Genetic Homogeneity of Clostridium botulinum Type A1 Strains with Unique Toxin Gene Clusters

ABSTRACT A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha−/orfX+) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha−/orfX+ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin.

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Genetic Diversity among Clostridium botulinum Strains Harboringbont/A2andbont/A3Genes

Applied and Environmental Microbiology ◽

10.1128/aem.02428-12 ◽

2012 ◽

Vol 78 (24) ◽

pp. 8712-8718 ◽

Cited By ~ 19

Author(s):

Carolina Lúquez ◽

Brian H. Raphael ◽

Lavin A. Joseph ◽

Sarah R. Meno ◽

Rafael A. Fernández ◽

...

Keyword(s):

Genetic Diversity ◽

Clostridium Botulinum ◽

Type A ◽

Gene Clusters ◽

Nucleotide Sequencing ◽

Toxin Gene ◽

Content Type ◽

Geographic Origins ◽

Diversity Studies ◽

Insight Into

ABSTRACTClostridium botulinumtype A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene,bont/A1, although all reportedbont/Agene variants have been associated with botulism cases. Our study provides insight into the genetic diversity ofC. botulinumtype A strains, which containbont/A2(n= 42) andbont/A3(n= 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by usingbontnucleotide sequencing, content analysis of thebontgene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences ofbontgenes obtained in this study showed 99.9 to 100% identity with otherbont/A2orbont/A3gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content.C. botulinumstrains harboringbont/A2andbont/A3genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE.

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Novel Structural Elements within the NonproteolyticClostridium botulinumType F Toxin Gene Cluster

Applied and Environmental Microbiology ◽

10.1128/aem.02422-10 ◽

2010 ◽

Vol 77 (5) ◽

pp. 1904-1906 ◽

Cited By ~ 10

Author(s):

N. Dover ◽

J. R. Barash ◽

K. K. Hill ◽

J. C. Detter ◽

S. S. Arnon

Keyword(s):

Gene Cluster ◽

Clostridium Botulinum ◽

Gene Clusters ◽

Gene Arrangement ◽

Toxin Gene ◽

Structural Elements ◽

Novel Gene ◽

First Time

ABSTRACTWe sequenced for the first time the complete neurotoxin gene cluster of a nonproteolyticClostridium botulinumtype F. The neurotoxin gene cluster contained a novel gene arrangement that, compared to otherC. botulinumneurotoxin gene clusters, lacked the regulatorybotRgene and contained an intergeniciselement between itsorfX2andorfX3genes.

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Differentiation of the Gene Clusters Encoding Botulinum Neurotoxin Type A Complexes in Clostridium botulinum Type A, Ab, and A(B) Strains

Applied and Environmental Microbiology ◽

10.1128/aem.70.12.7192-7199.2004 ◽

2004 ◽

Vol 70 (12) ◽

pp. 7192-7199 ◽

Cited By ~ 42

Author(s):

Giovanna Franciosa ◽

Francesca Floridi ◽

Antonella Maugliani ◽

Paolo Aureli

Keyword(s):

United States ◽

Clostridium Botulinum ◽

Botulinum Toxin Type A ◽

Type A ◽

Gene Clusters ◽

Botulinum Toxin Type ◽

The United States ◽

Toxin Gene ◽

Polymorphism Analysis ◽

Genes Encoding

ABSTRACT We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.

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Looking for the X Factor in Bacterial Pathogenesis: Association of orfX-p47 Gene Clusters with Toxin Genes in Clostridial and Non-Clostridial Bacterial Species

Toxins ◽

10.3390/toxins12010019 ◽

2019 ◽

Vol 12 (1) ◽

pp. 19 ◽

Cited By ~ 1

Author(s):

Maria B. Nowakowska ◽

François P. Douillard ◽

Miia Lindström

Keyword(s):

Gene Cluster ◽

In Silico ◽

Botulinum Neurotoxin ◽

Bacterial Species ◽

Gene Clusters ◽

Bacterial Pathogenesis ◽

Toxin Gene ◽

Toxin Complex ◽

Analytical Tools ◽

Bacillus Isolate

The botulinum neurotoxin (BoNT) has been extensively researched over the years in regard to its structure, mode of action, and applications. Nevertheless, the biological roles of four proteins encoded from a number of BoNT gene clusters, i.e., OrfX1-3 and P47, are unknown. Here, we investigated the diversity of orfX-p47 gene clusters using in silico analytical tools. We show that the orfX-p47 cluster was not only present in the genomes of BoNT-producing bacteria but also in a substantially wider range of bacterial species across the bacterial phylogenetic tree. Remarkably, the orfX-p47 cluster was consistently located in proximity to genes coding for various toxins, suggesting that OrfX1-3 and P47 may have a conserved function related to toxinogenesis and/or pathogenesis, regardless of the toxin produced by the bacterium. Our work also led to the identification of a putative novel BoNT-like toxin gene cluster in a Bacillus isolate. This gene cluster shares striking similarities to the BoNT cluster, encoding a bont/ntnh-like gene and orfX-p47, but also differs from it markedly, displaying additional genes putatively encoding the components of a polymorphic ABC toxin complex. These findings provide novel insights into the biological roles of OrfX1, OrfX2, OrfX3, and P47 in toxinogenesis and pathogenesis of BoNT-producing and non-producing bacteria.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00080-13 ◽

2013 ◽

Vol 79 (12) ◽

pp. 3856-3859 ◽

Cited By ~ 17

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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Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727

Journal of Bacteriology ◽

10.1128/jb.00262-15 ◽

2015 ◽

Vol 197 (15) ◽

pp. 2536-2544 ◽

Cited By ~ 24

Author(s):

Letizia Lo Grasso ◽

Sonia Maffioli ◽

Margherita Sosio ◽

Mervyn Bibb ◽

Anna Maria Puglia ◽

...

Keyword(s):

Gene Cluster ◽

Antibiotic Production ◽

Regulatory Protein ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Regulatory Mechanisms ◽

Antibiotic Biosynthesis ◽

Strain Atcc ◽

Protein Coding ◽

Content Type

ABSTRACTThe actinomyceteNonomuraeasp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by thedbvgene cluster, which contains 37 protein-coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation ofdbv6had no effect. In addition, overexpression ofdbv3led to higher levels of A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons,dbv14-dbv8anddbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4,dbv29,dbv36, anddbv37) and of six operons (dbv2-dbv1,dbv14-dbv8,dbv17-dbv15,dbv21-dbv20,dbv24-dbv28, anddbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription ofdbv4and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export, and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 directly regulates biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation.IMPORTANCEThis report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomyceteNonomuraeasp. strain ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of skin infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control two other similar glycopeptides (balhimycin and teicoplanin) have been elucidated, and beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.

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Neurotoxin Gene Clusters in Clostridium botulinum Type Ab Strains

Applied and Environmental Microbiology ◽

10.1128/aem.01009-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6094-6101 ◽

Cited By ~ 31

Author(s):

Carolina Lúquez ◽

Brian H. Raphael ◽

Susan E. Maslanka

Keyword(s):

Amino Acid ◽

Nucleotide Sequence ◽

Amino Acid Sequence ◽

Clostridium Botulinum ◽

Gene Diversity ◽

Gene Clusters ◽

Toxin Gene ◽

Predicted Amino Acid Sequence ◽

Botulinum Type ◽

Type Ab

ABSTRACT There is limited knowledge of the neurotoxin gene diversity among Clostridium botulinum type Ab strains. Only the sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene in C. botulinum type Ab strain CDC588 have been reported. In this study, we sequenced the entire bont/A- and bont/B-associated neurotoxin gene clusters of C. botulinum type Ab strain CDC41370 and the bont/A gene of strain CDC588. In addition, we analyzed the organization of the neurotoxin gene clusters in strains CDC588 and CDC1436. The bont/A nucleotide sequence of strain CDC41370 differed from those of the known bont/A subtypes A1 to A4 by 2 to 7%, and the predicted amino acid sequence differed by 4% to 14%. The bont/B nucleotide sequence in strain CDC41370 showed 99.7% identity to the sequence of subtype B1. The bont/A nucleotide sequence of strain CDC588 was 99.9% identical to that of subtype A1. Although all of the C. botulinum type Ab strains analyzed contained the two sets of neurotoxin clusters, similar to what has been found in other bivalent strains, the intergenic spacing of p21-orfX1 and orfX2-orfX3 varied among these strains. The type Ab strains examined in this study had differences in their toxin gene cluster compositions and bont/A and bont /B nucleotide sequences, suggesting that they may have arisen from separate recombination events.

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Genomic Characterization of Strains From a Cluster of Infant Botulism Type A in a Small Town in Colorado, United States

Frontiers in Microbiology ◽

10.3389/fmicb.2021.688240 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lori Gladney ◽

Jessica L. Halpin ◽

Carolina Lúquez

Keyword(s):

United States ◽

Environmental Samples ◽

Type A ◽

The United States ◽

Toxin Gene ◽

Nucleotide Polymorphisms ◽

Monophyletic Cluster ◽

Infant Botulism ◽

Genomic Characterization

Three cases of infant botulism were reported in a small Colorado town between 1981 and 1984. The first two cases occurred in 1981, 6 months apart, and the third case occurred in 1984. Clostridium botulinum type A was isolated from stool of all three case patients and from environmental samples of the patient’s homes. An epidemiological investigation and follow-up study were conducted from 1981 to 1986 and concluded the cases were likely related. In this study, we sought to determine whether the C. botulinum type A clinical isolates were related to each other and to isolates obtained from environmental samples. We performed whole genome sequencing (WGS) for 17 isolates associated with this potential cluster of infant botulism. Fifteen isolates were confirmed to be C. botulinum type A(B) and contained botulinum toxin gene subtypes A1 and B5 by WGS; these strains formed a monophyletic cluster in a phylogeny and were considered closely related to each other (0–18 high-quality single-nucleotide polymorphisms), but distinct from other C. botulinum type A(B) in Colorado and elsewhere in the United States. Results of our study suggest that the three infant botulism cases could have represented a cluster due to a C. botulinum type A(B) strain present in the environment.

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Identification of trans-AT polyketide clusters in two marine bacteria reveals cryptic similarities between distinct symbiosis factors

10.1101/2020.09.18.303172 ◽

2020 ◽

Author(s):

Dina Kačar ◽

Librada M Cañedo ◽

Pilar Rodríguez ◽

Elena Gonzalez ◽

Beatriz Galán ◽

...

Keyword(s):

Gene Cluster ◽

Marine Bacteria ◽

Genome Mining ◽

Evolutionary Relationship ◽

Gene Clusters ◽

Comparative Genomic ◽

Homologous Gene ◽

Modular Architecture ◽

Orphan Gene ◽

Strong Antitumor Activity

AbstractGlutaramide-containing polyketides are known as potent antitumoral and antimetastatic agents. However, the associated gene clusters have only been identified and studied in a few Streptomyces producers and sole Burkholderia gladioli symbiont. The new glutaramide-family polyketides, denominated sesbanimides D, E and F along with the previously known sesbanimide A and C, were isolated from two marine alphaproteobacteria Stappia indica PHM037 and Labrenzia aggregata PHM038. Structures of the isolated compounds were elucidated based on 1D and 2D homo and heteronuclear NMR analyses and ESI-MS spectrometry. All compounds exhibited strong antitumor activity in lung, breast and colorectal cancer cell lines. Subsequent whole genome sequencing and genome mining revealed the presence of the trans-AT PKS gene cluster responsible for the sesbanimide biosynthesis, described as sbn cluster, and the sesbanimide modular assembly is proposed. Interestingly, numerous homologous orphan gene clusters were localized in distantly related bacteria and used as comparative genomic assets for a more global characterization of sbn like-clusters. Strikingly, the modular architecture of downstream mixed type PKS/NRPS, SbnQ, revealed high similarity to PedH in pederin and Lab13 in labrenzin gene clusters, although those clusters are responsible for the production of structurally completely different molecules. The unexpected presence of SbnQ homologs in unrelated polyketide gene clusters across phylogenetically distant bacteria, raises intriguing questions about the evolutionary relationship between glutaramide-like and pederin-like pathways, as well as the functionality of their synthetic products.SignificanceGlutaramide-containing polyketides are still a largely understudied group of polyketides, produced mainly by the genera Streptomyces, with a great potential for antitumor drug production. Here, we describe genomes of two cultivable marine bacteria, Stappia indica PHM037 and Labrenzia aggregata PHM038, producers of the cytotoxic glutaramide-family polyketides sesbanimide A and C with chemical elucidation of newly identified analogs D, E and F. Genome mining revealed trans-AT PKS gene cluster responsible for sesbanimide biosynthesis. Although there are numerous homologous gene clusters present in remarkably different bacteria, this is the first time that the biosynthesis product has been reported. The comparative genome analysis reveals stunning, cryptic evolutionary relationship between sesbanimides, glutaramides from Streptomyces spp. and the pederin-family gene clusters.

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Comparative Genomics among Closely Related Streptomyces Strains Revealed Specialized Metabolite Biosynthetic Gene Cluster Diversity

Antibiotics ◽

10.3390/antibiotics7040086 ◽

2018 ◽

Vol 7 (4) ◽

pp. 86 ◽

Cited By ~ 15

Author(s):

Cláudia Vicente ◽

Annabelle Thibessard ◽

Jean-Noël Lorenzi ◽

Mabrouka Benhadj ◽

Laurence Hôtel ◽

...

Keyword(s):

Gene Cluster ◽

Sequence Data ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Core Gene ◽

Chemical Diversity ◽

Specific Gene ◽

Biosynthetic Gene ◽

Single Strain ◽

Specialized Metabolites

Specialized metabolites are of great interest due to their possible industrial and clinical applications. The increasing number of antimicrobial resistant infectious agents is a major health threat and therefore, the discovery of chemical diversity and new antimicrobials is crucial. Extensive genomic data from Streptomyces spp. confirm their production potential and great importance. Genome sequencing of the same species strains indicates that specialized metabolite biosynthetic gene cluster (SMBGC) diversity is not exhausted, and instead, a pool of novel specialized metabolites still exists. Here, we analyze the genome sequence data from six phylogenetically close Streptomyces strains. The results reveal that the closer strains are phylogenetically, the number of shared gene clusters is higher. Eight specialized metabolites comprise the core metabolome, although some strains have only six core gene clusters. The number of conserved gene clusters common between the isolated strains and their closest phylogenetic counterparts varies from nine to 23 SMBGCs. However, the analysis of these phylogenetic relationships is not affected by the acquisition of gene clusters, probably by horizontal gene transfer events, as each strain also harbors strain-specific SMBGCs. Between one and 15 strain-specific gene clusters were identified, of which up to six gene clusters in a single strain are unknown and have no identifiable orthologs in other species, attesting to the existing SMBGC novelty at the strain level.

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