Abstract:Based on a wrap-up of the research proposals received and awards made during 2011 through 2015 in the discipline of microbiology of the Department of Life Sciences, National Natural Science Foundation of China, this article presents a statistic analysis of award recipient institutions and main research trends, and attempts a prospective prioritization of the funding areas from the points of encouraging interdisciplinary research, optimizing funding instruments and strengthening talent training, with a view to providing reference for scientists and researchers in the field of microbiology.

Abstract:Quinolone antibacterial drugs, developing from the treatment of urinary tract infection in early time and now from the treatment of intestinal infection and respiratory infection, have been widely used in clinical, animal husbandry and aquaculture. Bacteria gradually become resistant to them and resistance mechanism is more and more complicated. Quinolone resistance mechanism is mainly divided into chromosome mediated resistance and plasmid mediated resistance, the latter plays an important role in spreading of antibiotic resistance. In 1998, plasmid mediated quinolone resistance mechanism was reported for the first time, namely the qnr gene mediated fluoroquinolone resistance mechanism. qnr genes can spread rapidly in different bacteria, which causes the infection difficult to control, makes the nosocomial infection popular in a wide range. In addition, qnr genes are usually associated with β-lactamase resistance gene. They exist in complex integron and integrate with the other varieties of resistance genes, which narrows the space of clinical medicine choose or drug combinations use to treat related bacterial infection and brings us a serious challenge. In this review, we provide a detailed overview for the historical discovery, classification, the resistance mechanisms of qnr genes, and the prevalence of those genes in China.

Abstract:Biofilm of Campylobacter jejuni was formed by cross-linking its extracellular secretion, polysaccharides, various extracellular proteins, nucleic acids etc to enhance its survival in hostile environments, especially for detergents, antibiotics and disinfectants. This paper elaborated C. jejuni biofilm formation and regulation mechanisms in the surface properties of the media, temperatures, gas environment, the regulation of gene etc, also analysed and discussed a variety of biofilm removal practical applications. We hope it can provide a reference for studies on biofilm control of C. jejuni.

Abstract:Some pathogenic microorganisms ubiquitous in the environment could cross kingdoms to infect diverse hosts. Several cross-kingdom human pathogens were summarized in this paper, including Serratia marcescens, Enterobacter cloacae and Pseudomonas aeuriginosa. They are ubiquitous in the nature and could cause plant diseases using the same or different infection strategies with which they infect humans and broaden host range. Among these bacteria, Klebsiella pneumoniae causes top rot disease of maize in the nature, revealing some plants in the environment could serve as a reservoir of various pathogens which might infect animals and probably humans when conditions are favorable, and even potentially harm food. Research on these cross-kingdom pathogens may play a very important role in the epidemiology of human, animal and plant diseases and be a hot topic in environment science.

Abstract:Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea, which is a second leading cause of death for the children under five years old from all over the world. The key factors of ETEC contain both colonization factors (CFs) and enterotoxins including heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). CFs mediated the binding of bacteria to the host intestinal epithelial cells, whereas LT and ST stimulated the over-secretion of body fluids and electrolytes, resulting in the destruction of the host fluid balance and leading diarrhea. The vaccine against CFs and enterotoxins could stimulate the host immune response, blocking ETEC adhesion and neutralizing enterotoxins, which is effective in the prevention of ETEC diarrhea. For the moment, depending on the stimulated immune response against LT, a cholera vaccine called Dukoral ® has been approved for use in some countries for the short-term protection and prevention of travelers' diarrhea. ETEC candidate vaccines are still in progress, which is designed to provide a long and wide-spectrum protection for ETEC infections. This paper briefly summarizes the advanced findings and key problems of vaccine development, and discusses prospects for future research.

Abstract:[Objective] To clone the biosynthetic gene clusters for secondary metabolites, we developed the genetic modification system and constructed a genomic library of Streptomyces luteosporeus NRRL 2401. [Methods] The genetic modification system was developed by using conjugal transfer vectors pSET152, pPM927 and pJTU1278 which were transferred from Escherichia coli ET12567/pUZ8002 to S. luteosporeus. The genomic library of S. luteosporeus NRRL 2401 was constructed by the fosmid vector pCClFOS^TM, with E. coli EPl300TM-T1R as the host strain. A PCR-based method was then developed for screening the biosynthetic gene clusters of secondary metabolites in the constructed genomic library. [Results] Vectors pSET152, pPM927 and pJTU1278 were successfully transferred into S. luteosporeus for genetic modification, with pSET152 presenting the highest transformation efficiency. The constructed genomic library of S. luteosporeus NRRL 2401 contained 2880 clones with an average -35 kb inserted DNA fragment in each clone, indicating the 99.99% coverage of the genome in the library. In this genomic library, we detected 9 clones containing possible indolmycin biosynthesis genes by the PCR-based screening method. [Conclusion] A stable, efficient genetic modification system and high-quality genomic library could be used for discovery of the biosynthetic gene clusters for secondary metabolites in S. luteosporeus NRRL 2401.

Abstract:[Objective] This study was carried out to obtain lead compounds targeting penicillin-binding protein 3 (PBP3) of Pseudomonas aeruginosa by virtual screening. [Methods] UCSF dock 6.5 was used for the virtual screening from a database containing 1.04 million small molecules. Hit compounds with simple structures were synthesized and then evaluated for their antibacterial activities. [Results] Grid score was used for the first round of screening, and 60000 small molecules whose scores lower than -30 kcal/mol were screened out from the database. These molecules were subjected to the second round of screening using amber score. Approximately 200 hit compounds with scores lower than -20 kcal/mol were analyzed and 4 of them were selected as lead compounds and then synthesized. The minimal inhibition concentrations (MICs) of the lead compounds were between 175-275 μg/mL, which were lower than that of Sulfadiazine (500 μg/mL) significantly. Meanwhile, these compounds were effective for both Gram-negative and Gram-positive bacteria. [Conclusion] The lead compounds had potential to become new antibacterial agents for conquering the drug resistance of P. aeruginosa.

Abstract:[Objective] Based on 16S rRNA and rpoC1 gene sequences, the phylogenetic relationship between Spirulina and Arthrospira were studied and compared. [Methods] We amplified, sequenced and analyzed 16S rRNA and rpoC1 of 84 strains. Then the phylogenetic trees were constructed and compared. [Results] The conserved sites percentage, average G+C content and sequence identity of rpoC1 were 49.7%, 47.7%, 76%-100% respectively, significantly lower than 79.4%, 55.6% and 91%-100% of 16S rRNA, and the heterogeneity degree was higher. The trees generated with two different genes showed similar topologies and thus inferred consistent phylogenetic relationships. Eighty-four experimental strains were divided into 3 groups belonging to 2 genera: F-351, F-904-2, F-1070 and TJBC14 were Spirulina and the rest were Arthrospira. Although morphospecies and geographical species could not be distinguished based on 16S rRNA and rpoC1 gene sequences, the bootstrap value of rpoC1 (100%) was higher than that of 16S rRNA (99%). Moreover, clustering effect of rpoC1 for Spirulina and Arthrospirai was better than 16S rRNA. [Conclusion] Spirulina and Arthrospira were different genera, rpoC1 gene has more advantage to distinguish the strains in the same genus than that of 16S rRNA gene.

Abstract:[Objective] In order to provide strains with high activity of anti-phytopathogenic fungi and antitumor activity, we studied the diversity and bioactivity of actinomycetes isolated from medicinal plant Taxus chinensis and rhizospheric soil. [Methods] Seven selective media were used to isolate actinomycetes. Experiments of anti-phytopathogenic fungi, cytotoxicity activity, and the 16S rRNA gene sequencing of them were carried out to evaluate the diversity and bioactivity. Strains with high activity were identified. [Results] A total of 277 actinomycetes were isolated, of which 111 strains were selected and assigned to 6 suborders, 7 families and 8 genera, in which Taxus chinensis can be divided into 10 groups. The bioactivity testing results indicated that: 30.9% isolates showed activity against at least one of the 12 phytopathogenic fungi; 44.1% strains and 33.3% strains showed cytotoxicity activity with inhibition rate above 40% against stomach cancer cell line SGC-7901 and lung cancer cell line NCI-H460 respectively. [Conclusion] Actinomycetes isolated from Taxus chinensis and rhizospheric soil is of high diversity and a good source for the selection of bioactive compounds. Taxus chinensis KLBMP 2170 is an excellent resource with antifungal and cytotoxicity activity for further studies.

Abstract:[Objective] This study was aimed to reveal the bacterial community characteristics of rearing water of marine recirculating aquaculture system for yellow grouper (Epinephelus awoara), and compare the differences between bacterial community structure of healthy rearing water and sick rearing water in order to elucidate the relationship between bacterial community and fish disease. [Methods] The next generation sequencing method was used, and the bacterial community structure and α-diversity indices (species richness, species evenness and phylogenetic diversity) between the rearing water of healthy and diseased groups were studied and compared. In addition, the traditional cultivation method was used to isolate suspected pathogens from the niduses of diseased yellow groupers. [Results] There was no significant difference between the α-diversities of healthy and diseased rearing water bodies, however, the results of principal coordinates analysis (PCoA) and the sample clustering of heatmap showed that the bacterial communities of healthy and diseased rearing water bodies were quite different. Although phyla Proteobacteria, Verrucomicrobia and Bacteroidetes were all the predominant ones in both communities, their relative abundance varied greatly. In diseased community, the relative abundances of α-Proteobacteria (25.07%) and γ-Protebacteria (22.74%) were similar, whereas the proportion of γ-Protebacteria (40.49%) was much higher than α-Proteobacteria (10.87%) in the healthy community. The differences of Verrucomicrobia and Bacteroidetes between the healthy and the diseased rearing water were also significant with relative abundances of 10.9% and 26.4%, and 20.9% and 12.3%, respectively. The significantly different families were Rhodobacteraceae and Rhodospirillaceae within class α-Proteobacteria; Alteromonadaceae and HTCC2188 within class γ-Protebacteria; Verrucomicrobiaceae within Verrucomicrobia; Cryomorphaceae within Bacteroidetes. The healthy and the diseased communities owned specific core microbes themselves. Glaciecola, HTCC, Sediminicola and Prevotella were the core genera in healthy rearing water, and Vibrio, Rubritalea and Oleibacter in diseased rearing water. Twenty strains of Vibrio spp. and one of Acinetobacter haemolyticus were isolated from skin, liver and spleen of diseased yellow grouper. [Conclusion] The shift of bacterial community structure and relative abundance of rearing water will help monitor the healthy status of recirculating aquaculture system. Our study provides theory and experimental basis to diagnosis and monitor of Vibrio disease for yellow grouper recirculating aquaculture system.

Abstract:[Objective] To explore effects of FtsZ mutants FtsZ^E75A, FtsZ^R78G and ftsZ^D82A on FtsZ self-assembly and interaction of FtsZ with MreB in Escherichia coli strains. [Methods] We constructed FtsZ and its mutant's plasmids by molecular clone and site-directed mutagenesis methods, and purified targeted proteins by affinity chromatography. QN6(ftsZ::yfp-cat), QN7(ftsZ^E75A::yfp-cat), QN8(ftsZ^R78G::yfp-cat) and QN9(ftsZ^D82A::yfp-cat) strains were constructed by linear DNA homologous recombination. We observed cellular localization pattern of FtsZ and its mutants in E. coli by living cell imaging experiments, examined interaction of FtsZ/FtsZ^*-FtsZ^* and FtsZ/FtsZ^*-MreB by Co-immunoprecipita-tion and bacteria two hybrid, and analyzed assembly characteristics of FtsZ mutants by Light scattering. [Results] The Yfp-labeled FtsZ^E75A, FtsZ^R78G and ftsZ^D82A mutant proteins failed to assemble into functional Z-ring structure and localize correctly in E. coli strains. Interaction of FtsZ with its mutants, or FtsZ^*-FtsZ^* and FtsZ^*-MreB interaction were weakened or completely disappeared. In addition, in vitro experiments show that E75A, R78G and D82A mutations decreased the polymerization efficiency of FtsZ monomer. [Conclusion] FtsZ E75, R78 and D82 are critical amino acids in the assembly, function of FtsZ protein and FtsZ-MreB interaction in E. coli strains.

Abstract:[Objective] Streptococcus suis (S. suis) is an emerging zoonotic pathogenic bacterium capable of infecting piglets and human and with sporadic infections in a variety of mammalian species. The aim of this study is to investigate the prevalence of S. suis in wild cats. [Methods] We isolated an S. suis strain from a wild cat. We tested the serotype of the isolated strain by anti-serum agglutination and PCR. We determined the sequence type (ST) of the isolated strain by multilocus sequence typing tests (MLST). We constructed the 16S rRNA phylogenetic tree of the isolation and S. suis strains in NCBI database to demonstrated genetic relationship of different strains. We measured the antibiotic resistance of the isolated strain by triple disk diffusion method. We detected the virulence of the isolated strain by mice infection experiments. [Results] We isolated an S. suis strain m70 from a wild cat, which belongs to serotype 9. MLST showed that m70 fell into a new ST. The 16S rRNA phylogenetic tree of m70 and S. suis strains in NCBI database demonstrated that m70 was in a separate cluster. m70 was resistant to tetracycline, intermediate to erythromycin, and sensitive to ampicillin, corresponding to clinical S. suis isolates in China. The mortality of mice infected with 108 CFU of m70 was achieved 60%-80% (3/5-4/5). The mean LD₅₀ of mice infected with m70 was 5.1×10⁷ CFU, while the mean LD₅₀ of virulent S. suis strain HA9801 was 3.9×10⁷ CFU. There is no significant difference between the LD₅₀ of the two strains(P<0.05). [Conclusion] We isolated an S. suis strain from a wild cat, which belongs to the prevalent serotype and was a virulent strain, indicating the potential of transmission of S. suis from wild cats to humans, especially some prevalent serotype strains.

Abstract:[Objective] To identify immunogenic proteins of Erysipelothrix rhusiopathiae C43065. [Methods] Antigens were extracted from E. rhusiopathiae C43065 by the alkaline extraction method. Proteins in the NaOH-extracted antigen were separated by SDS-PAGE and transferred to nitrocellulose membranes, and then Western blotting was performed with rabbit antiserum against the NaOH-extracted antigens. The immunogenic protein bands were identified by MALDI-TOF mass spectrometry. The genes encoding 5 major immunogenic proteins was amplified by PCR from the genomic DNA of E. rhusiopathiae C43065, and inserted into the pMD18-T vector and then sequenced. [Results] A total of 9 immunogenic surface proteins in the NaOH-extracted antigen from E. rhusiopathiae C43065 were successfully identified by MALDI-TOF mass spectrometry. Four of the proteins were putative virulence-associated proteins: enolase, ATP-binding cassette transporter, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase class-II. The genes encoding the chaperone protein GroEL, enolase, ATP-binding cassette transporter, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase class-II were 1614, 1296, 1260, 1005 and 867 bp in length, and the nucleotide sequences homologies of the genes between the C43065 strain and the previously reported E. rhusiopathiae Fujisawa strain was more than 98%. [Conclusion] Several putative virulence-associated proteins in the NaOH-extracted antigen of E. rhusiopathiae C43065 will be useful for elucidating the roles of these proteins in the pathogenesis of the organism.

Abstract:[Objective] To construct heptyl glycosyltransferase gene II (waaF) gene deletion mutant of Vibrio parahaemolyticus, and explore the function of the waaF gene in Vibrio parahaemolyticus. [Methods] The waaF gene deletion mutant was constructed by chitin-based transformation technology using clinical isolates, and then the growth rate, morphology and serotypes were identified. The different sources (O3, O5 and O10) waaF gene complementations were constructed through E. coli S17λpir strains conjugative transferring with Vibrio parahaemolyticus, and the function of the waaF gene was further verified by serotypes. [Results] The waaF gene deletion mutant strain was successfully constructed and it grew normally. The growth rate and morphology of mutant were similar with the wild type strains (WT), but the mutant could not occurred agglutination reaction with O antisera. The O3 and O5 sources waaF gene complementations occurred agglutination reaction with O antisera, but the O10 sources waaF gene complementations was not. [Conclusion] The waaF gene was related with O-antigen synthesis and it was the key gene of O-antigen synthesis pathway in Vibrio parahaemolyticus. The function of different sources waaF gene were not the same.

Abstract:[Objective] AuMan5A is a glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii. To improve its enzymatic properties, we have previously constructed a mutant with loop substitution, AuMan5A/Af, by substituting a loop of seven residues (³¹⁶KSPDGGN³²²) in its substrate binding groove with the corresponding region (PSPNDHF) of A. fumigatus GH family 5 β-mannanase. To reveal the correlation between the superior enzymatic properties of AuMan5A/Af and its residue Asp³²⁰, site-directed mutagenesis was used to obtain a new mutant enzyme AuMan5A/Af D^320G. [Methods] Using megaprimer PCR method, we constructed a new mutant-encoding gene, Auman5A/Af ^D320G by mutating an Asp³²⁰-encoding codon GAC of Auman5A/Af into a Gly³²⁰-encoding GGT. Then, Auman5A/Af ^D320G was extracellularly expressed in Pichia pastoris GS115, and the enzymatic properties of the expressed product were analyzed. [Results] Analytical results indicated that the optimal and melting temperature of AuMan5A/Af D^320G was 70.0 ℃ and 71.5 ℃, repectively, higher than those of AuMan5A (T_opt=65.0 ℃, T_m=64.5 ℃) and lower than those of AuMan5A/Af (T_opt=75.0 ℃, T_m=76.6 ℃); its half-life at 70.0 ℃ was 40 min, 10 min longer than that of AuMan5A but greatly shorter than 480 min of AuMan5A/Af. Besides, its specific activity was 2.7 fold and 0.3 fold that of AuMan5A and AuMan5A/Af, respectively, and its catalytic efficiency (k_cat/K_m) was 3.9 fold and 0.3 fold that of AuMan5A and AuMan5A/Af. [Conclusion] The mutation of Asp³²⁰ into Gly³²⁰ greatly affected the temperature characteristics and catalytic activity of AuMan5A/Af, demonstrating that Asp³²⁰ plays an improtant role in temperature characteristics, specific activity and catalytic efficiency improving of AuMan5A after loop substitution.

Abstract:[Objective] We aimed at characterizing a malic enzyme isoform V from Mucor circinelloides. [Methods] me1 gene encoding malic enzyme isoform V was amplified and cloned into expression vector pET28a. High-purity recombinant protein BLME1 was obtained by affinity chromatography using Ni-NTA column and characterized subsequently. [Results] The optimum conditions were pH at 8.0 and temperature at 33 ℃. Under optimum conditions, BLME1 activity achieved 92.8 U/mg. The K_m for L-malate and NADP⁺ were 0.74960±0.06120 mmol/L and 0.22070±0.01810 mmol/L, the V_max for L-malate and NADP⁺ were 72.820±1.077 U/mg and 86.110±1.665 U/mg, respectively. In addition, ions played important roles in BLME1 activity; several ions such as Mn²⁺, Mg²⁺, Co²⁺, Ni²⁺ could activate BLME1, whereas Ca²⁺, Cu²⁺ could be used as inhibitors. Additionally, the metabolic intermediates such as oxaloacetic acid and α-ketoglutaric acid inhibited the activity of BLME1, whereas succinic acid activated it. [Conclusion] A malic enzyme isoform V from Mucor circinelloides was characterized, providing the references for further studies on this enzyme.

Abstract:[Objective] To isolate and identify Streptococcus agalactiae phages and screen candidate phages to control infection caused by bovine S. agalactiae. [Methods] We used two methods for isolation of S. agalactiae phages, namely (1) isolation of phages from milk and environmental samples, and (2) isolation of phages via induction of lysogens with Mitomycin C. Double-layer agar culture method was used to purify phages. Then the newly obtained phages, with S. agalactiae phage JX01 isolated from mastitis milk, were comparatively analyzed in the following aspects: morphology of phages by transmission electron microscopy, host range of phages to 55 S. agalactiae strains and other Streptococcus strains, phages DNA using EcoR I, Xba I, Pst I and Sal I, the optical multiplicity of infection, absorption curve and one step growth curve, and the stability of phages at different storage conditions. [Results] The comparative analysis of the 3 novel phages LYGO9, HZ04 and pA11 (induced from S. agalctiae bovine clinical isolate HAJL2011070601) with JX01 showed that the 4 phages were classified as the member of Siphovirdae family. EcoR I, Sal I, Xba I and Pst I separately digested the 4 phages DNA provided 4, 3, 3 and 2 profiles, respectively. This suggested that they were different strains. All the 4 phages specifically infected bovine S. agalactiae isolates. LYGO9, pA11, JX01 and HZ04 could lyse 12, 13, 20 and 23 of 42 tested bovine S. agalctiae isolates, respectively. This clearly indicated that these 4 phages are closely related. [Conclusion] The 3 new phages which specifically lyse bovine S. agalactiae isolates are siphovirus phages. Phage LYGO9 was shown having a short latent period and a larger burst size.