scholarly journals The Bacillus cereus Group Is an Excellent Reservoir of Novel Lanthipeptides

2014 ◽  
Vol 81 (5) ◽  
pp. 1765-1774 ◽  
Author(s):  
Bingyue Xin ◽  
Jinshui Zheng ◽  
Ziya Xu ◽  
Xiaoling Song ◽  
Lifang Ruan ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Gene Cluster ◽  
Transmembrane Protein ◽  
Gene Clusters ◽  
Transcriptional Analysis ◽  
Exponential Growth Phase ◽  
Type I ◽  
Structural Genes ◽  
Gram Positive ◽  
Content Type

ABSTRACTLantibiotics are ribosomally synthesized peptides that contain multiple posttranslational modifications. Research on lantibiotics has increased recently, mainly due to their broad-spectrum antimicrobial activity, especially against some clinical Gram-positive pathogens. Many reports about various bacteriocins in theBacillus cereusgroup have been published, but few were about lantibiotics. In this study, we identified 101 putative lanthipeptide gene clusters from 77 out of 223 strains of this group, and these gene clusters were further classified into 20 types according to their gene organization and the homologies of their functional genes. Among them, 18 types were novel and have not yet been experimentally verified. Two novel lantibiotics (thuricin 4A-4 and its derivative, thuricin 4A-4D) were identified in the type I-1 lanthipeptide gene cluster and showed activity against all tested Gram-positive bacteria. The mode of action of thuricin 4A-4 was studied, and we found that it acted as a bactericidal compound. The transcriptional analysis of four structural genes (thiA1,thiA2,thiA3, andthiA4) in the thuricin 4A gene cluster showed that only one structural gene,thiA4, showed efficient transcription in the exponential growth phase; the other three structural genes did not. In addition, the putative transmembrane protein ThiI was responsible for thuricin 4A-4 immunity. Genome analysis and functional verification illustrated thatB. cereusgroup strains were a prolific source of novel lantibiotics.

scholarly journals Discovery of Gene Cluster for Mycosporine-Like Amino Acid Biosynthesis from Actinomycetales Microorganisms and Production of a Novel Mycosporine-Like Amino Acid by Heterologous Expression

2014 ◽  
Vol 80 (16) ◽  
pp. 5028-5036 ◽  
Author(s):  
Kiyoko T. Miyamoto ◽  
Mamoru Komatsu ◽  
Haruo Ikeda
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Dependent Manner ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria

ABSTRACTMycosporines and mycosporine-like amino acids (MAAs), including shinorine (mycosporine-glycine-serine) and porphyra-334 (mycosporine-glycine-threonine), are UV-absorbing compounds produced by cyanobacteria, fungi, and marine micro- and macroalgae. These MAAs have the ability to protect these organisms from damage by environmental UV radiation. Although no reports have described the production of MAAs and the corresponding genes involved in MAA biosynthesis from Gram-positive bacteria to date, genome mining of the Gram-positive bacterial database revealed that two microorganisms belonging to the orderActinomycetales,Actinosynnema mirumDSM 43827 andPseudonocardiasp. strain P1, possess a gene cluster homologous to the biosynthetic gene clusters identified from cyanobacteria. When the two strains were grown in liquid culture,Pseudonocardiasp. accumulated a very small amount of MAA-like compound in a medium-dependent manner, whereasA. mirumdid not produce MAAs under any culture conditions, indicating that the biosynthetic gene cluster ofA. mirumwas in a cryptic state in this microorganism. In order to characterize these biosynthetic gene clusters, each biosynthetic gene cluster was heterologously expressed in an engineered host,Streptomyces avermitilisSUKA22. Since the resultant transformants carrying the entire biosynthetic gene cluster controlled by an alternative promoter produced mainly shinorine, this is the first confirmation of a biosynthetic gene cluster for MAA from Gram-positive bacteria. Furthermore,S. avermitilisSUKA22 transformants carrying the biosynthetic gene cluster for MAA ofA. mirumaccumulated not only shinorine and porphyra-334 but also a novel MAA. Structure elucidation revealed that the novel MAA is mycosporine-glycine-alanine, which substitutesl-alanine for thel-serine of shinorine.

scholarly journals ApnI, a Transmembrane Protein Responsible for Subtilomycin Immunity, Unveils a Novel Model for Lantibiotic Immunity

2014 ◽  
Vol 80 (20) ◽  
pp. 6303-6315 ◽  
Author(s):  
Yun Deng ◽  
Cong-Zhi Li ◽  
Yi-Guang Zhu ◽  
Peng-Xia Wang ◽  
Qing-Dong Qi ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Transmembrane Protein ◽  
Single Gene ◽  
Gene Clusters ◽  
Cross Resistance ◽  
Specific Sequence ◽  
Recognition Mechanism ◽  
Content Type ◽  
Sequence Recognition ◽  
Novel Model

ABSTRACTSubtilomycin was detected from the plant endophytic strainBacillus subtilisBSn5 and was first reported fromB. subtilisstrain MMA7. In this study, a gene cluster that has been proposed to be related to subtilomycin biosynthesis was isolated from the BSn5 genome and was experimentally validated by gene inactivation and heterologous expression. Comparison of the subtilomycin gene cluster with other verified related lantibiotic gene clusters revealed a particular organization of the genesapnIandapnTdownstream ofapnAPBC, which may be involved in subtilomycin immunity. Through analysis of expression of theapnIand/orapnTgenes in the subtilomycin-sensitive strain CU1065 and inactivation ofapnIandapnTin the producer strain BSn5, we showed that the single geneapnI, encoding a putative transmembrane protein, was responsible for subtilomycin immunity. To our knowledge, evidence for lantibiotic immunity that is solely dependent on a transmembrane protein is quite rare. Further bioinformatic analysis revealed the abundant presence of ApnI-like proteins that may be responsible for lantibiotic immunity inBacillusandPaenibacillus. We cloned thepaeIgene, encoding one such ApnI-like protein, into CU1065 and showed that it confers resistance to paenibacillin. However, no cross-resistance was detected between ApnI and PaeI, even though subtilomycin and paenibacillin share similar structures, suggesting that the protection provided by ApnI/ApnI-like proteins involves a specific-sequence recognition mechanism. Peptide release/binding assays indicated that the recombinantB. subtilisexpressingapnIinteracted with subtilomycin. Thus, ApnI represents a novel model for lantibiotic immunity that appears to be common.

scholarly journals CerR, a Single-Domain Regulatory Protein of the LuxR Family, Promotes Cerecidin Production and Immunity inBacillus cereus

2017 ◽  
Vol 84 (5) ◽  
Author(s):  
Li Zhang ◽  
Kunling Teng ◽  
Jian Wang ◽  
Zheng Zhang ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Single Domain ◽  
Binding Motif ◽  
Peptide Antibiotics ◽  
Promoter Regions ◽  
Content Type ◽  
Regulatory Systems ◽  
Pharmaceutical Industries

ABSTRACTCerecidins are small lantibiotics fromBacillus cereusthat were obtained using a semi-in vitrobiosynthesis strategy and showed prominent antimicrobial activities against certain Gram-positive bacteria. However, the parental strainB. cereusAs 1.1846 is incapable of producing cerecidins, most probably due to the transcriptional repression of the cerecidin gene cluster. Located in the cerecidin gene cluster,cerRencodes a putative response regulator protein that belongs to the LuxR family transcriptional regulators. CerR (84 amino acids) contains only a conserved DNA binding domain and lacks a conventional phosphorylation domain, which is rarely found in lantibiotic gene clusters. To investigate its function in cerecidin biosynthesis,cerRwas constitutively expressed inB. cereusAs 1.1846. Surprisingly, Constitutive expression ofcerRenabled the production of cerecidins and enhanced self-immunity ofB. cereustoward cerecidins. Reverse transcription-PCR analysis and electrophoresis mobility shift assays indicated, respectively, that thecercluster was transcribed in two transcripts (cerAMandcerRTPFE) and that CerR regulated the cerecidin gene cluster directly by binding to the two predicted promoter regions ofcerAandcerR. DNase I footprinting experiments further confirmed that CerR specifically bound to the two promoter regions at a conserved inverted repeat sequence that was designated a CerR binding motif (cerRbox). The present study demonstrated that CerR, as the first single-domain LuxR family transcriptional regulator, serves as a transcriptional activator in cerecidin biosynthesis and activates the cerecidin gene cluster, which was otherwise cryptic inB. cereus.IMPORTANCELantibiotics with intriguing and prominent bioactivities are potential peptide antibiotics that could be applied in many areas, including food and pharmaceutical industries. The biosynthesis of lantibiotics is generally controlled by two-component regulatory systems consisting of histidine kinases and response regulators, while some unique and interesting regulatory systems are also revealed with the ever-increasing discovery of lantibiotic gene clusters among diverse microorganisms. Dissection of diverse lantibiotic regulation machineries would permit deep understanding of the biological functions of lantibiotics in different niches and even enable genetic activation of lantibiotic gene clusters that are otherwise cryptic. The significance of our study is to illuminate the regulatory mechanism of a special single-domain protein, CerR, in regulating cerecidin biosynthesis inBacillus cereus, providing a possible novel approach to activate cryptic lantibiotic clusters.

scholarly journals The Novel Transcriptional Regulator LmbU Promotes Lincomycin Biosynthesis through Regulating Expression of Its Target Genes in Streptomyces lincolnensis

2017 ◽  
Vol 200 (2) ◽  
Author(s):  
Bingbing Hou ◽  
Yanwei Lin ◽  
Haizhen Wu ◽  
Meijin Guo ◽  
Hrvoje Petkovic ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Target Genes ◽  
Regulatory Mechanism ◽  
Transcriptional Regulator ◽  
Gene Clusters ◽  
Saccharopolyspora Erythraea ◽  
Transcriptional Analysis ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Streptomyces Lincolnensis

ABSTRACT Lincomycin A is a clinically important antimicrobial agent produced by Streptomyces lincolnensis . In this study, a new regulator designated LmbU (GenBank accession no. ABX00623.1) was identified and characterized to regulate lincomycin biosynthesis in S. lincolnensis wild-type strain NRRL 2936. Both inactivation and overexpression of lmbU resulted in significant influences on lincomycin production. Transcriptional analysis and in vivo neomycin resistance (Neo r ) reporter assays demonstrated that LmbU activates expression of the lmbA , lmbC , lmbJ , and lmbW genes and represses expression of the lmbK and lmbU genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that LmbU can bind to the regions upstream of the lmbA and lmbW genes through the consensus and palindromic sequence 5′-CGCCGGCG-3′. However, LmbU cannot bind to the regions upstream of the lmbC , lmbJ , lmbK , and lmbU genes as they lack this motif. These data indicate a complex transcriptional regulatory mechanism of LmbU. LmbU homologues are present in the biosynthetic gene clusters of secondary metabolites of many other actinomycetes. Furthermore, the LmbU homologue from Saccharopolyspora erythraea (GenBank accession no. WP_009944629.1) also binds to the regions upstream of lmbA and lmbW , which suggests widespread activity for this regulator. LmbU homologues have no significant structural similarities to other known cluster-situated regulators (CSRs), which indicates that they belong to a new family of regulatory proteins. In conclusion, the present report identifies LmbU as a novel transcriptional regulator and provides new insights into regulation of lincomycin biosynthesis in S. lincolnensis . IMPORTANCE Although lincomycin biosynthesis has been extensively studied, its regulatory mechanism remains elusive. Here, a novel regulator, LmbU, which regulates transcription of its target genes in the lincomycin biosynthetic gene cluster ( lmb gene cluster) and therefore promotes lincomycin biosynthesis, was identified in S. lincolnensis strain NRRL 2936. Importantly, we show that this new regulatory element is relatively widespread across diverse actinomycetes species. In addition, our findings provide a new strategy for improvement of yield of lincomycin through manipulation of LmbU, and this approach could also be evaluated in other secondary metabolite gene clusters containing this regulatory protein.

scholarly journals Regulation of the Alkane Hydroxylase CYP153 Gene in a Gram-Positive Alkane-Degrading Bacterium, Dietzia sp. Strain DQ12-45-1b

2015 ◽  
Vol 82 (2) ◽  
pp. 608-619 ◽  
Author(s):  
Jie-Liang Liang ◽  
Jing-Hong JiangYang ◽  
Yong Nie ◽  
Xiao-Lei Wu
Keyword(s):  
Gene Cluster ◽  
Mutant Strain ◽  
Gene Transcription ◽  
Gene Promoter ◽  
Gene Clusters ◽  
Gram Positive ◽  
Content Type ◽  
Degrading Bacterium ◽  
Degrading Bacteria ◽  
Arac Family

ABSTRACTCYP153, one of the most common medium-chainn-alkane hydroxylases belonging to the cytochrome P450 superfamily, is widely expressed inn-alkane-degrading bacteria. CYP153 is also thought to cooperate with AlkB in degrading variousn-alkanes. However, the mechanisms regulating the expression of the protein remain largely unknown. In this paper, we studied CYP153 gene transcription regulation by the potential AraC family regulator (CypR) located upstream of the CYP153 gene cluster in a broad-spectrumn-alkane-degrading Gram-positive bacterium,Dietziasp. strain DQ12-45-1b. We first identified the transcriptional start site and the promoter of the CYP153 gene cluster. Sequence alignment of upstream regions of CYP153 gene clusters revealed high conservation in the −10 and −35 regions inActinobacteria. Further analysis of the β-galactosidase activity in the CYP153 gene promoter-lacZfusion cell indicated that the CYP153 gene promoter was induced byn-alkanes comprised of 8 to 14 carbon atoms, but not by derived decanol and decanic acid. Moreover, we constructed acypRmutant strain and found that the CYP153 gene promoter activities and CYP153 gene transcriptional levels in the mutant strain were depressed compared with those in the wild-type strain in the presence ofn-alkanes, suggesting that CypR served as an activator for the CYP153 gene promoter. By comparing CYP153 gene arrangements inActinobacteriaandProteobacteria, we found that the AraC family regulator is ubiquitously located upstream of the CYP153 gene, suggesting its universal regulatory role in CYP153 gene transcription. We further hypothesize that the observed mode of CYP153 gene regulation is shared by manyActinobacteria.

scholarly journals Genetic and Transcriptional Analyses of the Flagellar Gene Cluster in Actinoplanes missouriensis

2016 ◽  
Vol 198 (16) ◽  
pp. 2219-2227 ◽  
Author(s):  
Moon-Sun Jang ◽  
Yoshihiro Mouri ◽  
Kaoru Uchida ◽  
Shin-Ichi Aizawa ◽  
Masayuki Hayakawa ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Bacterial Genome ◽  
Promoter Sequence ◽  
Transcriptional Analysis ◽  
Flagellar Gene ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Short Period ◽  
Flagellar Genes

ABSTRACTActinoplanes missouriensis, a Gram-positive and soil-inhabiting bacterium, is a member of the rare actinomycetes. The filamentous cells produce sporangia, which contain hundreds of flagellated spores that can swim rapidly for a short period of time until they find niches for germination. These swimming cells are called zoospores, and the mechanism of this unique temporal flagellation has not been elucidated. Here, we report all of the flagellar genes in the bacterial genome and their expected function and contribution for flagellar morphogenesis. We identified a large flagellar gene cluster composed of 33 genes that encode the majority of proteins essential for assembling the functional flagella of Gram-positive bacteria. One noted exception to the cluster was the location of thefliQgene, which was separated from the cluster. We examined the involvement of four genes in flagellar biosynthesis by gene disruption,fliQ,fliC,fliK, andlytA. Furthermore, we performed a transcriptional analysis of the flagellar genes using RNA samples prepared fromA. missouriensisgrown on a sporangium-producing agar medium for 1, 3, 6, and 40 days. We demonstrated that the transcription of the flagellar genes was activated in conjunction with sporangium formation. Eleven transcriptional start points of the flagellar genes were determined using the rapid amplification of cDNA 5′ ends (RACE) procedure, which revealed the highly conserved promoter sequence CTCA(N15–17)GCCGAA. This result suggests that a sigma factor is responsible for the transcription of all flagellar genes and that the flagellar structure assembles simultaneously.IMPORTANCEThe biology of a zoospore is very interesting from the viewpoint of morphogenesis, survival strategy, and evolution. Here, we analyzed flagellar genes inA. missouriensis, which produces sporangia containing hundreds of flagellated spores each. Zoospores released from the sporangia swim for a short time before germination occurs. We identified a large flagellar gene cluster and an orphan flagellar gene (fliQ). These findings indicate that the zoospore flagellar components are typical of Gram-positive bacteria. However, the transcriptional analysis revealed that all flagellar genes are transcribed simultaneously during sporangium formation, a pattern differing from the orderly, regulated expression of flagellar genes in other bacteria, such asSalmonellaandEscherichia coli. These results suggest a novel regulatory mechanism for flagellar formation inA. missouriensis.

scholarly journals Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

2014 ◽  
Vol 58 (10) ◽  
pp. 6185-6196 ◽  
Author(s):  
Marius Spohn ◽  
Norbert Kirchner ◽  
Andreas Kulik ◽  
Angelika Jochim ◽  
Felix Wolf ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gene Cluster ◽  
High Performance ◽  
Pathogenic Bacteria ◽  
Genome Mining ◽  
Gene Clusters ◽  
Von Willebrand Disease ◽  
Biosynthesis Gene Cluster ◽  
Content Type ◽  
Bioinformatic Tools

ABSTRACTThe emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products isAmycolatopsis. However,Amycolatopsis japonicumdoes not produce an antibiotic under standard laboratory conditions. In contrast to mostAmycolatopsisstrains,A. japonicumis genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, thebbrgene fromAmycolatopsis balhimycina(bbrAba), intoA. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing ofA. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed thein silicoprediction that the recombinantA. japonicum/pRM4-bbrAbasynthesizes ristomycin A.

scholarly journals Restoration of Bioactive Lantibiotic Suicin from a RemnantlanLocus of Pathogenic Streptococcus suis Serotype 2

2013 ◽  
Vol 80 (3) ◽  
pp. 1062-1071 ◽  
Author(s):  
Jian Wang ◽  
Yong Gao ◽  
Kunling Teng ◽  
Jie Zhang ◽  
Shutao Sun ◽  
...  
Keyword(s):  
Gene Promoter ◽  
Streptococcus Suis ◽  
Gene Clusters ◽  
Disulfide Bridge ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
System A ◽  
Bactericidal Activities

ABSTRACTLantibiotics are ribosomally synthesized, posttranslationally modified antimicrobial peptides. Their biosynthesis genes are usually organized in gene clusters, which are mainly found in Gram-positive bacteria, including pathogenic streptococci. Three highly virulentStreptococcus suisserotype 2 strains (98HAH33, 05ZYH33, and SC84) have been shown to contain an 89K pathogenicity island. Here, on these islands, we unveiled and reannotated a putative lantibiotic locus designatedsuiwhich contains a virulence-associated two-component regulator,suiK-suiR. In silicoanalysis revealed that the putative lantibiotic modification genesuiMwas interrupted by a 7.9-kb integron and that other biosynthesis-related genes contained various frameshift mutations. By reconstituting the intactsuiMinEscherichia colitogether with a semi-in vitrobiosynthesis system, a putative lantibiotic named suicin was produced with bactericidal activities against a variety of Gram-positive strains, including pathogenic streptococci and vancomycin-resistant enterococci. Ring topology dissection indicated that the 34-amino-acid lantibiotic contained two methyllanthionine residues and one disulfide bridge, which render suicin in an N-terminal linear and C-terminal globular shape. To confirm the function ofsuiK-suiR, SuiR was overexpressed and purified.In vitroanalysis showed that SuiR could specifically bind to thesuiAgene promoter. Its coexpression withsuiKcould activatesuiAgene promoter inLactococcus lactisNZ9000. Conclusively, we obtained a novel lantibiotic suicin by restoring its production from the remnantsuilocus and demonstrated that virulence-associated SuiK-SuiR regulates its production.

scholarly journals Complete Genome Sequence of Bacillus cereus Sensu Stricto VKM B-370, Isolated from the Silkworm Bombyx mori

2021 ◽  
Vol 10 (20) ◽  
Author(s):  
Emma G. Piligrimova ◽  
Rustam M. Buzikov ◽  
Olesya A. Kazantseva ◽  
Andrey M. Shadrin
Keyword(s):  
Bacillus Cereus ◽  
Bombyx Mori ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Sensu Stricto ◽  
Host Strain ◽  
Gram Positive ◽  
Content Type ◽  
Silkworm Bombyx Mori

ABSTRACT Bacillus cereus is a Gram-positive rod-shaped spore-forming bacterium widespread in different environmental niches. Here, we report the complete genome sequence of Bacillus cereus VKM B-370 from the All-Russian Collection of Microorganisms, the host strain for bacteriophages vB_BtS_B83, vB_BcM_Sam46, vB_BcM_Sam112, and Izhevsk.

scholarly journals Plasmid-Borne Type E Neurotoxin Gene Clusters in Clostridium botulinum Strains

2013 ◽  
Vol 79 (12) ◽  
pp. 3856-3859 ◽  
Author(s):  
Zhen Zhang ◽  
Hannamari Hintsa ◽  
Ying Chen ◽  
Hannu Korkeala ◽  
Miia Lindström
Keyword(s):  
Gene Cluster ◽  
Gel Electrophoresis ◽  
Clostridium Botulinum ◽  
Southern Hybridization ◽  
Gene Clusters ◽  
Pulsed Field Gel Electrophoresis ◽  
Content Type ◽  
Pulsed Field ◽  
Large Plasmids

ABSTRACTA collection of 36Clostridium botulinumtype E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted tobotEandorfX1in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.

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