Abstract:[Background] The final step of the peptide nucleoside antibiotic Blasticidin S (BS) biosynthesis pathway is the leucylblasticidin S (LBS) hydrolyzation to BS. The intact cells of BS native producer Streptomyces griseochromogenes and the heterologous producer Streptomyces lividans WJ2 can catalyze this step. Earlier work showed that both strains encode three PepN homologues, of which PepN1 from each strain is mainly responsible for the hydrolysis of LBS and leucyldemethylblasticidin S (LDBS) at comparable catalytic efficiency. However, comparing with the native producer that only synthesizes BS, WJ2 can also generate LBS and LDBS. This result indicates that the competence of PepN1 is different in these two producers. [Objective] Investigation of the effects of the expression level and localization of PepN1 on the productivity and components of BS and derivatives in two producers. [Methods] The concentration of PepN1 in the cell lysate and in the cell culture medium of two BS producers at different growth time were tracked and compared by Western blot using PepN1 polyclonal antibody. The protein in the culture medium of the two strains was collected by ammonium sulfate precipitation, and the activity of hydrolyzed LDBS was assayed in vitro. [Results] On one hand, the content of PepN1 in the native strain didn’t change from the 2nd to 6th day, whereas PepN1 started to diminish from the 4th day and totally disappeared on the 6th day in the heterologous strain. On the other hand, PepN1 was detected in the culture medium of native producer rather than the heterologous strain, which was consistent with the different activity of two strain’s cell culture medium in hydrolyzing LDBS. [Conclusion] The BS native producer can continuously express PepN1 and export part of PepN1 outside of the cell. On the contrary, the heterologous producer WJ2 cease to synthesize PepN1 from the 4th day and it can’t be detected in the culture medium, leading to the accumulation of leucylated intermediates. Given that intact cells of two strains are capable of hydrolyzing LDBS and LBS, PepN1 that diminished from the cell lysate might secrete to the cell wall, but not release into the medium, this part of activity accounts for the generation of DBS and BS in WJ2.

Abstract:[Background] Burkholderia pseudomallei is an intracellular parasitic bacterium that can cause human disease and even death. The type III secretory system plays an important role in the bacterial invasion of epithelial cells, escaping host immunity and the secretion of virulence factors. The bopA gene is an important effector protein encoded by the TTSS-3 gene, and it plays an important role in the immune escape of the Burkholderia pseudomallei. [Objective] To construct Burkholderia pseudomallei bopA gene knockout mutant strain, and evaluate the biological characteristics of the mutant strain. [Methods] We constructed suicide plasmid pK18mobSacB-ΔbopA, and then transformed the plasmid into B. pseudomallei from Escherichia coli S17-1λpair by conjugation. bopA gene was knocked out by homologous recombination, and the mutant strain was selected by sucrose agar screening. The phenotypic variation of the mutant strain was finally evaluated at the cell and animal levels. [Results] We constructed B. pseudomallei bopA mutant strain successfully, and found the invasion rate, intracellular survival ability as well as the ability of colonization in vivo of mutant strain was significantly reduced. [Conclusion] We constructed B. pseudomallei bopA mutant strain by homologous recombination, to provide a basis for further understanding of this gene.

Abstract:[Background] Mitogen-activated protein kinase (MAPK) signaling pathway of plant pathogenic fungi is involved in sexual reproduction, cell wall integrity, mycelial infection, pathogenicity, stress response and other processes. The MAPK signaling pathway is involved in the growth, development, pathogenicity and stress response in Botrytis cinerea. However, the function of MAPK signaling pathway gene has not been fully elucidated in B. cinerea. [Objective] The objective of this study is to analyze the function of B. cinerea MAPK encoding genes bmp1 and bmp3 in growth, development, pathogenicity and response to oxidative stress, and to lay a foundation for clarifying the molecular mechanism of the MAPK signaling pathway in growth, development and pathogenicity in B. cinerea. [Methods] RNAi mutants of bmp1 and bmp3 genes were successfully constructed using RNAi technology. Compared with the wild-type strain BC22 (WT), phenotype, pathogenicity, and sensitivity to oxidative stress of bmp1 and bmp3 genes RNAi mutants were analyzed. [Results] The colony morphology and mycelia morphology of bmp1 and bmp3 gene RNAi mutants showed no obvious difference with WT. The bmp1 gene RNAi mutants grew slowly and produce fewer conidial. The growth rate of bmp3 gene RNAi mutants showed no obvious difference with WT. The bmp3 gene RNAi mutants did not produce conidia. RNAi mutants of bmp1 and bmp3 genes showed no pathogenicity and penetrating ability. RNAi mutants of bmp1 gene were significantly less inhibited than those of WT in the media containing H2O2, while the degree of inhibition was significantly higher in the media containing menadione than that of WT. RNAi mutants of bmp3 gene were significantly inhibited in H2O2 and menadione media than that of WT. [Conclusion] The bmp1 gene positively regulated growth, conidial formation, pathogenicity and penetrability, and was involved in the regulation of the response to oxidative stress in B. cinerea. The bmp3 gene positively regulates conidial formation, pathogenicity, penetrability, and response to oxidative stress in B. cinerea.

Abstract:[Background] Pseudomonas chlororaphis GP72 is a rhizobacteriaum that could produce phenazine antibiotics containing phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). Based on analyzing the whole-genome sequence, aurI/aurR regulatory system was found existed in GP72. [Objective] This article aims to study the regulation of aurI/aurR on phenazine production in GP72. [Methods] The aurI gene was heterologously expressed in Escherichia coli, then Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NTL4 were used in plate assays. The knockout strains and complemental strains of aurI and aurR were constructed, and the growth curve and phenazine production of mutants were measured by fermentation. A transcriptional fusion plasmid was used to assess transcription level of promoter of phenazine synthesis gene cluster. [Results] The results of plate assays showed that aurI could produce a variety of signal molecules that could produce blue pigment in NTL4 and purple pigment in CV026. Fermentation results showed phenazine production of knock-out gene mutants were raised four times, and phenazine production of the complemental strains of aurI and aurR were reduced to the wild type level. It was found that the β-galactosidase activity fused with promoter of phenazine synthesis genes in the knock out strains were higher than that of the wild type. [Conclusion] aurI/aurR is a pair of negative regulation genes for phenazine in GP72. It could inhibit the transcription level of promoter of phenazine synthesis gene cluster, thereby affecting phenazine production.

Abstract:[Background] The cutR/cutS two component system plays a significant role in secondary metabolite in Streptomyces. [Objective] The aim of the study is to investigate the function of the cutR/cutS two component system in the production of lomofungin in Streptomyces lomondensis S015. [Methods] HPLC was used to analysis fermentation production, meanwhile accessing quantitative real-time PCR to monitor the levels of gene expression. [Results] HPLC results indicated that the yield of lomofungin in S015ΔcutR and S015ΔcutS reach the total amount of 128.1±26.4 mg/L and 61.8±4.5 mg/L respectively, that is 11.5 and 5.5 times of the yield of wild type. Results of qPCR indicated that in S015ΔcutR mutant the relative gene expression of lomo14, lomo10, lphzB, lphzC, lphzE and lphzG reached respectively 1 151.7±88.8, 110.5±5.8, 129.3±7.7, 380.2±34.6, 348.2±42.1 and 299.8±38.2 times of the wild type, and in S015ΔcutS mutant the relative gene expression of lomo14, lomo10, lphzB, lphzC, lphzE and lphzG were respectively 4.3±0.5, 2.2±0.2, 9.3±0.9, 10.3±0.6, 20.7±1.5 and 20.4±0.8 times. [Conclusion] The study shows that the cutR and cutS negatively regulate several main synthetic genes and side-chain modification genes of lomofungin production in S. lomondensis, thereby reducing the production.

Abstract:[Background] Bimolecular fluorescence complementation (BiFC) is applied to explore the protein-protein interactions in some organisms such as Fusarium fujikuroi, but not yet applied in Aspergillus oryzae. [Objective] We used BiFC system to detect the interactions between proteins Fus3 and Ste12 in Aspergillus oryzae during growth and development. We used this system to specifically visualize the location of the target proteins in living cells. [Methods] Complex vectors were constructer by using the Multisite Gateway cloning system. Proteins Fus3 and Ste12 were fused to C- and N-terminal green fluorescence protein fragments, respectively, and then the hyphae of the obtained transformants were observed under a fluorescence microscope. [Results] The green fluorescence protein was observed in successfully transformed cells, indicating that the interaction occurred between Fus3 and Ste12 happened in A. oryzae. [Conclusion] The method to detect protein-protein interaction in A. oryzae proves its broader application possibility.

Abstract:[Background] Corynebacterium glutamicum is an important industrial platform strain. Efficient and convenient genetic manipulation tools is in urgent need for improving C. glutamicum strains in commercial applications. [Objective] Gene argR and argF of C. glutamicum ATCC 13032 was deleted by CRISPR-Cpf1-assisted-homologous recombination and Cre/loxP-assisted-homologous recombination knockout system respectively. To provide guidance for reasonable selection of gene knockout systems, the advantages and disadvantages of these two methods are investigated in detail. [Methods] Firstly, Cre/loxP-assisted-homologous recombination replaced the targeted gene in genome with kanR fragment containing loxP sites at 5′ and 3′ ends. Then recombinase Cre expressed by pDTW109 recognized and bound on the loxP sites, and catalyzed the homologous recombination between two loxP sites to remove the kanR fragment. Eventually, the pDTW109 was eliminated by elevating the temperature to 37 °C based on its temperature-sensitive characteristic. In the knockout of targeted gene by CRISPR-Cpf1-assisted genome editing, the pre-crRNA was processed by Cpf1 and the resultant crRNA guided Cpf1 to bind to the specific sequence and cleave the target DNA. The targeted gene was removed by homologous recombination. The recombinant plasmid was also eliminated by elevating the cultivation temperature. [Results] In the genes knockout of C. glutamicum, Cre/loxP-assisted system allowed a complete N rounds of iterative gene knockout in 8N+2 d, while CRISPR-Cpf1-assisted system only need 5N+2 d. Theoretically, the latter could achieve the simultaneous knockout of multiple genes, however suffers from disadvantage of low homologous recombination efficiency and high false-positive rates. [Conclusion] In comparison with Cre/loxP system, CRISPR-Cpf1 assisted genome editing is time-saving and labor-saving in gene knockout of C. glutamicum, and theoretically it can be employed in knockout of multiple genes at a time. Consequently, CRISPR-Cpf1-assisted system has higher overall efficiency. However, its editing efficiency still has great potentials for improvement.

Abstract:[Background] The interaction between species is an important driving force for the evolution of species. But it is challenging to determine the single nucleotide polymorphism (SNPs) that play an important role in the process of interaction between species and establish an accurate genotype-phenotype map. [Objective] To obtain the significant SNPs that play an important role in the interaction between Escherichia coli and Staphylococcus aureus and to detect how do these SNPs relate to each other over time. [Methods] Real-time quantitative PCR (qPCR) was used to determine the abundance of all strains. We reared E. coli and S. aureus in monoculture and compared to the difference of growth between the same strain in monoculture and co-culture. The phenotypes and genotypes of all strains were analyzed by systems mapping and the significant SNPs were found out. [Results] We obtained a three-dimensional Manhattan plot that identifies 54 significant combinations of SNPs derived from 41 SNPs in E. coli and 12 SNPs in S. aureus. Among them, 6 genes we obtained by annotation can directly or indirectly affect the growth of microorganisms, nhaR (E19056) was involved in the formation of biofilm, rhlE (E832164) is related to ribosome assembly, the expression of csiD (E2789300) can make cells face stress environment, alkB (E2309274) can participate in DNA damage repair, sucA (E759230) and yjjW (E4614704) are involved in the metabolic process of cells. [Conclusion] Systems mapping can detect the significant SNPs. The genetic effects of different SNPs are variable in the process of species interaction. Bacterial interaction was found to exert direct genetic effects, indirect genetic effects and interspecific epistatic effects.

Abstract:微生物遗传学是一门具有悠久历史的学科，微生物遗传学研究不仅为遗传学中一些基本理论的阐明奠定了基础，还有力推动了分子生物学的发展。从DNA是遗传物质的证明，到“一个基因一种酶”的假说，再到近年来的CRISPR/Cas9基因编辑技术，都离不开微生物遗传学的发展。随着基因组测序技术的迅猛发展，微生物遗传学也迎来了全面快速发展的时期，我国微生物遗传学研究在与微生物生理代谢、微生物组学、环境微生物学、合成生物学等学科的交叉融合中取得了长足进步。《微生物学通报》本期推出了“微生物遗传学主题刊”，旨在展现我国微生物遗传学研究的最新进展和成果，促进我国微生物遗传学的交流和发展。

Abstract:Drug tolerance is the capacity of genetically susceptible bacteria to survive the killing by bactericidal antibiotics. At population level, drug tolerance can result in slow killing kinetics or failure of sterilization by antibiotic treatment. Drug tolerance can stem from phenotypic heterogeneity or from environmentally induced stress response at population level. While various genes and pathways were shown to be implicated in bacterial drug tolerance, it is becoming clear that the common mechanism underlies the survival of antibiotic-tolerant cells is the alteration of cellular growth or metabolic state achieved by either regulation or genetic mutation, which counteracts or diminishes the killing effect induced by the drug-target interaction. Increasing clinic evidence shows that drug tolerance is a causative reason account for the requirement of lengthy treatment and the high relapse rate observed in persistent infections. Importantly, recent studies demonstrated that drug tolerance could accelerate the emergency of drug-resistant mutants. Therefore, deciphering the molecular mechanisms of antibiotic tolerance may shed light on our understanding of antibiotic killing and the adaptive evolution of antibiotic resistance, and could facilitate the development of new intervention agents and therapeutic strategies.

Abstract:Bacterial resistance is very common in China, multi-drug and even pan-resistant strains continue to emerge, posing a major threat to public health and food safety. Along with the development of human activities and animal husbandry, the medical industry and aquaculture have resulted in a large number of drug resistance/resistant bacteria in farmed animals and their related environments. The medical industry, animal breeding, and the natural environment are mutually interacting with each other in the spread and development of drug-resistant bacteria. Drug-resistant genes can be circulated in humans, animals and the environment by means of gene level transfer, increasing the risk of humans taking resistance genes. Faced with such public health problems, traditional singular health work systems are unable to effectively solve such challenges, and multi-disciplinary cooperation is urgently needed. In this paper, bacterial resistance in clinical, animal and environment of China and the transmission and genetic mechanism of drug-resistant bacteria are reviewed to provide reference for bacterial resistance research.

Abstract:Base editors are novel genome-editing tools developed in the past two years that comprise fusions between a catalytically disabled CRISPR/Cas endonuclease and a base deaminase to deaminate the exocyclic amine of the target bases, thereby leading to base substitutions in DNA or RNA. Two classes of base editors have been developed, namely DNA base editors and RNA base editors. Two types of DNA editors have been described: cytosine base editors (CBEs) convert C to T and adenine base editors (ABEs) convert A to G. Base editors do not create double-strand DNA breaks (DSBs) and do not rely on cellular non-homologous end joining (NHEJ) and homology-directed repair (HDR), so they minimize the generation of DSB-associated by products, such as small insertions or deletion (indels). RNA base editors based on CRISPR/Cas systems could achieve adenosine conversion to inosine. In this review, we summarize the development process, scope of application and editing features of base editors and highlight their recent applications in bacterial genome editing. Finally, we will also briefly discuss limitations and future directions of base editors for applications in bacteria.

Abstract:Kin discrimination provides a means for bacterial individuals to identify self from non-self, and is thus the basis and prerequisite for the competition and cooperation among bacterial cells. By kin discrimination, competitive non-self-neighbors are separated from each other or are eliminated from self-populations, whereas the kin groups perform some exclusive social behaviors, such as social movement, biofilm formation and fruiting-body morphogenesis. Kin discrimination is thus suggested to aid the co-survival of different kin groups in nature. Recently, kin discrimination is becoming a hot spot in bacteriological studies. This review summarizes recent progresses in the mechanisms for bacterial kin discrimination and interactions.

Abstract:Protein secretion systems are crucial tools that microbes use to interact with their surrounding environment. The type VI protein secretion system (T6SS) is widely distributed in Gram-negative bacteria and capable of delivering effector to both eukaryotic and prokaryotic cells. Here we discuss, from effector study perspective, the mechanism of substrate recognition and translocation by T6SS, focusing on the key discoveries of VgrG-PAAR (proline-alanine-alanine-aRginine) dependent secretion pathway, and its cognate effectors and chaperone proteins.

Abstract:In recent years, with the emergence and development of the high-throughput next generation sequencing technology, the cost of sequencing decreases continuously and the number of fungal species with complete genome sequence increases rapidly. Population genomics based on large-scale sequencing has also been gradually used in dissecting population structure, speciation, population divergence and locus-specific effects in fungi. In this review, we summarize the progress of population genomics in industrial fungi, pathogenic fungi, edible fungi, symbiotic fungi and genetic architecture dissection of phenotypic traits, and discuss future development.

Abstract:DNA replication is one of the fundamental processes of all lives. Errors produced by DNA replication and abnormalities in its regulation are major sources of genomic instability in cells, leading to abnormal cell growth, aging, tumorigenesis and even death. In order to ensure accurate and complete duplication of genomic DNA, DNA replication is strictly regulated in all eukaryotes. In the G1 phase, the core component of the DNA replicative helicase——Mcm2-7 is recruited to the origins. This is called replication licensing. After cells entering the S phase, the helicase co-activators Cdc45 and GINS are recruited to Mcm2-7, forming the helicase holoenzyme Cdc45-Mcm2-7-GINS (CMG) complex. Subsequently, numerous replicating proteins are recruited to the CMG platform under precise spatiotemporal control and assembled into a replication machine to initiate bidirectional replication. When two converging replication forks encounter each other, CMGs are displaced to terminate the progression of these forks. The last decade has witnessed the leap-forward breakthroughs in this field, particularly in the model organism Saccharomyces cerevisiae. Here, we summarize the recent advances in eukaryotic DNA replication with a focus on the motor of replisome, DNA helicase CMG.

Abstract:Pseudomonas aeruginosa is a major opportunistic pathogen in clinic and can produce a variety of virulence factors and high antibiotic resistance. Type III secretion system (T3SS) is an important virulence factor secretion system that facilitates the delivery of bacterial effector proteins into eukaryotic host cells. Successful evasion of phagocytosis and subversion of normal host cell physiology to promote pathogenesis depend primarily on the presence of T3SS. Research on T3SS will help us understand the pathogenesis of P. aeruginosa and provide a theoretical basis for clinical treatment and the development of novel antipathogenic drugs. In this review, the structure, function, regulation mechanism and targeted treatment of T3SS in P. aeruginosa are discussed.

Abstract:Gas-fermenting clostridia is a major class of chemoautotrophs that uses CO2 and CO to synthesize a variety of chemicals and fuels, thereby exhibiting good industrial application prospects. Wild-type gas-fermenting Clostridium species are inefficient in gas uptake, fixation and conversion and energy metabolism, and moreover, incompetent to produce high value products. In recent years, with the rapid development of omics, molecular genetic tools and biochemical techniques in gas-fermenting clostridia of which the physiology and metabolism as well as the underlying molecular mechanisms, metabolic design and engineering, and fermentation technologies have been widely studied. Here, we review the latest research progresses in gas-fermenting clostridia to provide information on these industrial microorganisms and bio-utilization of one-carbon gases.

Abstract:Symbiosis between rhizobia and legumes is not only very important for the sustainable agriculture but also known as one of the model systems to study the mutualism between prokaryotes and eukaryotes. For a long time, studies on symbiotic nitrogen fixation of rhizobia have mainly focused on the role of key symbiosis genes involved in biosynthesis and regulation of Nod factors and nitrogenase. However, receiving these key genes does not guarantee rhizobia an effective symbiosis with legumes. Along with the rapid development and application of comparative and functional genomics, accumulated evidences indicate that rhizobia have recruited many lineage-specific dispensable genes to establish effective symbiosis with legumes, further revealing the complexity of mutualistic symbiosis. Here we review the cumulative evidence from recent genomics, genetics and experimental evolution studies to discuss the evolution of this complex symbiotic trait.

Abstract:Sulfolobus islandicus is a commonly used type strain to study replication, cell cycle and CRISPR-Cas system in archaea. For this reason, it is important to establish a genetic manipulation system in Sulfolobus islandicus. Here, we describe the plasmid vector, selection marker and transformation methods, and discuss two currently widely used gene knockout systems. Meanwhile, we also address some major problems in genetic manipulation systems of Sulfolobus islandicus, and indicate development prospects.

Abstract:Actinomycetes are an important source of natural antibiotics, and have a wide variety of transcription factors that precisely control the biosynthesis of antibiotics as secondary metabolites. As an important family of prokaryotic single-component signaling systems, TetR family transcriptional regulators (TFRs) are involved in the regulation of multiple cellular activities such as antibiotic biosynthesis, drug efflux, primary metabolism. On the basis of the recent studies in our lab and the research advances published, we review here the molecular regulatory mechanism of TFRs involved in the biosynthesis of several important antibiotics from the perspective of regulated target genes, and outlined the ligands of TFRs. Finally, this review summarizes and indicates the application of TFRs in increasing antibiotic production, activating silent cryptic gene clusters and artificially exploiting synthetic biology elements.

Abstract:Tuberculosis (TB) is still one of the leading causes of death worldwide. Due to the ever-growing problem of Mycobacterium tuberculosis (Mtb) drug resistance, TB is still not well controlled. There is an urgent need to develop new technology for promoting Mtb basic research, and to reveal new mechanisms, identify new targets, and develop new anti-Mtb drugs. Bore this need in mind, a Mtb functional proteome microarray was constructed to promote basic research. Considering the high virulence of Mtb, long replication cycle, it is very difficult to perform Mtb related studies, thus efficient tool is need. The proteome microarray is a powerful tool for the researchers in the field of TB study. It has already been successfully applied in a wide range of studies. Better understandings of a variety of key biological processes have already been added, for example, Mtb-host interactions, small-molecule-protein binding and mechanism of antibiotic resistance. In order to help readers to better understand this powerful tool, we present several major applications of the Mtb functional proteome microarray in this review, we anticipate researchers will adopt this powerful tool for a variety of TB related studies, thus facilitate both basic and clinical research of TB.

Abstract:Atypical angucyclines are a class of aromatic polyketides with unique structures and formed via oxidative rearrangement reactions. Recent studies have demonstrated that their various skeletons are derived from a common biosynthetic intermediate dehydrorabelomycin. A unique family of oxygenases, atypical angucycline ring-opening oxygenases, catalyzes the oxidative carbon-carbon bond cleavage and rearrangement reactions of dehydrorabelomycin, which is a critical step in the biosynthesis of these compounds. Although these ring-opening oxygenases belong to the same protein family, and catalyze the oxidative ring-opening reaction of the same substrate dehydrorabelomycin, different rearrangement reactions render different structures of the products, which are consistent with the skeletons of their final products. The in-depth study on the catalysis of this family of oxygenases will not only contribute to the structural modification of known aromatic polyketides and the discovery of polyketides with novel skeletons, but also help to deepen our understanding of protein sequence evolution and functional diversities.