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As a pathogen, Photobacterium damselae subsp. damselae (PDD) is distributed widely in the marine environment, and the host species of PDD are diverse. Although in the past decade the number of reports on the pathogenicity of PDD to marine animals has gradually increased, it is necessary to study this further in order to establish targeted prevention and technical control measures to reduce harm to marine organisms. In this study, a high-virulence PDD strain (PDD1608) was selected to explore the effect of the virulence gene dly on the biological characteristics and pathogenicity. The dly-deleted mutant strain Δdly PDD1608::Cm was successfully constructed using the λRed recombination technique. The biological characteristics of the wild-type and mutant strains were compared, including growth, swarming motility, drug susceptibility, physiological and biochemical characteristics, biofilm formation ability, and hemolytic and phospholipase activity of extracellular products (ECP). Meanwhile, Oryzias melastigma was used as the target host, and the pathogenicity of wild-type and mutant strains and their ECPs to O.melastigma was detected by the artificial infection test. There was a positive correlation between the results of the bacterial solution and ECP challenge, indicating that deletion of the dly gene affects the virulence of the PDD strain. Deletion of the virulence gene dly resulted in slower growth of the PDD mutant strain; reduced swarming and hemolytic and phospholipase activity; no change in the drug susceptibility or physiological and biochemical characteristics of the wild type and mutant strains; compared with the wild type strain, significant different (P < 0.05) biofilm formation ability of the mutant strain; the artificial infection test showed that the pathogenicity of the mutant strain and its ECP decreased. The results of this study revealed that the virulence gene dly affects many biological characteristics of PDD strains, such as growth, swarming motility, and hemolytic and phospholipase activity, and it was found to be closely related to the pathogenicity of the PDD strain and ECP.The results of this study contribute to further study of the influence of the virulence gene dly on the biological characteristics of the PDD strain in order to determine the appropriate prevention and control measures to curtail its spread and infection. The artificial infection test of the model organism O.melastigma helps provide an animal reference model for further exploration of the pathogenic mechanism and infection process of the PDD strain on the host. The results of the ECP experiments provide a theoretical reference for the research and development of PDD subunit vaccines.

ABSTRACT: Quinoa (Chenopodium quinoa) has earned special attention worldwide due to its higher nutritional value and its adaptive ability to contrasting environments. Here, it was explored how quinoa seed germination is regulated. CqNLP1 gene was identified and cloned due to its higher expression level in quinoa seeds. The role of CqNLP1 in seed germination was studied based on model species as Arabidopsis sp. The function of NO3 - during seed germination of quinoa was analyzed. The results showed CqNLP1 gene can restore the germination rate of Arabidopsis mutant nlp8-1 and nlp8-2 strains, suggesting that CqNLP1 gene plays an important role in promoting seed germination. Appropriate level of NO3 - could improve the germination rate of quinoa seeds, promote the decomposition and utilization of soluble protein and ABA, increase the expression level of CqNLP1 and CYP707A2 during germination. The optimal NO3 - concentration to promote seed germination is 1mM.

Porcine pseudorabies has long existed in China and is a serious threat to the Chinese farming industry. To understand the prevalence and genetic variation of the porcine pseudorabies virus (PRV) and its pathogenicity in Yunnan Province, China, we collected 560 serum samples across seven Yunnan Province regions from 2020 to 2021 and detected anti-gE antibodies in these samples. Sixty-one clinical tissue samples were also collected from pigs with suspected PRV that were vaccinated with Bartha-K61. PRV-gE antibodies were found in 29.6% (166/560) of the serum samples. The PRV positivity rate in clinical tissue samples was 13.1% (8/61). Two isolates, PRV-KM and PRV-QJ, were obtained. The identity of the gB, gD, and gE genes between these isolates and the Chinese mutants exceeded 99.5%. These isolates and the classical Fa strain were used to infect 4-week-old rats intranasally to assess their pathogenicity. All infected rats showed the typical clinical and pathological features of PRV two days post-infection. The viral loads in the organs differed significantly among the infected groups. Viruses were detected in the saliva and feces at 12 h. Significant dynamic changes in total white blood cell counts (WBC), lymphocyte counts (Lym), and neutrophil counts (Gran) occurred in the blood of the infected groups at 24 and 48 h. These results show that mutant PRV strains are prevalent in Bartha-K61-vaccinated pigs in Yunnan Province, China. Moreover, rats shed PRV in their saliva and feces during early infection, indicating the need for rodent control in combatting PRV infections in Yunnan Province, China. [ABSTRACT FROM AUTHOR]

为了进一步提高裂壶藻突变株的DHA产量，以经过60Co-γ射线辐照诱变后所得高产DHA裂壶藻突变株1.6-7-1为研究对象，通过Plackett-Burman实验、最陡爬坡实验和响应面实验对其发酵培养基进行优化，同时通过发酵罐发酵培养研究不同溶氧水平对突变株代谢的影响。结果表明：葡萄糖、C5H8NNaO4和NaCl添加量对该突变株产DHA具有显著影响，其最佳添加量分别为葡萄糖125.46 g/L、C5H8NNaO4 12.44 g/L、NaCl 4 g/L，在此条件下该突变株DHA产量达6.01 g/L，相较于原始菌株提升了49.88%；在高溶氧水平下，突变株生物量高但油脂产量和DHA产量较低，可能是因为细胞优先用营养物质进行自身的生长繁殖，而过低的溶氧水平会抑制能量代谢，减慢细胞繁殖速度。综上，优化发酵条件可以提高突变株DHA产量。 Abstract：In order to further increase the DHA yield of Schizochytrium sp. mutant, the Schizochytrium sp. mutant 1.6-7-1 with high yield DHA obtained by 60Co-γ radiation mutagenesis was used as research object, and the fermentation medium of DHA production by the Schizochytrium sp. mutant was further optimized by Plackett-Burman experiment, steepest ascent experiment and response surface methodology. In addition, the effects of different dissolved oxygen levels on the metabolism of the mutant strain were investigated through fermentation cultivation in fermentation tank. The results showed that the dosages of glucose, sodium glutamate and NaCl had significant effects on the DHA production of the mutant strain, and their optimum dosages were glucose 125.46 g/L, sodium glutamate 12.44 g/L and NaCl 4 g/L. Under the optimum conditions, the DHA production could reach 6.01 g/L, which increased by 49.88% compared with the original strain.Under high dissolved oxygen conditions, the mutant strain exhibited higher biomass accumulation but lower oil and DHA production, which possibly caused by the preferential use of nutrients for the own growth and reproduction of cells. Conversely, low dissolved oxygen inhibited energy metabolism and slowed down cell proliferation. Therefore, optimizing fermentation conditions can increase the DHA yield of the mutant strain.

Abstract Background Pseudorabies (PR) (also called Aujeszky’s disease, AD) is a serious infectious disease affecting pigs and other animals worldwide. The emergence of variant strains of pseudorabies virus (PRV) since 2011 has led to PR outbreaks in China and a vaccine that antigenically more closely matches these PRV variants could represent an added value to control these infections. Methods The objective of this study was to develop new live attenuated and subunit vaccines against PRV variant strains. Genomic alterations of vaccine strains were based on the highly virulent SD-2017 mutant strain and gene-deleted strains SD-2017ΔgE/gI and SD-2017ΔgE/gI/TK, which constructed using homologous recombination technology. PRV gB-DCpep (Dendritic cells targeting peptide) and PorB (the outer membrane pore proteins of N. meningitidis) proteins containing gp67 protein secretion signal peptide were expressed using the baculovirus system for the preparation of subunit vaccines. We used experimental animal rabbits to test immunogenicity to evaluate the effect of the newly constructed PR vaccines. Results Compared with the PRV-gB subunit vaccine and SD-2017ΔgE/gI inactivated vaccines, rabbits (n = 10) that were intramuscularly vaccinated with SD-2017ΔgE/gI/TK live attenuated vaccine and PRV-gB + PorB subunit vaccine showed significantly higher anti-PRV-specific antibodies as well as neutralizing antibodies and IFN-γ levels in serum. In addition, the SD-2017ΔgE/gI/TK live attenuated vaccine and PRV-gB + PorB subunit vaccine protected (90–100%) rabbits against homologous infection by the PRV variant strain. No obvious pathological damage was observed in these vaccinated rabbits. Conclusions The SD-2017ΔgE/gI/TK live attenuated vaccine provided 100% protection against PRV variant challenge. Interestingly, the subunit vaccines with gB protein linked to DCpep and PorB protein as adjuvant may also be a promising and effective PRV variant vaccine candidate.

Pectinases have broad applications in food industries attributed to their role as a biocatalyst in degrading pectic compounds. The present work reports the purification and characterization of a pectinase from a mutant strain of Bacillus subtilis. A three-step enzyme purification involving ammonium sulfate precipitation, followed by ion-exchange chromatography (IEC) and size-exclusion chromatography (SEC), was evaluated to recover the enzyme effectively. The pectinase was purified up to 7.98-fold with 65.63% total recovery, resulting in maximum specific activity of 362.04 U/mg after three purification steps. The eluted fractions of IEC and SEC showed pectinase activities of 9520 U and 8288 U, respectively. The molecular weight of this extracellular pectinase was found to be 30 kDa after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimal conditions of temperature and pH for maximum pectinase activity were 37 °C and 4–5, respectively. The enzyme also exhibited high stability when incubated at optimal conditions for 60 min. The addition of calcium and magnesium ions elicited pectinase activity at 4 mM concentrations, while copper and zinc ions inhibited the pectinase activity. The Vmax and Km values were 362 U and 0.62 mg/ml, respectively, for the pectin substrate obtained from citrus peel. Further analysis on the efficiency of clarification in sweet lime, pineapple, and lime juice showed 79.25, 72.76, and 61.24% reductions in turbidity within 1-h incubation. Therefore, characteristics of pectinase suggest that it can be potentially used for industrial applications as a biocatalyst. [ABSTRACT FROM AUTHOR]

The currently ongoing COVID-19 outbreak remains a global health concern. Understanding the transmission modes of COVID-19 can help develop more effective prevention and control strategies. In this study, we devise a two-strain nonlinear dynamical model with the purpose to shed light on the effect of multiple factors on the outbreak of the epidemic. Our targeted model incorporates the simultaneous transmission of the mutant strain and wild strain, environmental transmission and the implementation of vaccination, in the context of shortage of essential medical resources. By using the nonlinear least-square method, the model is validated based on the daily case data of the second COVID-19 wave in India, which has triggered a heavy load of confirmed cases. We present the formula for the effective reproduction number and give an estimate of it over the time. By conducting Latin Hyperbolic Sampling (LHS), evaluating the partial rank correlation coefficients (PRCCs) and other sensitivity analysis, we have found that increasing the transmission probability in contact with the mutant strain, the proportion of infecteds with mutant strain, the ratio of probability of the vaccinated individuals being infected, or the indirect transmission rate, all could aggravate the outbreak by raising the total number of deaths. We also found that increasing the recovery rate of those infecteds with mutant strain while decreasing their disease-induced death rate, or raising the vaccination rate, both could alleviate the outbreak by reducing the deaths. Our results demonstrate that reducing the prevalence of the mutant strain, improving the clearance of the virus in the environment, and strengthening the ability to treat infected individuals are critical to mitigate and control the spread of COVID-19, especially in the resource-constrained regions.

Background: Pseudorabies (PR) (also called Aujeszky's disease, AD) is a serious infectious disease affecting pigs and other animals worldwide. The emergence of variant strains of pseudorabies virus (PRV) since 2011 has led to PR outbreaks in China and a vaccine that antigenically more closely matches these PRV variants could represent an added value to control these infections. Methods: The objective of this study was to develop new live attenuated and subunit vaccines against PRV variant strains. Genomic alterations of vaccine strains were based on the highly virulent SD-2017 mutant strain and gene-deleted strains SD-2017ΔgE/gI and SD-2017ΔgE/gI/TK, which constructed using homologous recombination technology. PRV gB-DCpep (Dendritic cells targeting peptide) and PorB (the outer membrane pore proteins of N. meningitidis) proteins containing gp67 protein secretion signal peptide were expressed using the baculovirus system for the preparation of subunit vaccines. We used experimental animal rabbits to test immunogenicity to evaluate the effect of the newly constructed PR vaccines. Results: Compared with the PRV-gB subunit vaccine and SD-2017ΔgE/gI inactivated vaccines, rabbits (n = 10) that were intramuscularly vaccinated with SD-2017ΔgE/gI/TK live attenuated vaccine and PRV-gB + PorB subunit vaccine showed significantly higher anti-PRV-specific antibodies as well as neutralizing antibodies and IFN-γ levels in serum. In addition, the SD-2017ΔgE/gI/TK live attenuated vaccine and PRV-gB + PorB subunit vaccine protected (90–100%) rabbits against homologous infection by the PRV variant strain. No obvious pathological damage was observed in these vaccinated rabbits. Conclusions: The SD-2017ΔgE/gI/TK live attenuated vaccine provided 100% protection against PRV variant challenge. Interestingly, the subunit vaccines with gB protein linked to DCpep and PorB protein as adjuvant may also be a promising and effective PRV variant vaccine candidate. [ABSTRACT FROM AUTHOR]

Porcine pseudorabies has long existed in China and is a serious threat to the Chinese farming industry. To understand the prevalence and genetic variation of the porcine pseudorabies virus (PRV) and its pathogenicity in Yunnan Province, China, we collected 560 serum samples across seven Yunnan Province regions from 2020 to 2021 and detected anti-gE antibodies in these samples. Sixty-one clinical tissue samples were also collected from pigs with suspected PRV that were vaccinated with Bartha-K61. PRV-gE antibodies were found in 29.6% (166/560) of the serum samples. The PRV positivity rate in clinical tissue samples was 13.1% (8/61). Two isolates, PRV-KM and PRV-QJ, were obtained. The identity of the gB, gD, and gE genes between these isolates and the Chinese mutants exceeded 99.5%. These isolates and the classical Fa strain were used to infect 4-week-old rats intranasally to assess their pathogenicity. All infected rats showed the typical clinical and pathological features of PRV two days post-infection. The viral loads in the organs differed significantly among the infected groups. Viruses were detected in the saliva and feces at 12 h. Significant dynamic changes in total white blood cell counts (WBC), lymphocyte counts (Lym), and neutrophil counts (Gran) occurred in the blood of the infected groups at 24 and 48 h. These results show that mutant PRV strains are prevalent in Bartha-K61-vaccinated pigs in Yunnan Province, China. Moreover, rats shed PRV in their saliva and feces during early infection, indicating the need for rodent control in combatting PRV infections in Yunnan Province, China.

Chrysomycin A (CA), a promising antibiotic agent, usually coexists with two analog chrysomycins B (CB) and C (CC) produced by several wild-type (WT) Streptomyces strains. With the aim to increase CA production, UV mutagenesis-based breeding had been employed on a marine-derived strain Streptomyces sp. 891 in our previous study and afforded an improved strain 891-B6 with enhanced CA yield. By comparative transcriptome analysis, significant differences in chrysomycin BGC-related gene expression between the WT strain 891 and the mutant strain 891-B6 were unveiled in the current study. Among 25 up-regulated genes in mutant 891-B6, chryA, chryB, chryC, chryF, chryG, chryK, chryP, and chryQ, responsible for the biosynthesis of benzonaphthopyranone aglycone, and chryD, chryE, and chryU in charge of production of its deoxyglycoside, were characterized. Furthermore, the expression of genes chryOII, chryOIII, and chryOIV responsible for the formation of 8-vinyl in CA from 8-ethyl in CB were greatly enhanced in strain 891-B6. These findings provide molecular mechanisms for increased yield of CA and decreased yield of CB for mutant 891-B6, which has potential application in industrial CA production. [ABSTRACT FROM AUTHOR]

Pectin lyase has several applications in different industrial areas. The enzyme has been produced by batch fermentation while the production of mutant pectin lyase has been improved by using carrot-koji fermentation. The present work deals with the stimulation in pectin lyase activity by a UV-irradiated Aspergillus oryzae mutant-auxotroph under carrot-koji process. The physical mutagenesis was induced using ultraviolet radiations. The effect of different distance from UV source (5-30 cm) and different exposure time (10-60 min) was investigated. The final mutant derivative UV-t30 was able to produce 9.26 U/ml of pectin lyase which was significantly higher than the wild-type. Two stage submerged fermentation was carried out while using carrot peelings as a substrate. The wild-type ISL-9 and mutant strain UV-t30 of A. oryzae showed the highest production by using 2 and 1.5 g carrot peelings, respectively 48 h after incubation (seeded with 8% inoculum). The enzyme was activated by CaCl2 and (NH4)2SO4 whereas it was inhibited in the presence of Tween-80. The enzyme was further activated by mutant strain UV-t30 while inhibited by wild-type ISL-9 using KNO3. After optimization of parameters for enzyme activity, the potent mutant showed a 1.3-fold increase in the enzyme activity as compared to the wild-type. The study proved that carrot peel has nutrients which enabled A. oryzae to produce pectin lyase in koji process. [ABSTRACT FROM AUTHOR]

Global warming poses new challenges for insects to adapt to higher temperatures. Trehalose is the main blood sugar in insects and plays an important role in energy metabolism and stress resistance. The transmembrane transport of trehalose mainly depends on the trehalose transporter (TRET1). Plutella xylostella (L.) is a worldwide agricultural pest; however, the effects of the trehalose transport mechanism and trehalose distribution in tissues on the development, reproduction and temperature adaptation of P. xylostella have yet to be reported. In this study, PxTret1-like was cloned and analyzed regarding its expression pattern. It was found that the expression of PxTret1-like was affected by ambient temperature. The knockout mutation of PxTret1-like was generated using a CRISPR/Cas9 system by targeted knockout. The trehalose content and trehalase activity of mutant P. xylostella increased at different developmental stages. The trehalose content increased in the fat body of the fourth-instar P. xylostella, and decreased in the hemolymph, and there was no significant change in glucose in the fat body and hemolymph. Mutant strains of P. xylostella showed a significantly reduced survival rate, fecundity and ability to withstand extreme temperatures. The results showed that PxTret1-like could affect the development, reproduction and temperature adaptability of P. xylostella by regulating the trehalose content in the fat body and hemolymph. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) was caused by a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 utilizes human angiotensin converting enzyme 2 (hACE2) as the cellular receptor of its spike glycoprotein (SP) to gain entry into cells. Consequently, we focused on the potential of repurposing clinically available drugs to block the binding of SARSCoV-2 to hACE2 by utilizing a novel artificial-intelligence drug screening approach. Based on the structure of S-RBD and hACE2, the pharmacophore of SARS-CoV-2-receptor-binding-domain (S-RBD) -hACE2 interface was generated and used to screen a library of FDA-approved drugs. A total of 20 drugs were retrieved as S-RBD-hACE2 inhibitors, of which 16 drugs were identified to bind to S-RBD or hACE2. Notably, tannic acid was validated to interfere with the binding of S-RBD to hACE2, thereby inhibited pseudotyped SARS-CoV-2 entry. Experiments involving competitive inhibition revealed that tannic acid competes with S-RBD and hACE2, whereas molecular docking proved that tannic acid interacts with the essential residues of S-RBD and hACE2. Based on the known antiviral activity and our findings, tannic acid might serve as a promising candidate for preventing and treating SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) nucleic acid detection is the gold standard for the laboratory diagnosis of coronavirus disease 2019 (COVID‐19). However, this method has high requirements for practitioners' skills and testing sites, so it is not easy to popularize and promote the application in places other than large hospitals. In addition, the detection flux of SARS‐CoV‐2 nucleic acid is small, and the whole detection process takes much time, which cannot meet the actual needs of rapid screening in large quantities. The WHO conditionally approved a batch of SARS‐CoV‐2 antigen reagents for clinical application to alleviate this contradiction. SARS‐CoV‐2 antigen detection offers a trade‐off among clinical performance, speed and accessibility. With the gradual increase in clinical application, the accumulated clinical data show that the sensitivity and specificity of the SARS‐CoV‐2 antigen assay are over 80% and 97%, respectively, which can basically meet the requirements of the WHO. However, the sensitivity of the SARS‐CoV‐2 Antigen Assay among asymptomatic people in low prevalence areas of COVID‐19 cannot meet the standard, leading to a large number of missed diagnoses. In addition, the detection ability of SARS‐CoV‐2 antigen reagent for different SARS‐CoV‐2 mutant strains differs greatly, especially for those escaping the COVID‐19 vaccines. In terms of results interpretation, it is highly reliable to exclude SARS‐CoV‐2 infection based on the high negative predictive value of the SARS‐CoV‐2 antigen assay. However, in the low prevalence environment, the probability of false positives of the SARS‐CoV‐2 antigen assay is high, so the positive results need to be confirmed by the SARS‐CoV‐2 nucleic acid reagent. The SARS‐CoV‐2 antigen assay is only a supplement to SARS‐CoV‐2 nucleic acid detection and can never completely replace it. To date, SARS‐CoV‐2 nucleic acid detection continues to be the standard laboratory method for COVID‐19 diagnosis. [ABSTRACT FROM AUTHOR]

Background: The fusion protein H65, composed of Mycobacterium tuberculosis (TB) ESX-secreted antigens, has improved the bacillus Calmette–Guerin-induced immune protection in a mouse model of bovine TB when formulated in the liposomal adjuvant CAF01. In this study, we aimed to evaluate the protective efficacy of an attenuated Mycobacterium bovis strain – a mutant in mce2 and phoP genes – combined with H65+CAF01 immunization. We evaluated the protection of MbΔmce2-phoP alone or combined with H65+CAF01 against M. bovis challenge in mice. Methods: Groups of BALBc mice were inoculated with the vaccine candidates or phosphate buffered saline (PBS), and 6 weeks after the last immunization, the animals were aerogenically challenged with virulent M. bovis. Bacterial load in organs was counted after 45 days of the challenge. One-way analysis of variance and Bonferroni's posttest were used for statistical analysis. Results: All vaccinated mice showed reduced bacterial loads in lungs compared to unvaccinated animals. However, the protection level was similar between vaccinated groups. Conclusions: The MbΔmce2-phoP strain combined with three doses of H65+CAF01 induced equivalent protection than the MbΔmce2-phoP strain alone. Thus, the use of combined vaccination strategies requires a careful analysis of the potential interactions of each of their components with the host's immune system.

Chrysomycin A (CA), a promising antibiotic agent, usually coexists with two analog chrysomycins B (CB) and C (CC) produced by several wild-type (WT) Streptomyces strains. With the aim to increase CA production, UV mutagenesis-based breeding had been employed on a marine-derived strain Streptomyces sp. 891 in our previous study and afforded an improved strain 891-B6 with enhanced CA yield. By comparative transcriptome analysis, significant differences in chrysomycin BGC-related gene expression between the WT strain 891 and the mutant strain 891-B6 were unveiled in the current study. Among 25 up-regulated genes in mutant 891-B6, chryA, chryB, chryC, chryF, chryG, chryK, chryP, and chryQ, responsible for the biosynthesis of benzonaphthopyranone aglycone, and chryD, chryE, and chryU in charge of production of its deoxyglycoside, were characterized. Furthermore, the expression of genes chryOII, chryOIII, and chryOIV responsible for the formation of 8-vinyl in CA from 8-ethyl in CB were greatly enhanced in strain 891-B6. These findings provide molecular mechanisms for increased yield of CA and decreased yield of CB for mutant 891-B6, which has potential application in industrial CA production.

Further research is required to determine the impact of these findings on protective vaccine efficacy with the various vaccines in use as the evolving UK B.1.1.7 lineage strain will likely acquire the E484K mutation [[20]]. In another study, it was noted that the UK B.1.1.7 strain did not reduce the effectiveness of the vaccine, and, the South African 1.351 strain had slightly reduced effectiveness [[18]]. Coronavirus, SARS-CoV-2, COVID-19, vaccine, mutant strain, mutant virus, variant virus Keywords: Coronavirus; SARS-CoV-2; COVID-19; vaccine; mutant strain; mutant virus; variant virus EN Coronavirus SARS-CoV-2 COVID-19 vaccine mutant strain mutant virus variant virus 635 638 4 09/04/21 20210601 NES 210601 1. [Extracted from the article]

Vaccination is the best approach to prevent brucellosis. The existing vaccines such as Rev1 are associated with disadvantages such as abortion in pregnant animals. They also induce antibodies to the O-polysaccharide which may complicate diagnosis. Therefore, it is necessary to generate an appropriate vaccine. The aim of this study is the improvement of the available mutant strain of Brucella melitensis Rev1, in which the prosamine synthetize coding sequence from the O-PS region has been replaced with kanR coding sequence, via the replacement of kanR with GFP reporter encoding sequence by using homologous recombination. The syntactic deletion cassette, containing the GFP encoding sequence flanked with the upstream and downstream sequences of kanR encoding sequence from Rev1 mutant strain, was cloned into pBluescriptIISK(-) vector by SacI and KpnI, and the suicide vector pBluescriptSK-∆kanR/gfp was electroporated into mutant strain. The constructs were confirmed by using PCR, restriction mapping and DNA sequencing. The loss of kanR function in transfected cells was confirmed by using the GFP phenotyping with flow cytometry and fluorescence microscopy, and following the phenotypic characterization of the mutant strain on selective media and the genomic DNA analysis with specific primers. This kanR-free Rev1 mutant strain lack the O-polysaccharide and have a reduced ability to enter and survive in the host cell. Therefore, it could be evaluated as a potential vaccine candidate if eliciting protective immunity. [ABSTRACT FROM AUTHOR]

Strain improvement has become an important strategy in large scale microbial fermentation processes to increase the production yield of enzymes at minimal cost. Here, ultraviolet (UV) irradiation-induced mutant strain of Bacillus cereus, identified using 16S rRNA molecular gene sequencing was subjected to cellulolytic potential screening before cultivating it for carboxymethyl cellulase (CMCase) production under submerged fermentation using a cost-effective plantain stalk–based medium. The enzyme was homogenously purified by a combination of ammonium sulphate precipitation and chromatographic techniques on both DEAE Sephadex A-50 anion-exchange and Sephadex G-100 size-exclusion columns. Sodium dodecyl sulphate polyacrylamide gel electrophoresis was used to determine the molecular weight. Remarkably, this 56 kDa purified cellulase was most active at 70°C and pH 6.0. The CMCase retained over 60% of initial activity after pre-incubation at 60°C for 3 h and was also very stable in both acidic and mild basic medium (pH 3.0-9.0). Cellulase activity was enhanced by Mn2+, Na+, Mg2+ and K+ but inhibited by Pb2+, Hg2+ and Cu2+ at 5 mM. The Km and Vmax of the enzyme with carboxymethyl cellulose are respectively, 3.25 mg/mL and 29.76 U/min/mg. The purified cellulase from B. cereus FOA-2 possesses remarkable properties for suitable application in biotechnological industries.

O-acetyl-homoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis, which catalyzes the conversion of o-acetyl-homoserine (OAH) to homocysteine. In our previous study, we found that OAHS of Streptococcus suis serotype 2 (SS2) can interact with the porcine blood-brain barrier (BBB) model, but whether OAHS regulates the penetration of BBB during SS2 infection is still unclear. To explore the role of OAHS in SS2 infection, OAHS-deficient SS2 mutant strain (SC19-ΔOAHS) and gene complemental strain (SC19-cΔOAHS) were constructed. Compared to the parent strain, with the loss of oahs , the chain length of SC19-ΔOAHS was shortened, the virulence was significantly reduced, the survival rate of mice infected with SC19-ΔOAHS was obviously increased accompanied by the relieved clinical symptoms. And the survival ability of SC19-ΔOAHS in whole blood was also remarkably decreased. Interestingly, the adhesion of SC19-ΔOAHS to endothelial cells was markedly increased, but the deficiency of OAHS significantly inhibited the strain penetrating BBB both in vivo and in vitro. Most of these phenomena can be reversed by the complemental strain (SC19-cΔOAHS). Further study showed that the deficiency of OAHS severely reduced SC19-induced endothelial cell apoptosis, tight junctions (TJs) protein impairment and the expression of SS2 virulence factor Enolase (Eno), involved in the destruction of BBB. Additionally, SC19-ΔOAHS immunized mice were able to resist SC19 or JZLQ022 infection. In conclusion, we confirmed that OAHS promoted the pathogenicity by enhancing host's BBB permeability and immune escape, and SC19- ΔOAHS is a potential live vaccine. ● The importance of OAHS to the virulence of SS2 was revealed. ● For the first time, we found that OAHS is associated with SS2-induced blood-brain barrier destruction. ● SC19-ΔOAHS has potential as a live vaccine. The mice immunized with SC19-ΔOAHS are able to resist homotype infection. [ABSTRACT FROM AUTHOR]

Background: The fusion protein H65, composed of Mycobacterium tuberculosis (TB) ESX-secreted antigens, has improved the bacillus Calmette–Guerin-induced immune protection in a mouse model of bovine TB when formulated in the liposomal adjuvant CAF01. In this study, we aimed to evaluate the protective efficacy of an attenuated Mycobacterium bovis strain – a mutant in mce2 and phoP genes – combined with H65+CAF01 immunization. We evaluated the protection of MbΔmce2-phoP alone or combined with H65+CAF01 against M. bovis challenge in mice. Methods: Groups of BALBc mice were inoculated with the vaccine candidates or phosphate buffered saline (PBS), and 6 weeks after the last immunization, the animals were aerogenically challenged with virulent M. bovis. Bacterial load in organs was counted after 45 days of the challenge. One-way analysis of variance and Bonferroni's posttest were used for statistical analysis. Results: All vaccinated mice showed reduced bacterial loads in lungs compared to unvaccinated animals. However, the protection level was similar between vaccinated groups. Conclusions: The MbΔmce2-phoP strain combined with three doses of H65+CAF01 induced equivalent protection than the MbΔmce2-phoP strain alone. Thus, the use of combined vaccination strategies requires a careful analysis of the potential interactions of each of their components with the host's immune system. [ABSTRACT FROM AUTHOR]

Abstract Brucellosis, caused by Brucella spp., is an important zoonosis worldwide. Vaccination is an effective strategy for protection against Brucella infection in livestock in developing countries and in wildlife in developed countries. However, current vaccine strains including S19 and RB51 are pathogenic to humans and pregnant animals, limiting their use. In this study, we constructed the Brucella abortus (B. abortus) S2308 mutant strain Δ22915, in which the putative lytic transglycosylase gene BAB_RS22915 was deleted. The biological properties of mutant strain Δ22915 were characterized and protection of mice against virulent S2308 challenge was evaluated. The mutant strain Δ22915 showed reduced survival within RAW264.7 cells and survival in vivo in mice. In addition, the mutant strain Δ22915 failed to escape fusion with lysosomes within host cells, and caused no observable pathological damage. RNA-seq analysis indicated that four genes associated with amino acid/nucleotide transport and metabolism were significantly upregulated in mutant strain Δ22915. Furthermore, inoculation of ∆22915 at 105 colony forming units induced effective host immune responses and long-term protection of BALB/c mice. Therefore, mutant strain ∆22915 could be used as a novel vaccine candidate in the future to protect animals against B. abortus infection.

Although the improved strain HR‐5 of Pyropia haitanensis has improvement in yield and quality, it shows earlier maturation and shortened growth period. In this study, blades of HR‐5 strain were treated with 60Co‐γ ray irradiation, and a mutant strain ST‐2 was selected and characterized. The blade length of ST‐2 strain was 401.4 cm on day 70 post‐culture, which was 1.9‐fold longer than that of the parental strain HR‐5. The blades of HR‐5 strain matured earlier and most were matured on day 55, while the ST‐2 strain was not matured until day 90. When blades of HR‐5 and ST‐2 strains were firstly cultured at 23°C for 40 days and their blade discs cultured at 29°C for another 30 days, the mean length of ST‐2 strain was 2.0‐fold longer than that of HR‐5 strain. Besides, the contents of Chl. a and phycobiliprotein and the conchospore numbers in ST‐2 were comparable to that in HR‐5 strain. In summary, compared with HR‐5, we observed delayed maturation, increased length, weight and high‐temperature resistance of the blades, high content of photosynthetic pigments, and high production of conchospores in ST‐2, suggesting it might be a better strain for large‐scale production. [ABSTRACT FROM AUTHOR]

• One monomeric aspartokinase (AK), generally multimer, has been identified. • Identification of mutation sites and construction of mutant strains. • Characterization of mutant strains. • Molecular dynamics simulation revealed the mechanism of enzyme activity increase. Aspartokinase (AK) is known to be inhibited by the synergistic feedback of threonine and lysine in the relevant biosynthesis pathway in Corynebacterium , thereby hindering downstream metabolite accumulation. In this study, site-directed mutagenesis was performed to modify AK, and the mutant's characteristics were found to be different, laying the foundation for the downstream metabolite accumulation. Position 379 was identified as the key inhibitor-binding amino acid residue via homologous sequencing and comparison with the 3AAW conformation (Protein Data Bank). The mutant strain T379L, with 9.16-fold higher enzymatic activity, was obtained via site-directed mutagenesis and high-throughput screening. An analysis of the properties of the mutant enzyme revealed that the optimal temperature increased from 26 to 35 °C, the optimal pH stayed at 8.0, and the enzyme stability decreased from 4.5 to 3.32 h. The inhibition of T379L was weaker at various inhibitor concentrations; however, the inhibition turned into activation at low concentrations of inhibitors: Lys, Lys + Thr, or Lys + Met. Molecular dynamics simulations revealed that, with an increase in the enzymatic activity of the mutant T379L, the affinity between AK and Asp increased, and the catalytic residues (S192 and D193) shifted toward Asp. [ABSTRACT FROM AUTHOR]

Docosahexaenoic acid (DHA), a n-3 long-chain polyunsaturated fatty acid, is critical for physiological activities of the human body. Marine eukaryote Aurantiochytrium sp. is considered a promising source for DHA production. Mutational studies have shown that ultraviolet (UV) irradiation (50 W, 30 s) could be utilized as a breeding strategy for obtaining high-yield DHA-producing Aurantiochytrium sp. After UV irradiation (50 W, 30 s), the mutant strain X2 which shows enhanced lipid (1.79-fold, 1417.37 mg/L) and DHA (1.90-fold, 624.93 mg/L) production, was selected from the wild Aurantiochytrium sp. Instead of eicosapentaenoic acid (EPA), 9.07% of docosapentaenoic acid (DPA) was observed in the mutant strain X2. The comparative transcriptomic analysis showed that in both wild type and mutant strain, the fatty acid synthesis (FAS) pathway was incomplete with key desaturases, but genes related to the polyketide synthase (PKS) pathway were observed. Results presented that mRNA expression levels of CoAT, AT, ER, DH, and MT down-regulated in wild type but up-regulated in mutant strain X2, corresponding to the increased intercellular DHA accumulation. These findings indicated that CoAT, AT, ER, DH, and MT can be exploited for high DHA yields in Aurantiochytrium.

Metalaxyl is one of the main fungicides used to control pepper blight caused by Phytophthora capsici. Metalaxyl resistance of P. capsici, caused by the long-term intense use of this fungicide, has become one of the most serious challenges facing pest management. To reveal the potential resistance mechanism of P. capsici to fungicide metalaxyl, a metalaxyl-resistant mutant strain SD1-9 was obtained under laboratory conditions. The pathogenicity test showed that mutant strain SD1-9 had different pathogenicity to different host plants with or without the treatment of metalaxyl compared with that of the wild type SD1. Comparative transcriptome sequencing of mutant strain SD1-9 and wild type SD1 led to the identification of 3845 differentially expressed genes, among them, 517 genes were upregulated, while 3328 genes were down-regulated in SD1-9 compared to that in the SD1. The expression levels of 10 genes were further verified by real-time RT-PCR. KEGG analysis showed that the differentially expressed genes were enriched in the peroxisome, endocytosis, alanine and tyrosine metabolism. The expression of the candidate gene XLOC_020226 during 10 life history stages was further studied, the results showed that expression level reached a maximum at the zoospores stage and basically showed a gradually increasing trend with increasing infection time in pepper leaves in SD1-9 strain, while its expression gradually increased in the SD1 strain throughout the 10 stages, indicated that XLOC_020226 may be related to the growth and pathogenicity of P. capsici. In summary, transcriptome analysis of plant pathogen P. capsici strains with different metalaxyl resistance not only provided database of the genes involved in the metalaxyl resistance of P. capsici, but also allowed us to gain novel insights into the potential resistance mechanism of P. capsici to metalaxyl in peppers.

Sonchus ( Sonchus oleraceus ) originated from Europe and is now cultivated worldwide. The wild resources of sonchus are very abundant, and it has rich nutritional and medicinal value. In this study, 15 sonchus samples with typical symptoms showing leaf curling, vein thickening, and enations were collected from Guigang and Baise City of Guangxi, China. Diseased sonchus were identified by PCR detection, whole genome sequence amplification, and phylogenetic and recombination analysis. The results showed that all the samples were confirmed infected by begomoviruses, and three full-length viral genomes were obtained from 15 sonchus, named GG7-13, GG8-6, and BS63-5. The full genome lengths were 2,584, 2,735, and 2,746 nt, respectively. The nucleotide identities among the three isolates ranged from 92.67 to 99.93%. All of them shared the highest identities (greater than 91.69%) with other isolates of ageratum yellow vein China virus (AYVCNV) (available on GenBank). According to the guidelines of classification of begomoviruses, the virus isolates obtained in this study are different isolates of AYVCNV; a phylogenetic tree analysis showed that these isolates formed a large branch with three other Guangxi isolates of AYVCNV, indicating their close evolution. The genome structures of GG8-6 and BS63-5 are consistent with the monopartite genome virus of the begomoviruses, and both have six open reading frames (ORFs), while GG7-13 has a 151-nt deletion between C2 and C3, resulting in a mutant strain of only five ORFs. This study is the first report on S. oleraceus infected by ageratum yellow vein China virus., Competing Interests: The author(s) declare no conflict of interest.

Herpes simplex virus is a prevalent pathogen of humans of various age groups. The fact that no prophylactic or therapeutic vaccine is currently available suggests a significant need to further investigate the immune mechanisms induced by the virus and various vaccine candidates. We previously generated an HSV-1 mutant strain, M3, with partial deletions in ul7 , ul41 and LAT that produced an attenuated phenotype in mice. In the present study, we performed a comparative analysis to characterize the immune responses induced by M3 versus wild-type HSV-1 in a mouse model. Infection with wild-type HSV-1 triggered an inflammatory-dominated response and adaptive immunity suppression and was accompanied by severe pathological damage. In contrast, infection with M3 induced a systematic immune response involving full activation of both innate and adaptive immunity and was accompanied by no obvious pathological changes. Furthermore, the immune response induced by M3 protected mice from lethal challenge with wild-type strains of HSV-1 and restrained virus proliferation and impaired latency. These data are useful for further HSV-1 vaccine development using a mutant strain construction strategy. [ABSTRACT FROM AUTHOR]

In June 2012, health authorities in Papua New Guinea detected an increase in febrile illnesses in Vanimo. Chikungunya virus of the Eastern/Central/Southern African genotype harboring the E1:A226V mutation was identified. This ongoing outbreak has spread to ≥8 other provinces and has had a harmful effect on public health.

Herpes simplex virus type 1(HSV-1) presents a conundrum to public health worldwide because of its specific pathogenicity and clinical features. Some experimental vaccines, such as the recombinant viral glycoproteins, exhibit the viral immunogenicity of a host-specific immune response, but none of these has achieved a valid epidemiological protective efficacy in the human population. In the present study, we constructed an attenuated HSV-1 strain M3 through the partial deletion of UL7, UL41, and the latency-associated transcript (LAT) using the CRISPR/Cas9 system. The mutant strain exhibited lowered infectivity and virulence in macaques. Neutralization testing and ELISpot detection of the specific T-cell responses confirmed the specific immunity induced by M3 immunization and this immunity defended against the challenges of the wild-type strain and restricted the entry of the wild-type strain into the trigeminal ganglion. These results in rhesus macaques demonstrated the potential of the attenuated vaccine for the prevention of HSV-1 in humans.

The decontamination of Aflatoxin B 1 (AFB 1 ) by immobilized cells of a new mutant strain, prepared on a base of HSCAS (hydrated sodium calcium aluminosilicate), was studied. Novel strains were induced by UV irradiation, from which 50 were screened according to their degradation efficacy on AFB 1, compared with the wild strain (FS-Z1). The FS-UV1 strain exhibited highest degradation efficacy, which was confirmed by 18SrDNA to be Aspergillus niger . The results indicate that both immobilized cells and this mutant strain which are incubated for 48 h at 30 °C, would considerably remediate AFB 1 in nutrient broth culture, by 95.32% and 82.43%, respectively. By the application of samples of contaminated cottonseed meal, with results of 93.46%∼96.82%, the degradation rate was also validated. The results of Ames test indicate the mutagenic activity of treated AFB 1 is greatly abated, with treated controls. The Application of LC-q-TOFMS (liquid-chromatography, quadrupole, time-of-flight mass spectrometry) deduces the structure and molecular formulas of the degradation products. In the vivo study, the damages of photomicrographic evidence are decreased in kidney and liver and the serum biochemical parameters is improved, in response to preventative treatment with immobilized cells. This is the application of HSCAS-prepared, immobilized A. niger cells to degrade AFB 1 of contaminated samples. The investigation in this paper offers a novel path for economical, time-saving biodegradation of AFB 1 in foods and feeds. [ABSTRACT FROM AUTHOR]

The currently ongoing COVID-19 outbreak remains a global health concern. Understanding the transmission modes of COVID-19 can help develop more effective prevention and control strategies. In this study, we devise a two-strain nonlinear dynamical model with the purpose to shed light on the effect of multiple factors on the outbreak of the epidemic. Our targeted model incorporates the simultaneous transmission of the mutant strain and wild strain, environmental transmission and the implementation of vaccination, in the context of shortage of essential medical resources. By using the nonlinear least-square method, the model is validated based on the daily case data of the second COVID-19 wave in India, which has triggered a heavy load of confirmed cases. We present the formula for the effective reproduction number and give an estimate of it over the time. By conducting Latin Hyperbolic Sampling (LHS), evaluating the partial rank correlation coefficients (PRCCs) and other sensitivity analysis, we have found that increasing the transmission probability in contact with the mutant strain, the proportion of infecteds with mutant strain, the ratio of probability of the vaccinated individuals being infected, or the indirect transmission rate, all could aggravate the outbreak by raising the total number of deaths. We also found that increasing the recovery rate of those infecteds with mutant strain while decreasing their disease-induced death rate, or raising the vaccination rate, both could alleviate the outbreak by reducing the deaths. Our results demonstrate that reducing the prevalence of the mutant strain, improving the clearance of the virus in the environment, and strengthening the ability to treat infected individuals are critical to mitigate and control the spread of COVID-19, especially in the resource-constrained regions.

Mn (II)-oxidizing microorganisms are thought to widely participate in the deposition of manganese oxides. However, the underlying physiological functions of Mn (II) oxidation by microorganisms have been poorly understood. In this study, we investigated the production of biogenic manganese oxides under laboratory conditions. The metabolic efficiency of Mn (II)-oxidizing strain MB266 screened from soil, and its mutant strain MB32 without Mn (II)-oxidizing capability, was monitored by microcalorimetry. The heat output directly yielded from Mn (II) oxidation was calculated. Powder X-ray diffraction (XRD), transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS) and X-ray absorption near-edge structure spectrocopy (XANES) were used to characterize biogenic manganese products. The results showed that the product of biogenic manganese oxidation was a weakly crystallized bixbyite-like phase. The metabolic thermal effects and biomass yields of the strain MB266 and the mutant strain MB32 did not differ significantly when cultured in Mn (II)-free medium. However, when cultured in the medium amended with 1 mM Mn (II), MB266 exhibited the greater metabolic efficiency than MB32, suggesting the benefit of bacterial Mn (II)-oxidizing ability in the presence of Mn (II), which might be the one of the underlying physiological functions of possessing the trait. In addition, much lower contribution of the heat derived directly from Mn (II) oxidation to the total heat production suggested Mn (II)-oxidizing bacteria mainly derive energy by decomposing organic substrates for chemoheterotrophic growth rather than by oxidizing Mn (II). [ABSTRACT FROM AUTHOR]

Highlights: [•] PEC inactivation of two strains was the fastest compared with PC and EC. [•] E. coli BW25113 showed a significant increase of resistance to PEC and PC treatment. [•] Adding halide ions could remarkably enhance the inactivation efficiencies during PEC. [•] Mechanism was proposed to enucleate the enhancement of bactericidal efficiency. [Copyright &y& Elsevier]

Mycoplasma pneumoniae (M. pneumoniae) is the causative agent of both upper and lower respiratory infections that can lead to pneumonia, extrapulmonary complications and devastating sequela. With the increasing rate of macrolide-resistant strains, the severe clinical consequence of refractory mycoplasma pneumonia in children health calls for the need of vaccine research for this pathogen. In this report, the immunomodulatory effectiveness of a live attenuated M. pneumoniae vaccine was evaluated. The vaccine strain was a mutant strain of M. pneumoniae , MUT129, obtained after multiple passages of M129 strain in PPLO broth. The SNP/InDel detection results showed that mutations were present in genes encoding the adhesion organelle-associated proteins and lipoproteins of M. pneumoniae MUT129. Upon intranasal challenge of BALB/c mice with 1 × 107 CFU of MUT129, there were very small amount of Mycoplasma antigens and almost no M. pneumoniae present in the lung tissues of BALB/c mice. Besides, there was almost no inflammatory cell infiltration in the lung tissue. Results of the M. pneumoniae challenge study showed that mice immunized with MUT129 presented with less inflammation, lower detectable number of M. pneumoniae in the lungs when compared with the unimmunized mice. These results indicated that the live attenuated vaccine can efficiently prevent the proliferation of M. pneumonia in the lungs, reduce but not completely prevent the pulmonary inflammatory response. • An attenuated natural mutant of Mycoplasma pneumoniae (named MUT129 strain) was generated. • The attenuated MUT129 strain triggered a strong immune protection against M. pneumoniae infection. • This study provide initial evidence for the suitability of MUT129 strain as an attenuated vaccine candidate. [ABSTRACT FROM AUTHOR]

Based on the generation of Pantoea agglomerans BH18 library using Tn7 transposon system, mutant screens were conducted for improvement the ability of hydrogen production of this strain. These mutants were used to test for ability of hydrogen production in the initial pH of 5.0. In contrast to wild type strain BH18, a transposon mutant, named as strain TB108, was screened for high hydrogen-producing capability and acid tolerance in the initial pH of 5.0. The factors required for hydrogen production of the aciduric transposon-mutagenized strain TB108 were determined. The mutant strain TB108 similar as wild type strain BH18 was able to produce hydrogen over a wide range of salt concentration from 0.4% to 6%. Under the marine conditions with the initial pH of 5.0 and glucose concentration of 10 g/L, the total hydrogen production of the mutant TB108 was (1.36 ± 0.04) mol H2/mol glucose (mean ± S.E.), increasing by 55% compared with wild type. In addition, the mutant strain TB108 could produce hydrogen using many carbon sources such as fructose, glucose, sucrose, sorbitol and so on. This result demonstrated that the mutant strain with high acid tolerance is beneficial for improvement of hydrogen production. [ABSTRACT FROM AUTHOR]

In June 2012, health authorities in Papua New Guinea detected an increase in febrile illnesses in Vanimo. Chikungunya virus of the Eastern/Central/Southern African genotype harboring the E1:A226V mutation was identified. This ongoing outbreak has spread to ≥8 other provinces and has had a harmful effect on public health. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) was caused by a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 utilizes human angiotensin converting enzyme 2 (hACE2) as the cellular receptor of its spike glycoprotein (SP) to gain entry into cells. Consequently, we focused on the potential of repurposing clinically available drugs to block the binding of SARS-CoV-2 to hACE2 by utilizing a novel artificial-intelligence drug screening approach. Based on the structure of S-RBD and hACE2, the pharmacophore of SARS-CoV-2-receptor-binding-domain (S-RBD) -hACE2 interface was generated and used to screen a library of FDA-approved drugs. A total of 20 drugs were retrieved as S-RBD-hACE2 inhibitors, of which 16 drugs were identified to bind to S-RBD or hACE2. Notably, tannic acid was validated to interfere with the binding of S-RBD to hACE2, thereby inhibited pseudotyped SARS-CoV-2 entry. Experiments involving competitive inhibition revealed that tannic acid competes with S-RBD and hACE2, whereas molecular docking proved that tannic acid interacts with the essential residues of S-RBD and hACE2. Based on the known antiviral activity and our findings, tannic acid might serve as a promising candidate for preventing and treating SARS-CoV-2 infection., Competing Interests: The authors 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., (Copyright © 2022 Chen, Wang, Wang, Yao, Wang, Huang, Xiang, Zhou, Xue, Li, Gao, Wang, Chu and Wang.)

Abstract: In this study we characterise the genomic and transcriptomic variability of a natural deletion strain of Mycobacterium avium subspecies paratuberculosis (MAP) prevalent in Spanish Guadarrama goats. Using a pan-genome microarray including MAP and M. avium subspecies hominissuis 104 genomes (MAPAC) we demonstrate the genotype to be MAP Type II with a single deletion of 19 contiguous ORFs (16kb) including a complete mammalian cell entry (mce7_1) operon and adjacent proline–glutamic acid (PE)/proline–proline–glutamic acid (PPE) genes. A deletion specific PCR test was developed and a subsequent screening identified four goat herds infected with the variant strain. Each was located in central Spain and showed epidemiological links suggestive of transmission between herds. A majority of animals infected with the variant manifested a paucibacillary form of the disease. Comparisons between virulent complete genome compliment strains isolated from multibacillary diseased goats and the MAP variant strain during entry into activated macrophages demonstrated an increased sensitivity in the variant to intracellular killing in human and ovine macrophages. As PPE and mce genes are associated with mycobacterial virulence and pathogenesis we investigated the interplay of these gene sets during cell entry using the MAPAC array. This showed significant differential transcriptome profiles compared to full genome complement MAP controls that included changes in other undeleted mce operons and PE/PPE genes, esx-like signalling operons and stress response/fatty acid metabolism pathways. This strain represents the first report of a MAP Type II genotype with significant natural genomic deletions which remains able to cause disease and is transmissible in goats. [Copyright &y& Elsevier]

Listeria monocytogenes is a food-borne pathogen able to cause serious disease in human and animals. Listeriolysin O (LLO), a major virulence factor secreted by this bacterium, is a vacuole-specific lysin that facilitates bacterial entrance into the host cytosol. Thus, LLO plays a key role in the translocation and intracellular spread of L. monocytogenes. To study the effect of LLO on virulence and immunopotency, a LLO-deficient L. monocytogenes mutant was constructed using a shuttle vector followed by homologous recombination. The mutant strain had lost hemolytic activity, which resulted in an extremely reduced virulence, 5 logs lower than that of the parent strain, yzuLM4, in BALB/c mice. The number of bacteria detected in the spleens and livers of mice infected with the mutant was greatly reduced, and the bacteria were rapidly eliminated by the host. Kinetics studies in this murine model of infection showed that the invasion ability of the mutant strain was much lower than that of the parent strain. Moreover, immunization with the mutant strain conferred protective immunity against listerial infection. In particular, stimulation with Ag85B240-259, strong specific Th1 type cellular immunity was elicited by vaccination C57BL/6 mice with hly deficient strain delivering Mycobacterium tuberculosis fusion antigen Ag85B-ESAT-6 via intravenous inoculation. These results clearly show that highly attenuated LLO-deficient L. monocytogenes is an attractive vaccine carrier for delivering heterologous antigens. [ABSTRACT FROM AUTHOR]

Abstract: A new mutant strain of fungus Trichoderma viride T 100-14 was cultivated on 1% microcrystalline cellulose (Avicel) for 120h and the resulting culture filtrate was prepared for protein identification and purification. To identify the predominant catalytic components, cellulases were separated by an adapted two-dimensional electrophoresis technique. The apparent major spots were identified by high performance liquid chromatography electrospray ionization mass (HPLC–ESI-MS). Seven of the components were previously known, i.e., the endoglucanases Cel7B (EG I), Cel12A (EG III), Cel61A (EG IV), the cellobiohydrolases Cel7A (CBH I), Cel6A (CBH II), Cel6B (CBH IIb) and the β-glucosidase. The seven major components in the fermentation broth of T. viride T 100-14 probably constitute the essential enzymes for crystalline cellulose hydrolysis and they were further purified to electrophoretic homogeneity by a series of chromatography column. Hydrolysis studies of the purified elements revealed that three of the cellulases were classified as cellobiohydrolases due to their main activities on p-nitrophenyl-β-d-cellobioside (pNPC). Three of the cellulases, with the abilities of hydrolyzing both carboxymethyl-cellulose (CMC) and Avicel indicate their endoglucanase activities. It deserved noting that the β-glucosidase from the T 100-14 displayed an extremely high activity on p-nitrophenyl-β-d-glycopyranoside (pNPG), which suggested it was a good candidate for the conversion of cellobiose to glucose. [Copyright &y& Elsevier]

Abstract: A mutant with reduced pigmentation was isolated from Aureobasidium pullulans A.p.-3 by physical mutagenesis. The ability to synthesize pullulan and melanin was compared between the mutant and the parent strain. Morphological differences between the strains were observed under scanning and transmission microscope (SEM and TEM). The chemical structure of the pullulan synthesized by the strains evaluated was compared using FT-IR spectroscopy with that of pullulan available commercially. The amount of pullulan synthesized by the mutant and parent strains was similar whereas the melanin content was significantly reduced. The chemical structure of the pullulan was the same as that of the pullulan from commercial sources. The changes in the appearance of mutant capsules were observed under SEM. Smaller amount, lower size of the melanin molecules and their dislocation influenced the brightness of mutant chlamydospores. [Copyright &y& Elsevier]

Abstract: An 8.3-kDa secretory antigen of Mycobacterium bovis bacillus Calmette-Guérin (BCG), called SA5K, was previously identified and characterized in our laboratory. Sequence analysis of the BCG sa5k gene, including the corresponding promoter region, showed that it is identical to the homologous gene in Mycobacterium tuberculosis (Rv1174c). No significant homology with other proteins was found and the physiologic role of SA5K for mycobacteria remains unknown. In the present study, a BCG mutant strain (BCGsa5k::aph) was constructed by allelic exchange involving the replacement of the sa5k gene with a kanamycin-inactivated copy. Mutant and parental strains showed similar growth rates in liquid medium, suggesting that the loss of the sa5k gene does not affect the in vitro growth of BCG. Nevertheless, BCGsa5k::aph showed a reduced ability to grow in human macrophages compared with the wild-type BCG, suggesting that SA5K is involved in intracellular survival/multiplication mechanisms. The mutant strain was also attenuated in vivo in a mouse infection model, showing impaired growth/survival in spleen and liver and fewer and smaller granulomatous lesions compared to the parental strain. Complementation of the mutation restored the parental phenotype. Taken together, results presented in this study suggest a role for SA5K in the growth capacity of BCG both in an intracellular milieu and in infected mice. [Copyright &y& Elsevier]