Journal of Microbiology

Protocol

A guide to genome mining and genetic manipulation of biosynthetic gene clusters in Streptomyces: Heonjun Jeong, YeonU Choe, Jiyoon Nam, Yeon Hee Ban; J. Microbiol. 2025;63(4):e2409026. Published online April 29, 2025; DOI: https://doi.org/10.71150/jm.2409026

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Abstract PDF: Streptomyces are a crucial source of bioactive secondary metabolites with significant clinical applications. Recent studies of bacterial and metagenome-assembled genomes have revealed that Streptomyces harbors a substantial number of uncharacterized silent secondary metabolite biosynthetic gene clusters (BGCs). These BGCs represent a vast diversity of biosynthetic pathways for natural product synthesis, indicating significant untapped potential for discovering new metabolites. To exploit this potential, genome mining using comprehensive strategies that leverage extensive genomic databases can be conducted. By linking BGCs to their encoded products and integrating genetic manipulation techniques, researchers can greatly enhance the identification of new secondary metabolites with therapeutic relevance. In this context, we present a step-by-step guide for using the antiSMASH pipeline to identify secondary metabolite-coding BGCs within the complete genome of a novel Streptomyces strain. This protocol also outlines gene manipulation methods that can be applied to Streptomyces to activate cryptic clusters of interest and validate the functions of biosynthetic genes. By following these guidelines, researchers can pave the way for discovering and characterizing valuable natural products.

Research Support, Non-U.S. Gov'ts

Molecular Characterization of Atoxigenic Aspergillus flavus Isolates Collected in China: Dandan Wei , Lu Zhou , Jonathan Nimal Selvaraj , Chushu Zhang , Fuguo Xing , Yueju Zhao , Yan Wang , Yang Liu; J. Microbiol. 2014;52(7):559-565. Published online May 30, 2014; DOI: https://doi.org/10.1007/s12275-014-3629-8

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Aspergillus flavus strains were isolated from peanut fields of Liaoning, Shandong, Hubei and Guangdong Provinces in China, and identified through phenotypic and molecular approaches. Of the 323 A. flavus strains isolated, 76 strains did not produce aflatoxins detectable by UPLC. The incidence of atoxigenic A. flavus strains decreased with increase in temperature and increased with increase in latitude in different geographical locations. Amplification of all the aflatoxin genes in the aflatoxin gene cluster in the atoxigenic isolates showed that there were 25 deletion patterns (A-Y), with 22 deletion patterns identified for the first time. Most of the atoxigenic A. flavus isolates with gene deletions (97%) had deletions in at least one of the four genes (aflT, nor-1, aflR, and hypB), indicating that these four genes could be targeted for rapid identification of atoxigenic strains. The atoxigenic isolates with gene deletions, especially the isolates with large deletions, are potential candidates for aflatoxin control.

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High sequence variations in the region containing genes encoding a cellular morphogenesis protein and the repressor of sexual development help to reveal origins of Aspergillus oryzae
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Identification and Characterization of Ectoine Biosynthesis Genes and Heterologous Expression of the ectABC Gene Cluster from Halomonas sp. QHL1, a Moderately Halophilic Bacterium Isolated from Qinghai Lake: Derui Zhu , Jian Liu , Rui Han , Guoping Shen , Qifu Long , Xiaoxing Wei , Deli Liu; J. Microbiol. 2014;52(2):139-147. Published online February 1, 2014; DOI: https://doi.org/10.1007/s12275-014-3389-5

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Abstract

The moderately halophilic bacterium Halomonas sp. QHL1 was identified as a member of the genus Halomonas by 16S rRNA gene sequencing. HPLC analysis showed that strain QHL1 synthesizes ectoine in its cytoplasm. The genes involved in the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC. Subsequently, the ectB gene from this strain was amplified by PCR, and the entire ectABC gene cluster (3,580 bp) was cloned using genome walking. Analysis showed that the ectA (579 bp), ectB (1269 bp), and ectC (390 bp) genes were organized in a single transcriptional unit and were predicted to encode three peptides of 21.2 kDa, 46.4 kDa, and 14.7 kDa, respectively. Two putative promoters, a δ70-dependent promoter and a δ38-controlled promoter, as well as several conserved motifs with unknown function were identified. Individual ectA, ectB, and ectC genes, and the entire ectABC gene cluster were inserted into the expression plasmid pET-28a(+) to generate the recombinant plasmids pET-28a(+)-ectA, pET-28a(+)-ectB, pET-28a(+)-ectC and pET-28a(+)-ectABC, respectively. Heterologous expression of these proteins in Escherichia coli BL21 (DE3) was confirmed by SDS-PAGE. The recombinant E. coli strain BL21 (pET-28a (+)-ectABC) displayed a higher salt tolerance than native E. coli cells but produced far less ectoine than the wild-type QHL1 strain.

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Comparative Genome Analysis of a Novel Alkaliphilic Actinobacterial Species Nesterenkonia haasae
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Lisa Stiller, Erwin Galinski, Elisabeth Witt
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Constanze Paulus, Yuriy Rebets, Josef Zapp, Christian Rückert, Jörn Kalinowski, Andriy Luzhetskyy
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Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis
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TBC: A Clustering Algorithm Based on Prokaryotic Taxonomy: Jae-Hak Lee , Hana Yi , Yoon-Seong Jeon , Sungho Won , Jongsik Chun; J. Microbiol. 2012;50(2):181-185. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1214-6

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Abstract: High-throughput DNA sequencing technologies have revolutionized the study of microbial ecology. Massive sequencing of PCR amplicons of the 16S rRNA gene has been widely used to understand the microbial community structure of a variety of environmental samples. The resulting sequencing reads are clustered into operational taxonomic units that are then used to calculate various statistical indices that represent the degree of species diversity in a given sample. Several algorithms have been developed to perform this task, but they tend to produce different outcomes. Herein, we propose a novel sequence clustering algorithm, namely Taxonomy-Based Clustering (TBC). This algorithm incorporates the basic concept of prokaryotic taxonomy in which only comparisons to the type strain are made and used to form species while omitting full-scale multiple sequence alignment. The clustering quality of the proposed method was compared with those of MOTHUR, BLASTClust, ESPRITTree, CD-HIT, and UCLUST. A comprehensive comparison using three different experimental datasets produced by pyrosequencing demonstrated that the clustering obtained using TBC is comparable to those obtained using MOTHUR and ESPRIT-Tree and is computationally efficient. The program was written in JAVA and is available from http://sw. ezbiocloud.net/tbc.

Ruminococcus faecis sp. nov., Isolated from Human Faeces: Min-Soo Kim , Seong Woon Roh , Jin-Woo Bae; J. Microbiol. 2011;49(3):487-491. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0505-7

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Abstract: Bacterial strain Eg2T, an anaerobic, Gram-positive, non-motile, and non-spore-forming coccus, was isolated from human faeces. The optimal temperature for its growth was 37°C. Oxidase activity was negative, but catalase activity was positive. The strain was able to hydrolyze esculin and to produce acids from the fermentation of several substrates, including glucose. Lactic and acetic acids were the main products of glucose fermentation. The major fatty acids present in this strain were C16:0, C14:0, and C18:1 cis11 DMA. The G+C content was 43.4 mol%. Based on the 16S rRNA gene sequence, strain Eg2T was closely related to species of the genus Ruminococcus (96.3% similarity to R. torques and 96.2% similarity to R. lactaris), and its taxonomic position was placed within the Clostridium cluster XIVa. Based on phenotypic, chemotaxonomic, genotypic, and phylogenetic evidence, we propose that this novel strain be assigned to the genus Ruminococcus and be named Ruminococcus faecis sp. nov. The type strain is Eg2T (=KCTC 5757T =JCM 15917T).

Genetic Diversity of Chromosomal Metallo-β-Lactamase Genes in Clinical Isolates of Elizabethkingia meningoseptica from Korea: Jong Hwa Yum , Eun Young Lee , Sung-Ho Hur , Seok Hoon Jeong , Hyukmin Lee , Dongeun Yong , Yunsop Chong , Eun-Woo Lee , Patrice Nordmann , Kyungwon Lee; J. Microbiol. 2010;48(3):358-364. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9308-5

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Abstract: This study was performed to characterize the chromosomal metallo-β-lactamases (MBLs) of Elizabethkingia meningoseptica isolated from Korea and to propose a clustering method of BlaB and GOB MBLs based on their amino acid similarities. Chromosomal MBL genes were amplified by PCR from 31 clinical isolates of E. meningoseptica. These PCR products were then cloned into a vector and electrotransformed into E. coli DH5α. Nucleotide sequencing was performed by the dideoxy chain termination method using PCR products or cloned DNA fragments. Antimicrobial susceptibilities were determined by the agar dilution method. PCR experiments showed that all 31 E. meningoseptica isolates contained both the blaB and the blaGOB genes. DNA sequence analysis revealed that E. meningoseptica isolates possessed seven types of blaB gene, including five novel variants (blaB-9 to blaB-13) and 11 types of blaGOB gene, including 10 novel variants (blaGOB-8 to blaGOB-17). The most common combination of MBL was BlaB-12 plus GOB-17 (n=19). Minimum inhibitory concentrations of imipenem and meropenem for the electrotransformants harboring novel BlaB and GOB MBLs were two- or four-fold higher than those for the recipient E. coli DH5α. BlaB and GOB MBLs were grouped in three and six clusters including fifteen novel variants, respectively, based on amino acid similarities.

Cloning and Characterization of the Gene Cluster for Biosynthesis of Ectoine from Nesterenkonia halobia DSM 20541: Bo Zhang , Xin Bao , Lei Wang , Su Sheng Yang; J. Microbiol. 2008;46(3):309-318. Published online July 5, 2008; DOI: https://doi.org/10.1007/s12275-008-0001-x

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Abstract: The ectABC genes encoding the biosynthesis of ectoine were identified from Nesterenkonia halobia DSM 20541. The intergenic regions of the ectABC genes from N. halobia DSM 20541 were more loosely spaced than those that had been reported before. The amino acid sequence deduced from ectABC of the strain was highly homologous to the EctABC of Brevibacterium linens BL2 (EctA 50%, EctB 70%, and EctC 68% identities). The osmoprotection of ectABC was studied in the Escherichia coli KNabc and E. coli XL1-Blue. The results revealed that ectABC could shorten the lag phase and enhance the final OD600 of E. coli XL1-Blue in MM63 medium containing 0.68 M NaCl, and could initiate KNabc growth in 0.2 M NaCl. Ectoine was proven to be accumulated in E. coli KNabc/pGEM-Nect using HPLC-UV, and validated by LC-MSD-Trap-VL.

Genetic Characterization of the Escherichia coli O66 Antigen and Functional Identification of its wzy Gene: Jiansong Cheng , Bin Liu , David A. Bastin , Weiqing Han , Lei Wang , Lu Feng; J. Microbiol. 2007;45(1):69-74.; DOI: https://doi.org/2488 [pii]

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Abstract: Escherichia coli is a clonal species, and occurs as both commensal and pathogenic strains, which are normally classified on the basis of their O, H, and K antigens. The O-antigen (O-specific polysaccharide), which consists of a series of oligosaccharide (O-unit) repeats, contributes major antigenic variability to the cell surface. The O-antigen gene cluster of E. coli O66 was sequenced in this study. The genes putatively responsible for the biosynthesis of dTDP-6-deoxy-L-talose and GDP-mannose, as well as those responsible for the transfer of sugars and for O-unit processing were identified based on their homology. The function of the wzy gene was confirmed by the results of a mutation test. Genes specific for E. coli O66 were identified via PCR screening against representatives of 186 E. coli and Shigella O type strains. The comparison of intergenic sequences located between galF and the O-antigen gene cluster in a range of E. coli and Shigella showed that this region may perform an important function in the homologous recombination of the O-antigen gene clusters.

A Method for Comparing Multiple Bacterial Community Structures from 16S rDNA Clone Library Sequences: Inae Hur , Jongsik Chun; J. Microbiol. 2004;42(1):9-13.; DOI: https://doi.org/2008 [pii]

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Abstract: Culture-independent approaches, based on 16S rDNA sequences, are extensively used in modern microbial ecology. Sequencing of the clone library generated from environmental DNA has advantages over fingerprint-based methods, such as denaturing gradient gel electrophoresis, as it provides precise identification and quantification of the phylotypes present in samples. However, to date, no method exists for comparing multiple bacterial community structures using clone library sequences. In this study, an automated method to achieve this has been developed, by applying pair wise alignment, hierarchical clustering and principle component analysis. The method has been demonstrated to be successful in comparing samples from various environments. The program, named CommCluster, was written in JAVA, and is now freely available, at http://chunlab.snu.ac.kr/commcluster/.

Numerical classification of actinomycetes isolated from volcanic soil: Kim, Seung Bum , Lee, Soon Dong , Kim, Seon Young , Oh, Hyung Myung , Kang, Sa Ouk , Hah, Yung Chil; J. Microbiol. 1996;34(2):105-116.

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Abstract: Of actinomycetes isolated from volcanic compost soils, 115 representative strains which showed distinctive morphologicla features were numerically classified, compared with reference strains of Streptomyces. One hundred and twenty unit characters were tested and the average probability of error was 4.27%. The cluster analysis resulted in two groups: group A included strains of actinomycetes except streptomycetes. Group A was divided into 2 major clusters (over 5 strains), 10-diaminopimelic acid. Group B was divided into 5 clusters, of which 4 clusters contained mesodiminopimelic acid and 1 cluster LL-diaminopimelic acid. The major clusters of group A showed higher abilities of substrate utilization and degradation, and higher resistance to inhibitors, whereas the minor and single member clusters of group A showed relatively higher antimicrobial activities. On the other hand, all clusters of group B showed relatively lower abilities of substrate utilization and degradation and lower resistance to inhibitors.

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