTIVES: 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. RESULTS: 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. CONCLUSION: The combined modification strategy used in this study is a novel approach to improve the production of guanosine in industrial bacterial strains.