Interkingdom gene transfer of a hybrid NPS/PKS from bacteria to filamentous Ascomycota

Nonribosomal peptides (NRPs) and polyketides (PKs) are ecologically important secondary metabolites produced by bacteria and fungi using multidomain enzymes called nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), respectively. Previous phylogenetic analyses of fungal NRPSs and PKSs have suggested that a few of these genes were acquired by fungi via horizontal gene transfer (HGT) from bacteria, including a hybrid NPS/PKS found in Cochliobolus heterostrophus (Dothideomycetes, Ascomycota). Here, we identify this hybrid gene in fungi representing two additional classes of Ascomycota (Aspergillus spp., Microsporum canis, Arthroderma spp., and Trichophyton spp., Eurotiomycetes; Chaetomium spp. and Metarhizium spp., Sordariomycetes) and use phylogenetic analyses of the most highly conserved domains from NRPSs (adenylation (A) domain) and PKSs (ketoacyl synthase (KS) domain) to examine the hypothesis that the hybrid NPS7/PKS24 was acquired by fungi from bacteria via HGT relatively early in the evolution of the Pezizomycotina. Our results reveal a unique ancestry of the A domain and KS domain in the hybrid gene relative to known fungal NRPSs and PKSs, provide strong evidence for HGT of the hybrid gene from a putative bacterial donor in the Burkholderiales, and suggest the HGT event occurred early in the evolution of the filamentous Ascomycota.     

Characterization of the Streptomyces violaceoruber SANK95570 plasmids pSV1 and pSV2

The gene coding for recA in the oral pathogen Actinobacillus actinomycetemcomitans SUNY 465 was cloned and sequenced. The DNA sequence coded for a 352-amino acid protein that was homologous to RecA of a variety of bacterial species. A derivative of a non-replicating mobilizable plasmid was constructed for directed mutagenesis in A. actinomycetemcomitans. A recA-deficient strain of A. actinomycetemcomitans was developed by homologous recombination of an internal recA fragment contained on the mobilizable suicide vector. The recA mutant strain was more sensitive to UV radiation and showed a reduced recombinatorial proficiency than the isogenic parent strain. These data suggest that recA of A. actinomycetemcomitans SUNY 465 is involved in the repair of DNA damage caused by UV irradiation and homologous recombination as determined for other bacteria.     

Site-directed modification of the adenylation domain of the fusaricidin nonribosomal peptide synthetase for enhanced production of fusaricidin analogs

Fusaricidins produced by Paenibacillus polymyxa DBB1709 are lipopeptide antibiotics active against fungi and Gram-positive bacteria. The cyclic hexapeptide structures of fusaricidins are synthesized by fusaricidin synthetase, a non-ribosomal peptide synthetase. The adenylation domain of the third module (FusA-A3) can recruit L: -Tyr, L: -Val, L: -Ile, L: -allo-Ile, or L: -Phe, which diversifies the fusaricidin structures. Since the L: -Phe-incorporated fusaricidin analog (LI-F07) exhibits more potent antimicrobial activity than other analogs, we modified a specificity-conferring sequence in the substrate binding pocket of FusA-A3 to direct the enhanced production of LI-F07. Base on comparison to the adenylation domain of gramicidin S synthetase 1 and tyrocidine synthetase 1, both of which mainly activate L: -Phe, six mutant strains with altered FusA-A3 were generated using site-directed mutagenesis. M3 (I239W, I299V), M5 (I299V, G322A, V330I), and M6 (S239W, I299V, G322A, V330I) mutants produced significantly more LI-F07 than the wild-type strain.     

The 1.75 A crystal structure of acetyl-CoA synthetase bound to adenosine-5'-propylphosphate and coenzyme A

Acetyl-coenzyme A synthetase catalyzes the two-step synthesis of acetyl-CoA from acetate, ATP, and CoA and belongs to a family of adenylate-forming enzymes that generate an acyl-AMP intermediate. This family includes other acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular nonribosomal peptide synthetases. We have determined the X-ray crystal structure of acetyl-CoA synthetase complexed with adenosine-5'-propylphosphate and CoA. The structure identifies the CoA binding pocket as well as a new conformation for members of this enzyme family in which the approximately 110-residue C-terminal domain exhibits a large rotation compared to structures of peptide synthetase adenylation domains. This domain movement presents a new set of residues to the active site and removes a conserved lysine residue that was previously shown to be important for catalysis of the adenylation half-reaction. Comparison of our structure with kinetic and structural data of closely related enzymes suggests that the members of the adenylate-forming family of enzymes may adopt two different orientations to catalyze the two half-reactions. Additionally, we provide a structural explanation for the recently shown control of enzyme activity by acetylation of an active site lysine.     

Function of MbtH homologs in nonribosomal peptide biosynthesis and applications in secondary metabolite discovery

Mycobacterium tuberculosis encodes mycobactin, a peptide siderophore that is biosynthesized by a nonribosomal peptide synthetase (NRPS) mechanism. Within the mycobactin biosynthetic gene cluster is a gene that encodes a 71-amino-acid protein MbtH. Many other NRPS gene clusters harbor mbtH homologs, and recent genetic, biochemical, and structural studies have begun to shed light on the function(s) of these proteins. In some cases, MbtH-like proteins are required for biosynthesis of their cognate peptides, and non-cognate MbtH-like proteins have been shown to be partially complementary. Biochemical studies revealed that certain MbtH-like proteins participate in tight binding to NRPS proteins containing adenylation (A) domains where they stimulate adenylation reactions. Expression of MbtH-like proteins is important for a number of applications, including optimal production of native and genetically engineered secondary metabolites produced by mechanisms that employ NRPS enzymes. They also may serve as beacons to identify gifted actinomycetes and possibly other bacteria that encode multiple functional NRPS pathways for discovery of novel secondary metabolites by genome mining.     

Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis

The gene thiI encodes a protein (ThiI) that plays a role in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine, but the reaction catalyzed by ThiI remains undetermined. Based upon sequence alignments, ThiI shares a unique "P-loop" motif with the PPi synthetase family, four enzymes that catalyze adenylation and subsequent substitution of carbonyl oxygens. To test whether or not this motif is critical for ThiI function, the Asp in the motif was converted to Ala (D189A), and a screen for in vivo 4-thiouridine production revealed the altered enzyme to be inactive. Further scrutiny of sequence data and the crystal structures of two members of the PPi synthetase family implicated Lys321 in the proposed adenylation function of ThiI, and the critical nature of Lys321 has been demonstrated by site-directed mutagenesis and genetic screening. Our results, then, indicate that ThiI catalyzes the adenylation of a substrate at the expense of ATP, a narrowing of possible reactions that provides a strong new basis for deducing the early steps in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine.     

非核糖体肽合成酶基因腺苷酰化结构域序列克隆及分析

微生物许多非核糖体肽类次生代谢产物主要是由非核糖体肽合成酶(NRPS)催化合成。参考Gontang发布的非核糖体肽合成酶(NRPS)通用引物设计扩增NRPS腺苷酰化结构域基因序列的特异引物,从海洋链霉菌L1的基因组DNA中扩增获得一个715 bp的NRPS基因序列。测序结果及比对分析表明该片段属于NRPS腺苷酰化结构域部分序列。对其拟翻译的氨基酸序列组成成分、理化性质进行分析,显示其包含AFD class I超基因家族核心结合区,为NRPS腺苷酰化结构域(A结构域)所在区域。对氨基酸序列的二级结构预测和三级结构模拟,发现与数据库中肠菌素合酶F组分的结构相似。为后续研究A结构域的特异性及完整NRPS基因簇克隆提供了参考。     

Phylogenetic analysis of carbamoylphosphate synthetase genes: complex evolutionary history includes an internal duplication within a gene which can root the tree of life

Carbamoylphosphate synthetase (CPS) catalyzes the first committed step in pyrimidine biosynthesis, arginine biosynthesis, or the urea cycle. Organisms may contain either one generalized or two specific CPS enzymes, and these enzymes may be heterodimeric (encoded by linked or unlinked genes), monomeric, or part of a multifunctional protein. In order to help elucidate the evolution of CPS, we have performed a comprehensive phylogenetic analysis using the 21 available complete CPS sequences, including a sequence from Sulfolobus solfataricus P2 which we report in this paper. This is the first report of a complete CPS gene sequence from an archaeon, and sequence analysis suggests that it encodes an enzyme similar to heterodimeric CPSII. We confirm that internal similarity within the synthetase domain of CPS is the result of an ancient gene duplication that preceded the divergence of the Bacteria, Archaea, and Eukarya, and use this internal duplication in phylogenetic tree construction to root the tree of life. Our analysis indicates with high confidence that this archaeal sequence is more closely related to those of Eukarya than to those of Bacteria. In addition to this ancient duplication which created the synthetase domain, our phylogenetic analysis reveals a complex history of further gene duplications, fusions, and other events which have played an integral part in the evolution of CPS.      

Crystal structure and enantiomer selection by D-alanyl carrier protein ligase DltA from Bacillus cereus

Ubiquitous D-alanylation of lipoteichoic acids modulates the surface charge and ligand binding of the gram-positive cell wall. Disruption of the bacterial DltABCD gene involved in teichoic acid alanylation, as well as inhibition of the DltA protein, has been shown to increase a gram-positive bacterium's susceptibility to antibiotics. The DltA D-alanyl carrier protein ligase promotes a two-step process starting with adenylation of D-alanine. We have determined the 2.0 A resolution crystal structure of a DltA protein from Bacillus cereus in complex with the D-alanine adenylate intermediate of the first reaction. Despite the low level of sequence similarity, the DltA structure resembles known structures of adenylation domains such as the acetyl-CoA synthetase. The enantiomer selection appears to be enhanced by the medium-sized side chain of Cys-269. The Ala-269 mutant protein shows marked loss of such selection. The network of noncovalent interactions between the D-alanine adenylate and DltA provides structure-based rationale for aiding the design of tight-binding DltA inhibitors for combating infectious gram-positive bacteria such as the notorious methicillin-resistant Staphylococcus aureus.     

Crystal structure of DltA. Implications for the reaction mechanism of non-ribosomal peptide synthetase adenylation domains

DltA, the D-alanine:D-alanyl carrier protein ligase responsible for the initial step of lipoteichoic acid D-alanylation in Gram-positive bacteria, belongs to the adenylation domain superfamily, which also includes acetyl-CoA synthetase and the adenylation domains of non-ribosomal synthetases. The two-step reaction catalyzed by these enzymes (substrate adenylation followed by transfer to the reactive thiol group of CoA or the phosphopantheinyl prosthetic group of peptidyl carrier proteins) has been suggested to proceed via large scale rearrangements of structural domains within the enzyme. The structures of DltA reported here reveal the determinants for D-Ala substrate specificity and confirm that the peptidyl carrier protein-activating domains are able to adopt multiple conformational states, in this case corresponding to the thiolation reaction. Comparisons of available structures allow us to propose a mechanism whereby small perturbations of finely balanced metastable structural states would be able to direct an ordered formation of non-ribosomal synthetase products.     

The linear pentadecapeptide gramicidin is assembled by four multimodular nonribosomal peptide synthetases that comprise 16 modules with 56 catalytic domains

Linear gramicidin is a membrane channel forming pentadecapeptide that is produced via the nonribosomal pathway. It consists of 15 hydrophobic amino acids with alternating l- and d-configuration forming a beta-helix-like structure. It has an N-formylated valine and a C-terminal ethanolamine. Here we report cloning and sequencing of the entire biosynthetic gene cluster as well as initial biochemical analysis of a new reductase domain. The biosynthetic gene cluster was identified on two nonoverlapping fosmids and a 13-kilobase pair (kbp) interbridge fragment covering a region of 74 kbp. Four very large open reading frames, lgrA, lgrB, lgrC, and lgrD with 6.8, 15.5, 23.3, and 15.3 kbp, were identified and shown to encode nonribosomal peptide synthetases with two, four, six, and four modules, respectively. Within the 16 modules identified, seven epimerization domains in alternating positions were detected as well as a putative formylation domain fused to the first module LgrA and a putative reductase domain attached to the C-terminal module of LgrD. Analysis of the substrate specificity by phylogenetic studies using the residues of the substrate-binding pockets of all 16 adenylation domains revealed a good agreement of the substrate amino acids predicted with the sequence of linear gramicidin. Additional biochemical analysis of the three adenylation domains of modules 1, 2, and 3 confirmed the colinearity of this nonribosomal peptide synthetase assembly line. Module 16 was predicted to activate glycine, which would then, being the C-terminal residue of the peptide chain, be reduced by the adjacent reductase domain to give ethanolamine, thereby releasing the final product N-formyl-pentadecapeptide-ethanolamine. However, initial biochemical analysis of this reductase showed only a one-step reduction yielding the corresponding aldehyde in vitro.     

The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life

RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the "stringent" response and regulator of cellular metabolism. The classical "long" RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppGpp-synthesizing and -hydrolyzing RSHs have also been discovered in disparate bacteria and animals respectively. However, there is considerable confusion in terms of nomenclature and no comprehensive phylogenetic and sequence analyses have previously been carried out to classify RSHs on a genomic scale. We have performed high-throughput sensitive sequence searching of over 1000 genomes from across the tree of life, in combination with phylogenetic analyses to consolidate previous ad hoc identification of diverse RSHs in different organisms and provide a much-needed unifying terminology for the field. We classify RSHs into 30 subgroups comprising three groups: long RSHs, small alarmone synthetases (SASs), and small alarmone hydrolases (SAHs). Members of nineteen previously unidentified RSH subgroups can now be studied experimentally, including previously unknown RSHs in archaea, expanding the "stringent response" to this domain of life. We have analyzed possible combinations of RSH proteins and their domains in bacterial genomes and compared RSH content with available RSH knock-out data for various organisms to determine the rules of combining RSHs. Through comparative sequence analysis of long and small RSHs, we find exposed sites limited in conservation to the long RSHs that we propose are involved in transmitting regulatory signals. Such signals may be transmitted via NTD to CTD intra-molecular interactions, or inter-molecular interactions either among individual RSH molecules or among long RSHs and other binding partners such as the ribosome.     

Detection of putative peptide synthetase genes in Trichoderma species: application of this method to the cloning of a gene from T. harzianum CECT 2413

Some of the secondary metabolites produced by Trichoderma, such as the peptaibols and other antibiotics, have a peptide structure and in their biosynthesis are involved proteins belonging to the Non-Ribosomal Peptide Synthetase family. In the present work, a PCR-mediated strategy was used to clone a region corresponding to an adenylation domain of a peptide synthetase (PS) gene from 10 different strains of Trichoderma. In addition, and using the fragment isolated by PCR from T. harzianum CECT 2413 as a probe, a fragment of 19.0 kb corresponding to a PS-encoding gene named salps1, including a 1.5 kb fragment of the promoter, was cloned and sequenced. The cloned region of salps1 contains four complete, and a fifth incomplete, modules, in which are found the adenylation, thiolation and condensation domains, but also an additional epimerization domain at the C-terminal end of the first module. The analysis of the Salps1 protein sequence, taking into consideration published data, suggests that it is neither a peptaibol synthetase nor a protein involved in siderophore biosynthesis. The presence of two breaks in the open reading frame and the expression of this gene under nitrogen starvation conditions suggest that salps1 could be a pseudogene.     

Cloning and characterization of a new hetero-gene cluster of nonribosomal peptide synthetase and polyketide synthase from the cyanobacterium Microcystis aeruginosa K-139

Two nonribosomal peptide synthetase genes responsible for the biosynthesis of microcystin and micropeptin in Microcystis aeruginosa K-139 have been identified. A new nonribosomal peptide synthetase gene, psm3, was identified in M. aeruginosa K-139. The gene is a cluster extending 30 kb and comprising 13 bidirectionally transcribed open reading frames arranged in two putative operons. psm3 encodes four adenylation proteins, one polyketide synthase, and several unique proteins, especially Psm3L consisting of halogenase, acyl-CoA binding protein-like protein, and acyl carrier protein. Alignment of the binding pocket of the adenylation domain and an ATP-PPi exchange analysis using a recombinant protein with the adenylation domain of Psm3B showed that Psm3G and Psm3B activate aspartic acid and tyrosine, respectively. Although disruption of psm3 did not reveal the product produced by Psm3, we identified microviridin B and aeruginosin K139 in the cells of M. aeruginosa K-139. The above-mentioned results indicated that M. aeruginosa possesses at least five nonribosomal peptide synthetase gene clusters.     

Biochemical and crystallographic analysis of substrate binding and conformational changes in acetyl-CoA synthetase

The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140 degrees rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.     

Serratia marcescens gene required for surfactant serrawettin W1 production encodes putative aminolipid synthetase belonging to nonribosomal peptide synthetase family

Serrawettin W1 produced by Serratia marcescens is a surface active exolipid having various functions supporting behaviors of bacteria on surface environments. Through the genetic analyses of serrawettin-less mutants of S. marcescens 274, the swrW gene encoding putative serrawettin W1 synthetase was identified. Homology analysis of the putative SwrW demonstrated the presence of condensation, adenylation, thiolation, and thioesterase domains which are characteristic for nonribosomal peptide synthetase (NRPS). NRPSs have been known as multi-modular enzymes. Linear alignment of these modules specifying respective amino acids will enable peptide bond formation resulting in a specific amino acid sequence. Putative SwrW was uni-modular NRPS specifying only L-serine. Possible steps in this simple unimodular NRPS for biosynthesis of serrawettin W1 [ cyclo-(D-3-hydroxydecanoyl-L-seryl) (2) ] were predicted by referring to the ingenious enzymatic activity of gramicidin S synthetase (multi-modular NRPS) of Brevibacillus brevis.     

Phylogenetic characterization of Centipeda periodontii, Selenomonas sputigena and Selenomonas species by 16S rRNA gene sequence analysis.

The nearly complete 16S rRNA gene sequences for oral Gram-negative anaerobic motile bacteria, Centipeda periodontii, Selenomonas sputigena and Selenomonas species (formerly S. sputigena type strain), were determined in order to unveil their relationship to other oral motile bacteria. To determine the phylogenetic characterization of these bacteria, their 16S rRNA gene sequences were obtained and compared with those from the ribosomal sequence databases previously reported. The 16S rRNA gene sequences of these bacteria were similar to those of Selenomonas ruminantium and Schwartzia succinivorans isolated from rumens, and to Pectinatus cerevisiiphilus isolated from spoiled beer. Among oral bacteria, the nucleotide sequence analysis of these bacteria revealed high nucleotide similarity to Veillonella species, whereas low similarity to oral motile bacteria such as Campylobacter species. Phylogenetic analysis clearly confirmed that C. periodontii and two Selenomonas species were classified as relatives of a group besides Selenomonas, Schwartzia, and Pectinatus species, and not as close relatives to oral motile bacteria, such as Campylobacter species. These results suggest that such oral Gram-negative anaerobic motile bacteria are close relatives of oral bacteria.     

Characterization of a novel riboswitch-regulated lysine transporter in Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is an opportunistic pathogen that resides primarily in the mammalian oral cavity. In this environment, A. actinomycetemcomitans faces numerous host- and microbe-derived stresses, including intense competition for nutrients and exposure to the host immune system. While it is clear that A. actinomycetemcomitans responds to precise cues that allow it to adapt and proliferate in the presence of these stresses, little is currently known about the regulatory mechanisms that underlie these responses. Many bacteria use noncoding regulatory RNAs (ncRNAs) to rapidly alter gene expression in response to environmental stresses. Although no ncRNAs have been reported in A. actinomycetemcomitans, we propose that they are likely important for colonization and persistence in the oral cavity. Using a bioinformatic and experimental approach, we identified three putative metabolite-sensing riboswitches and nine small regulatory RNAs (sRNAs) in A. actinomycetemcomitans during planktonic and biofilm growth. Molecular characterization of one of the riboswitches revealed that it is a lysine riboswitch and that its target gene, lysT, encodes a novel lysine-specific transporter. Finally, we demonstrated that lysT and the lysT lysine riboswitch are conserved in over 40 bacterial species, including the phylogenetically related pathogen Haemophilus influenzae.     

4-Nitrotryptophan is a substrate for the non-ribosomal peptide synthetase TxtB in the thaxtomin A biosynthetic pathway

Thaxtomin A, a cyclic dipeptide with a nitrated tryptophan moiety, is a phytotoxic pathogenicity determinant in scab-causing Streptomyces species that inhibits cellulose synthesis by an unknown mechanism. Thaxtomin A is produced by the action of two non-ribosomal peptide synthetase modules (TxtA and TxtB) and a complement of modifying enzymes, although the order of biosynthesis has not yet been determined. Analysis of a thaxtomin dual module knockout mutant and single module knockout mutants revealed that 4-nitrotryptophan is an intermediate in thaxtomin A biosynthesis prior to backbone assembly. The 4-nitrotryptophan represents a novel substrate for non-ribosomal peptide synthetases. Through identification of N -methyl-4-nitrotryptophan in a single module knockout and the use of adenylation domain specificity prediction software, TxtB was identified as the non-ribosomal peptide synthetase module specific for 4-nitrotryptophan.     

The barbamide biosynthetic gene cluster: a novel marine cyanobacterial system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS) origin involving an unusual trichloroleucyl starter unit

Barbamide was extracted from the marine cyanobacterium Lyngbya majuscula strain 19L as a chlorinated lipopeptide for its potent molluscicidal activity. Precursor incorporation studies indicated that it is derived from acetate, L-phenylalanine, L-leucine and L-cysteine. The gene cluster responsible for biosynthesis of barbamide (bar) was cloned and characterized in this study. DNA sequence analysis of cosmid pLM49 revealed a cluster of 12 open reading frames (barA-barK) extending 26 kb including the expected polyketide synthase and non-ribosomal peptide synthetase modules and tailoring genes. The genetic architecture and domain organization of the bar cluster supports the assignment based on the apparent co-linearity of the systems. The activity assay of adenylation domains of barD (A(D)), barE (A(E)) and barG (A(G2) for module 2) in an amino acid-dependent ATP-pyrophosphate exchange experiment supports the conclusion that barbamide is synthesized from acetate, L-phenylalanine, L-cysteine and L-leucine with trichloroleucine as a direct precursor by a mixed polyketide synthase/non-ribosomal polypeptide synthetase. Assembly of barbamide includes unique biochemical mechanisms for chlorination, one-carbon truncation during chain elongation, E-double bond formation and thiazole ring formation.