Genome editing reveals that pSCL4 is required for chromosome linearity in Streptomyces clavuligerus

Streptomyces clavuligerus is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid’s parB gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.

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Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase

Applied and Environmental Microbiology ◽

10.1128/aem.02608-17 ◽

2018 ◽

Vol 84 (6) ◽

Cited By ~ 34

Author(s):

Kaifeng Li ◽

Dongbo Cai ◽

Zhangqian Wang ◽

Zhili He ◽

Shouwen Chen

Keyword(s):

Genome Editing ◽

Bacillus Licheniformis ◽

High Efficiency ◽

Genetic Manipulation ◽

Single Gene ◽

Strain Improvement ◽

Future Research ◽

Widespread Application ◽

Content Type ◽

Dna Fragment

ABSTRACT Bacillus strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B. licheniformis DW2 with overexpression driven by the P43 promoter. The yvmC gene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragment bacABC (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene aprN , which codes for nattokinase in Bacillus subtilis , was inserted into the chromosome of B. licheniformis with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-S sacC strain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-S sacC . Finally, the engineered strain DWc9nΔ7 (Δ epr Δ wprA Δ mpr Δ aprE Δ vpr Δ bprA Δ bacABC ), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n in B. licheniformis . This tool could be applied to strain improvement for future research. IMPORTANCE As important industrial bacteria, Bacillus strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to be an efficient and precise tool in previous reports. The significance of our research is the development of an efficient, more precise, and systematic genome editing method for single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration in Bacillus licheniformis via Cas9 nickase. We also applied this method to the genetic engineering of the host strain for protein expression.

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Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System

mSphere ◽

10.1128/msphere.00130-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 68

Author(s):

Kyunghun Min ◽

Yuichi Ichikawa ◽

Carol A. Woolford ◽

Aaron P. Mitchell

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Genetic Analysis ◽

Genome Editing ◽

Drug Targets ◽

Gene Deletion ◽

Genetic Manipulation ◽

Content Type ◽

Biological Features ◽

Link Type

ABSTRACT The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.

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Treponema phagedenis (ex Noguchi 1912) Brumpt 1922 sp. nov., nom. rev., isolated from bovine digital dermatitis

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.004027 ◽

2020 ◽

Vol 70 (3) ◽

pp. 2115-2123 ◽

Cited By ~ 11

Author(s):

Peter Kuhnert ◽

Isabelle Brodard ◽

Maher Alsaaod ◽

Adrian Steiner ◽

Michael H. Stoffel ◽

...

Keyword(s):

Type Strain ◽

Type Species ◽

Sequence Data ◽

Whole Genome Sequence ◽

Valid Species ◽

Species Identity ◽

Digital Dermatitis ◽

Content Type ◽

Link Type ◽

Specific Pcr

‘ Treponema phagedenis ’ was originally described in 1912 by Noguchi but the name was not validly published and no type strain was designated. The taxon was not included in the Approved Lists of Bacterial Names and hence has no standing in nomenclature. Six Treponema strains positive in a ‘ T. phagedenis ’ phylogroup-specific PCR test were isolated from digital dermatitis (DD) lesions of cattle and further characterized and compared with the human strain ‘ T. phagedenis ’ ATCC 27087. Results of phenotypic and genotypic analyses including API ZYM, VITEK2, MALDI-TOF and electron microscopy, as well as whole genome sequence data, respectively, showed that they form a cluster of species identity. Moreover, this species identity was shared with ‘ T. phagedenis ’-like strains reported in the literature to be regularly isolated from bovine DD. High average nucleotide identity values between the genomes of bovine and human ‘ T. phagedenis ’ were observed. Slight genomic as well as phenotypic variations allowed us to differentiate bovine from human isolates, indicating host adaptation. Based on the fact that this species is regularly isolated from bovine DD and that the name is well dispersed in the literature, we propose the species Treponema phagedenis sp. nov., nom. rev. The species can phenotypically and genetically be identified and is clearly separated from other Treponema species. The valid species designation will allow to further explore its role in bovine DD. The type strain for Treponema phagedenis sp. nov., nom. rev. is B43.1T (=DSM 110455T=NCTC 14362T) isolated from a bovine DD lesion in Switzerland.

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Teredinibacter haidensis sp. nov., Teredinibacter purpureus sp. nov. and Teredinibacter franksiae sp. nov., marine, cellulolytic endosymbiotic bacteria isolated from the gills of the wood-boring mollusc Bankia setacea (Bivalvia: Teredinidae) and emended description of the genus Teredinibacter

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.004627 ◽

2021 ◽

Author(s):

Marvin A. Altamia ◽

J. Reuben Shipway ◽

David Stein ◽

Meghan A. Betcher ◽

Jennifer M. Fung ◽

...

Keyword(s):

Type Strain ◽

Type Species ◽

Sequence Data ◽

Amino Acid Identity ◽

Whole Genome Sequence ◽

Phenotypic Characterization ◽

Rrna Gene ◽

Bacterial Strains ◽

Content Type ◽

Link Type

Here, we describe three endosymbiotic bacterial strains isolated from the gills of the shipworm, Bankia setacea (Teredinidae: Bivalvia). These strains, designated as Bs08T, Bs12T and Bsc2T, are Gram-stain-negative, microaerobic, gammaproteobacteria that grow on cellulose and a variety of substrates derived from lignocellulose. Phenotypic characterization, phylogeny based on 16S rRNA gene and whole genome sequence data, amino acid identity and percentage of conserved proteins analyses, show that these strains are novel and may be assigned to the genus Teredinibacter . The three strains may be differentiated and distinguished from other previously described Teredinibacter species based on a combination of four characteristics: colony colour (Bs12T, purple; others beige to brown), marine salt requirement (Bs12T, Bsc2T and Teredinibacter turnerae strains), the capacity for nitrogen fixation (Bs08T and T. turnerae strains) and the ability to respire nitrate (Bs08T). Based on these findings, we propose the names Teredinibacter haidensis sp. nov. (type strain Bs08T=ATCC TSD-121T=KCTC 62964T), Teredinibacter purpureus sp. nov. (type strain Bs12T=ATCC TSD-122T=KCTC 62965T) and Teredinibacter franksiae sp. nov. (type strain Bsc2T=ATCC TSD-123T=KCTC 62966T).

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Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

mBio ◽

10.1128/mbio.00308-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 69

Author(s):

Ian R. Monk ◽

Jai J. Tree ◽

Benjamin P. Howden ◽

Timothy P. Stinear ◽

Timothy J. Foster

Keyword(s):

Staphylococcus Aureus ◽

Plasmid Dna ◽

High Efficiency ◽

Recombinant Dna ◽

Genetic Manipulation ◽

Bacterial Pathogen ◽

Type I ◽

Content Type ◽

E Coli ◽

Adenine Methylation

ABSTRACTStaphylococcus aureusis a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates ofS. aureus. Here, we describe the construction and characterization of the IMXXB series ofEscherichia colistrains that mimic the type I adenine methylation profiles ofS. aureusclonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of majorS. aureuslineages.IMPORTANCEThe genetic manipulation of clinicalS. aureusisolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset ofS. aureusstrains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite ofE. colistrains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer intoS. aureus. The IMXXB series ofE. colistrains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation ofS. aureus.

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Enteractinococcus coprophilus gen. nov., sp. nov., of the family Micrococcaceae , isolated from Panthera tigris amoyensis faeces, and transfer of Yaniella fodinae Dhanjal et al. 2011 to the genus Enteractinococcus as Enteractinococcus fodinae comb. nov.

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.034249-0 ◽

2012 ◽

Vol 62 (Pt_11) ◽

pp. 2710-2716 ◽

Cited By ~ 24

Author(s):

Yan-Ru Cao ◽

Yi Jiang ◽

Rong-Xian Jin ◽

Li Han ◽

Wen-Xiang He ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Type Strain ◽

New Genus ◽

Sequence Data ◽

Rrna Gene ◽

Panthera Tigris ◽

Content Type ◽

Link Type ◽

The Family ◽

Phosphatidylinositol Mannosides

A novel actinobacterium, designated strain YIM 100590T, was isolated from Panthera tigris amoyensis faeces collected from Yunnan Wild Animal Park in Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequence data showed that strain YIM 100590T is a member of the family Micrococcaceae . Cells were coccoid to oval (0.7–1.5 µm in diameter) occurring singly or in clusters. Growth was observed at 10–37 °C (optimum 28 °C) and at pH 7.0–11.0 (optimum pH 8.0). The major fatty acids were iso-C15 : 0 (32.22 %), anteiso-C15 : 0 (31.64 %) and iso-C16 : 0 (17.38 %). The peptidoglycan was of A4α type (l-Lys–Gly–l-Glu). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, dimannosyl diacylglycerol, an unknown glycolipid and two unknown phospholipids. The quinone system comprised menaquinones MK-7 (91.9 %) and MK-8 (8.3 %). The DNA G+C content of strain YIM 100590T was 56.2 mol%. Chemotaxonomic data indicated that the strain belongs to the family Micrococcaceae . On the basis of morphological and chemotaxonomic data and phylogenetic analysis, strain YIM 100590T is considered to represent a novel species of a new genus within the family Micrococcaceae , for which the name Enteractinococcus coprophilus gen. nov., sp. nov. is proposed. The type strain of Enteractinococcus coprophilus is YIM 100590T ( = DSM 24083T = JCM 17352T). Yaniella fodinae DSM 22966T was transferred to the new genus as Enteractinococcus fodinae comb. nov. (type strain G5T = DSM 22966T = JCM 17931T = MTCC 9846T).

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Transforming the Untransformable: Application of Direct Transformation To Manipulate Genetically Staphylococcus aureus and Staphylococcus epidermidis

mBio ◽

10.1128/mbio.00277-11 ◽

2012 ◽

Vol 3 (2) ◽

Cited By ~ 242

Author(s):

Ian R. Monk ◽

Ishita M. Shah ◽

Min Xu ◽

Man-Wah Tan ◽

Timothy J. Foster

Keyword(s):

Staphylococcus Aureus ◽

Staphylococcus Epidermidis ◽

High Efficiency ◽

Genetic Manipulation ◽

Laboratory Strain ◽

Small Subset ◽

Type I ◽

Major Barrier ◽

Content Type ◽

Functional Genomic Analysis

ABSTRACTThe strong restriction barrier present inStaphylococcus aureusandStaphylococcus epidermidishas limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain ofS. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiencyEscherichia colicloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates ofS. aureusandS. epidermidis. We also show that the loss of restriction (both type I and IV) in anS. aureusUSA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens.IMPORTANCEStaphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding ofStaphylococcus aureusandStaphylococcus epidermidisinfections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.

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Complete and Circularized Genome Assemblies of the Kroppenstedtia eburnea Genus Type Strain and the Kroppenstedtia pulmonis Species Type Strain with MiSeq and MinION Sequence Data

Microbiology Resource Announcements ◽

10.1128/mra.00650-20 ◽

2020 ◽

Vol 9 (44) ◽

Author(s):

Christopher A. Gulvik ◽

Dhwani Batra ◽

Lori A. Rowe ◽

Milli Sheth ◽

Ben W. Humrighouse ◽

...

Keyword(s):

Type Strain ◽

Sequence Data ◽

Gene Clusters ◽

Illumina Miseq ◽

Putative Gene ◽

Content Type ◽

Oxford Nanopore ◽

Species Type ◽

Genome Assemblies ◽

Oxford Nanopore Technologies

ABSTRACT Kroppenstedtia eburnea DSM 45196T and Kroppenstedtia pulmonis W9323T are aerobic, Gram-positive, filamentous, chemoorganotrophic thermoactinomycetes. Here, we report on the complete and circular genome assemblies generated using Illumina MiSeq and Oxford Nanopore Technologies MinION reads. Putative gene clusters predicted to be involved in the production of secondary metabolites were also identified.

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The Pathway-Specific Regulator ClaR of Streptomyces clavuligerus Has a Global Effect on the Expression of Genes for Secondary Metabolism and Differentiation

Applied and Environmental Microbiology ◽

10.1128/aem.00916-15 ◽

2015 ◽

Vol 81 (19) ◽

pp. 6637-6648 ◽

Cited By ~ 19

Author(s):

Yolanda Martínez-Burgo ◽

Rubén Álvarez-Álvarez ◽

Antonio Rodríguez-García ◽

Paloma Liras

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Aerial Mycelium ◽

Gene Clusters ◽

Streptomyces Clavuligerus ◽

Plasmid Copy Number ◽

Wild Type ◽

Plasmid Copy ◽

Content Type ◽

Expression Of Genes

ABSTRACTStreptomyces clavuligerusclaR::aphis aclaR-defective mutant, but in addition to itsclaRdefect it also carries fewer copies of the resident linear plasmids pSCL2 and pSCL4 (on the order of 4 × 105-fold lower than the wild-type strain), as shown by qPCR. To determine the function of ClaR without potential interference due to plasmid copy number, a new strain,S. clavuligerusΔclaR::aac, withclaRdeleted and carrying the wild-type level of plasmids, was constructed. Transcriptomic analyses were performed inS. clavuligerusΔclaR::aacandS. clavuligerusATCC 27064 as the control strain. The new ΔclaRmutant did not produce clavulanic acid (CA) and showed a partial expression of genes for the early steps of the CA biosynthesis pathway and a very poor expression (1 to 8%) of the genes for the late steps of the CA pathway. Genes for cephamycin C biosynthesis were weakly upregulated (1.7-fold at 22.5 h of culture) in the ΔclaRmutant, but genes for holomycin biosynthesis were expressed at levels from 3- to 572-fold higher than in the wild-type strain, supporting the observed overproduction of holomycin byS. clavuligerusΔclaR::aac. Interestingly, three secondary metabolites produced by gene clusters SMCp20, SMCp22, and SMCp24, encoding still-cryptic compounds, had partially or totally downregulated their genes in the mutant, suggesting a regulatory role for ClaR wider than previously reported. In addition, theamfRgene was downregulated, and consequently, the mutant did not produce aerial mycelium. Expression levels of about 100 genes in the genome were partially up- or downregulated in the ΔclaRmutant, many of them related to the upregulation of the sigma factor-encodingrpoEgene.

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Carboxyethylarginine Synthase Genes Show Complex Cross-Regulation in Streptomyces clavuligerus

Applied and Environmental Microbiology ◽

10.1128/aem.02600-12 ◽

2012 ◽

Vol 79 (1) ◽

pp. 240-249 ◽

Cited By ~ 2

Author(s):

Thomas Kwong ◽

Kapil Tahlan ◽

Cecilia L. Anders ◽

Susan E. Jensen

Keyword(s):

Deletion Mutant ◽

Chromosomal Location ◽

Streptomyces Clavuligerus ◽

Single Amino Acid ◽

Wild Type ◽

Content Type ◽

Single Amino Acid Residue ◽

Mild Reduction ◽

Low Levels ◽

A Site

ABSTRACTCarboxyethylarginine synthase is the first dedicated enzyme of clavam biosynthesis inStreptomyces clavuligerusand is present in two isoforms encoded by two separate genes. When grown on a liquid soy medium, strains withceaS1deleted showed only a mild reduction of clavam biosynthesis, while disruption ofceaS2abolished all clavam biosynthesis. Creation of an in-frameceaS2deletion mutant to avoid polarity did not restore clavam production, nor did creation of a site-directed mutant altered only in a single amino acid residue important for activity. Reverse transcriptase PCR analyses of these mutants indicated that the failure to produce clavam metabolites could be traced to reduced or abolished transcription ofceaS1in theceaS2mutants, despite the location ofceaS1on a replicon completely separate from that ofceaS2. Western analyses further showed that the CeaS1 protein (as well as the CeaS2 protein) was absent from theceaS2mutants. Complementation experiments were able to restore clavam production partially, but only by virtue of restoring CeaS2 production. CeaS1 was still absent from the complemented strains. While this dependence of CeaS1 production on the expression ofceaS2from its native chromosomal location was seen in all of theceaS2mutants, the effect was limited to growth in liquid medium. When the same mutants were grown on solid soy medium, clavam production was restored and CeaS1 was produced, albeit at low levels compared to the wild type.

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