Efforts toward broadening the spectrum of arylomycin antibiotic activity

New antibiotics are needed, and one source may be ‘latent’ antibiotics, natural products whose once broad-spectrum activity is currently limited by the evolution of resistance in nature. We have identified a potential class of latent antibiotics, the arylomycins, which are lipopeptides with a C-terminal macrocycle that target signal peptidase and whose spectrum is limited by a resistance-conferring mutation in many bacteria. Herein, we report the synthesis and evaluation of several arylomycin derivatives, and demonstrate that both C-terminal homologation with a glycyl aldehyde and addition of a positive charge to the macrocycle increase the activity and spectrum of the arylomycin scaffold.     

Type I signal peptidase and protein secretion in Staphylococcus aureus

Staphylococcus aureus is an important human pathogen whose virulence relies on the secretion of many different proteins. In general, the secretion of most proteins in S. aureus, as well as other bacteria, is dependent on the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein to the general secretory pathway. The arylomycins are a class of natural product antibiotics that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. While wild-type S. aureus (NCTC 8325) is naturally resistant to the arylomycins, sensitivity is conferred via a point mutation in its SPase. Here, we use a synthetic arylomycin along with a sensitized strain of S. aureus and multidimensional protein identification technology (MudPIT) mass spectrometry to identify 46 proteins whose extracellular accumulation requires SPase activity. Forty-four possess identifiable Sec-type signal peptides and thus are likely canonically secreted proteins, while four also appear to possess cell wall retention signals. We also identified the soluble C-terminal domains of two transmembrane proteins, lipoteichoic acid synthase, LtaS, and O-acyteltransferase, OatA, both of which appear to have noncanonical, internal SPase cleavage sites. Lastly, we identified three proteins, HtrA, PrsA, and SAOUHSC_01761, whose secretion is induced by arylomycin treatment. In addition to elucidating fundamental aspects of the physiology and pathology of S. aureus, the data suggest that an arylomycin-based therapeutic would reduce virulence while simultaneously eradicating an infection.     

Type I signal peptidase and protein secretion in Staphylococcus epidermidis

Bacterial protein secretion is a highly orchestrated process that is essential for infection and virulence. Despite extensive efforts to predict or experimentally detect proteins that are secreted, the characterization of the bacterial secretome has remained challenging. A central event in protein secretion is the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein for secretion via the general secretory pathway, and the arylomycins are a class of natural products that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. Here, using an arylomycin derivative, along with two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identify 11 proteins whose secretion from stationary-phase Staphylococcus epidermidis is dependent on SPase activity, 9 of which are predicted to be translated with canonical N-terminal signal peptides. In addition, we find that the presence of extracellular domains of lipoteichoic acid synthase (LtaS) and the β-lactam response sensor BlaR1 in the medium is dependent on SPase activity, suggesting that they are cleaved at noncanonical sites within the protein. In all, the data define the proteins whose stationary-phase secretion depends on SPase and also suggest that the arylomycins should be valuable chemical biology tools for the study of protein secretion in a wide variety of different bacteria.     

Gene arrangement in the upstream region of Clostridium botulinum type E and Clostridium butyricum BL6340 progenitor toxin genes is different from that of other types

The cluster of genes encoding the botulinum progenitor toxin and the upstream region including p21 and p47 were divided into three different gene arrangements (class I–III). To determine the gene similarity of the type E neurotoxin (BoNT/E) complex to other types, the gene organization in the upstream region of the nontoxic-nonhemagglutinin gene (ntnh) was investigated in chromosomal DNA from Clostridium botulinum type E strain Iwanai and C. butyricum strain BL6340. The gene cluster of type E progenitor toxin (Iwanai and BL6340) was similar to those of type F and type A (from infant botulism in Japan), but not to those of types A, B, and C. Though genes for the hemagglutinin component and P21 were not discovered, genes encoding P47, NTNH, and BoNT were found in type E strain Iwanai and C. butyricum strain BL6340. However, the genes of ORF-X₁ (435 bp) and ORF-X₂ (partially sequenced) were present just upstream of that of P47. The orientation of these genes was in inverted direction to that of p47. The gene cluster of type E progenitor toxin (Iwanai and BL6340) is, therefore, a specific arrangement (class IV) among the genes encoding components of the BoNT complex.     

Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (≤3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.     

Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters

Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. We sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ⁷⁰ family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. This TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.     

A Single Module Type I Polyketide Synthase Directs de Novo Macrolactone Biogenesis during Galbonolide Biosynthesis in Streptomyces galbus

Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-¹³C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed β-keto group modifications.     

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

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.     

A novel host-specific restriction system associated with DNA backbone S-modification in Salmonella

A novel, site-specific, DNA backbone S-modification (phosphorothioation) has been discovered, but its in vivo function(s) have remained obscure. Here, we report that the enteropathogenic Salmonella enterica serovar Cerro 87, which possesses S-modified DNA, restricts DNA isolated from Escherichia coli, while protecting its own DNA by site-specific phosphorothioation. A cloned 15-kb gene cluster from S. enterica conferred both host-specific restriction and DNA S-modification on E. coli. Mutational analysis of the gene cluster proved unambiguously that the S-modification prevented host-specific restriction specified by the same gene cluster. Restriction activity required three genes in addition to at least four contiguous genes necessary for DNA S-modification. This functional overlap ensures that restriction of heterologous DNA occurs only when the host DNA is protected by phosphorothioation. Meanwhile, this novel type of host-specific restriction and modification system was identified in many diverse bacteria. As in the case of methylation-specific restriction systems, targeted inactivation of this gene cluster should facilitate genetic manipulation of these bacteria, as we demonstrate in Salmonella.     

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

A collection of 36 Clostridium botulinum type E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted to botE and orfX1 in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.     

Neurotoxin Gene Clusters in Clostridium botulinum Type Ab Strains

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 {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}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 {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}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 {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}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.Clostridium botulinum is a gram-positive anaerobic bacterium that produces an extremely potent toxin, the botulinum neurotoxin (BoNT). There are seven serologically distinct types of BoNT (serotypes A through G). Although most strains of C. botulinum express a single toxin serotype, some isolates have been shown to produce more than one, namely, Ab, Af, Ba, and Bf (11). In addition, many strains designated type A by mouse bioassay harbor nucleotide sequences for both type A and B toxins (6). These strains have been designated A(B) to indicate the presence of the bont/B gene without type B-specific toxicity.Based on phylogenetic analysis of the neurotoxin gene sequences, four subtypes have been identified within serotype A and five subtypes within serotype B (12). The neurotoxin gene nucleotide sequences of these subtypes differ by up to 8%, and the predicted amino acid sequences differ by up to 16%. In addition, the genes encoding components of the toxin complexes are arranged in clusters that differ in composition and organization (14) (Fig. ​(Fig.1).1). The toxin gene cluster of subtype A1 (termed ha cluster) includes the gene encoding the nontoxic nonhemagglutinin (ntnh), a regulatory gene (botR), and an operon encoding three hemagglutinins (ha70, ha33, and ha17). The toxin gene clusters containing bont/A2 or bont/A3 (termed orfX cluster) include the ntnh and p21 (analogous to botR) genes and several genes of unknown function (orfX1, orfX2, orfX3, and p47). Type Ba and A(B) strains contain two sets of neurotoxin cluster genes in which ha70, ha33, and ha17 are associated with the bont/B gene, and orfX1, orfX2, orfX3, and p47 are associated with the bont/A gene. In addition, some A1 strains contain a neurotoxin gene cluster that is similar to those in A2 and A3, but the bont/A nucleotide sequence is 99.9% identical to that in other A1 strains. These strains have been designated HA⁻ Orfx⁺ A1 (14). The neurotoxin gene cluster in type B strains includes the ntnh, botR, ha70, ha33, and ha17 genes. Notably, no differences in the neurotoxin gene cluster arrangements among the subtypes within serotype B have been reported.Open in a separate windowFIG. 1.Toxin gene cluster arrangements for BoNT type A-producing strains, including Ab, A(B), and Ba strains.Although several studies have described the organization and the nucleotide sequences of the neurotoxin gene cluster components among type A and B strains [including type Ba and A(B) strains], there is limited information regarding the diversity of the neurotoxin cluster genes among C. botulinum type Ab strains. The nucleotide sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene of C. botulinum type Ab strain CDC588 have been previously reported; strain CDC1436 harbors a bont/A2 gene, and both strains CDC1436 and CDC588 harbor a bont/bvB gene (12, 15). Four additional type Ab strains from Italy have been analyzed by a restriction fragment length polymorphism method to determine the bont/A and bont/B subtypes (7, 9). To the best of our knowledge, the complete nucleotide sequences of the neurotoxin gene clusters in C. botulinum type Ab strains have not been reported. Thus, the objective of this study was to analyze the neurotoxin gene cluster composition in three C. botulinum type Ab strains ({"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370, CDC588, and CDC1436) available in the CDC strain collection. We report differences in bont/A gene sequence among type Ab strains, including the identification of a novel bont/A nucleotide sequence in strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370, and describe differences in the organization of the neurotoxin gene clusters among these strains.     

Polarity of meiotic gene conversion is 5′ to 3′ within the niaD gene of Aspergillus nidulans

We have examined polarity of meiotic gene conversion in the niiA-niaD gene cluster of Aspergillus nidulans in two-point crosses. The type and position of the mutations represented by the niaD alleles and the correlation between the relative frequency of gene conversion and the physical position of these mutations were determined. We show that polarity of meiotic gene conversion is 5′ to 3′ (transcribed strand) within the niaD gene. Additional crosses involving a niiA allele and a niaD allele show little polarity of gene conversion, which suggests that the recombination events leading to restoration of the niaD gene are initiated upstream of the coding region of the niaD gene but within the niiA-niaD gene cluster, possibly within the intergenic promoter region.     

Molecular organization of the xcp gene cluster in Pseudomonas putida: absence of an xcpX (gspK) homologue

A DNA fragment containing xcp (gsp) gene homologues, required for extracellular protein secretion by the general secretory pathway (GSP) in various Gram-negative bacteria, was cloned from Pseudomonas putida (Pp) strain WCS358 and sequenced. The results presented here and those previously reported (de Groot, A., Krijger, J.-J., Filloux, A., Tommassen, J., 1996. Characterization of type II protein secretion (xcp) genes in the plant growth-stimulating Pseudomonas putida, strain WCS358 Mol. Gen. Genet. 250, 491–504) complete the sequence of the xcp gene cluster of Pp. Unlike that of Pseudomonas aeruginosa (Pa), the xcp gene cluster of Pp contains a gspN homologue. More surprisingly, in contrast to all known gsp gene clusters, the xcpX (gspK) homologue is not found. In addition, genes flanking the xcp cluster of Pp are not related to those flanking the xcp genes of Pa. Overall, the xcp gene products of Pp are as much related to those of Pa as to gsp gene products of enterobacterial species, suggesting that the xcp clusters of Pp and Pa have evolved separately.     

Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition

Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of large gene clusters like type I polyketide/nonribosomal peptide pathways for heterologous expression is no longer a bottleneck. Here, we apply recombineering to engineer either the epothilone (epo) or myxochromide S (mchS) gene cluster for transpositional delivery and expression in heterologous hosts. The 58-kb epo gene cluster was fully reconstituted from two clones by stitching. Then, the epo promoter was exchanged for a promoter active in the heterologous host, followed by engineering into the MycoMar transposon. A similar process was applied to the mchS gene cluster. The engineered gene clusters were transferred and expressed in the heterologous hosts Myxococcus xanthus and Pseudomonas putida. We achieved the largest transposition yet reported for any system and suggest that delivery by transposon will become the method of choice for delivery of large transgenes, particularly not only for metabolic engineering but also for general transgenesis in prokaryotes and eukaryotes.     

Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

A metabolomics approach exploring the function of the ESX-3 type VII secretion system of M. smegmatis

The genome of Mycobacterium, including Mycobacterium tuberculosis, contains five copies of a cluster of genes encoding a novel type VII secretion system, named the ESX gene cluster region. This ESX-3 gene cluster is essential for in vitro growth and is thought to play a role in iron and zinc homeostasis, however, its exact functionality remains an enigma. A metabolomics research approach was subsequently used to compare the metabolite profiles of a M. smegmatis ESX-3 knockout strain to that a wild type parental strain, in order to elucidate its functionality from a metabolic perspective. Statistical analysis of the GC–MS generated data showed a clear separation between the wild type and knockout sample groups, based on the analysed metabolite profiles of these organisms. Of all the metabolite markers identified, various amino acids and metabolite pathways related to these, appeared to be most affected by the ESX-3 knockout, especially those with enzymes regulated by iron and zinc, supporting previous genomics and proteomics generated hypotheses and findings. This study is the first to demonstrate the capacity of using metabolomics, in conjunction with previous genomics and proteomic findings, to identify underlying metabolic changes and confirm previous hypotheses related to the functionality of ESX-3 in Mycobacterium growth and survival.     

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

Mycosporines 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 order Actinomycetales, Actinosynnema mirum DSM 43827 and Pseudonocardia sp. strain P1, possess a gene cluster homologous to the biosynthetic gene clusters identified from cyanobacteria. When the two strains were grown in liquid culture, Pseudonocardia sp. accumulated a very small amount of MAA-like compound in a medium-dependent manner, whereas A. mirum did not produce MAAs under any culture conditions, indicating that the biosynthetic gene cluster of A. mirum was 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 avermitilis SUKA22. 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. avermitilis SUKA22 transformants carrying the biosynthetic gene cluster for MAA of A. mirum accumulated not only shinorine and porphyra-334 but also a novel MAA. Structure elucidation revealed that the novel MAA is mycosporine-glycine-alanine, which substitutes l-alanine for the l-serine of shinorine.