Zwittermicin A biosynthetic cluster.

The goal of this study was to identify the biosynthetic cluster for zwittermicin A, a novel, broad spectrum, aminopolyol antibiotic produced by Bacillus cereus. The nucleotide sequence of 2.7kb of DNA flanking the zwittermicin A self-resistance gene, zmaR, from B. cereus UW85 revealed three open reading frames (ORFs). Of these ORFs, two had sequence similarity to acyl-CoA dehydrogenases and polyketide synthases, respectively. Insertional inactivation demonstrated that orf2 is necessary for zwittermicin A production and that zmaR is necessary for high-level resistance to zwittermicin A but is not required for zwittermicin A production. Expression of ZmaR was temporally associated with zwittermicin A production. The results suggest that zmaR is part of a cluster of genes that is involved in zwittermicin A biosynthesis, representing the first biosynthetic pathway for an aminopolyol antibiotic.     

Zwittermicin A resistance gene from Bacillus cereus.

Zwittermicin A is a novel aminopolyol antibiotic produced by Bacillus cereus that is active against diverse bacteria and lower eukaryotes (L.A. Silo-Suh, B.J. Lethbridge, S.J. Raffel, H. He, J. Clardy, and J. Handelsman, Appl. Environ. Microbiol. 60:2023-2030, 1994). To identify a determinant for resistance to zwittermicin A, we constructed a genomic library from B. cereus UW85, which produces zwittermicin A, and screened transformants of Escherichia coli DH5alpha, which is sensitive to zwittermicin A, for resistance to zwittermicin A. Subcloning and mutagenesis defined a genetic locus, designated zmaR, on a 1.2-kb fragment of DNA that conferred zwittermicin A resistance on E. coli. A DNA fragment containing zmaR hybridized to a corresponding fragment of genomic DNA from B. cereus UW85. Corresponding fragments were not detected in mutants of B. cereus UW85 that were sensitive to zwittermicin A, and the plasmids carrying zmaR restored resistance to the zwittermicin A-sensitive mutants, indicating that zmaR was deleted in the zwittermicin A-sensitive mutants and that zmaR is functional in B. cereus. Sequencing of the 1.2-kb fragment of DNA defined an open reading frame, designated ZmaR. Neither the nucleotide sequence nor the predicted protein sequence had significant similarity to sequences in existing databases. Cell extracts from an E. coli strain carrying zmaR contained a 43.5-kDa protein whose molecular mass and N-terminal sequence matched those of the protein predicted by the zmaR sequence. The results demonstrate that we have isolated a gene, zmaR, that encodes a zwIttermicin A resistance determinant that is functional in both B. cereus and E. coli.     

Cloning, mutagenesis, and physiological effect of a hydroxypyruvate reductase gene from Methylobacterium extorquens AM1.

The gene encoding the serine cycle hydroxypyruvate reductase of Methylobacterium extorquens AM1 was isolated by using a synthetic oligonucleotide with a sequence based on a known N-terminal amino acid sequence. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced a serine cycle hydroxypyruvate reductase null mutant. This mutant had lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds, showing that the hydroxypyruvate reductase operating in the serine cycle is not involved in the conversion of acetyl coenzyme A to glycine as previously proposed. A second hydroxypyruvate-reducing enzyme with a low level of activity was found in M. extorquens AM1; this enzyme was able to interconvert glyoxylate and glycollate. The gene encoding hydroxypyruvate reductase was shown to be located about 3 kb upstream of two other serine cycles genes encoding phosphoenolpyruvate carboxylase and malyl coenzyme A lyase.     

Crystal structure of the novel PaiA N-acetyltransferase from Thermoplasma acidophilum involved in the negative control of sporulation and degradative enzyme production

GCN5-related N-acetyltransferases (GNATs) are the most widely distributed acetyltransferase systems among all three domains of life. GNATs appear to be involved in several key processes, including microbial antibiotic resistance, compacting eukaryotic DNA, controlling gene expression, and protein synthesis. Here, we report the crystal structure of a putative GNAT Ta0374 from Thermoplasma acidophilum, a hyperacidophilic bacterium, that has been determined in an apo-form, in complex with its natural ligand (acetyl coenzyme A), and in complex with a product of reaction (coenzyme A) obtained by cocrystallization with spermidine. Sequence and structural analysis reveals that Ta0374 belongs to a novel protein family, PaiA, involved in the negative control of sporulation and degradative enzyme production. The crystal structure of Ta0374 confirms that it binds acetyl coenzyme A in a way similar to other GNATs and is capable of acetylating spermidine. Based on structural and docking analysis, it is expected that Glu53 and Tyr93 are key residues for recognizing spermidine. Additionally, we find that the purification His-Tag in the apo-form structure of Ta0374 prevents binding of acetyl coenzyme A in the crystal, though not in solution, and affects a chain-flip rotation of "motif A" which is the most conserved sequence among canonical acetyltransferases.     

Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases.

We have deduced the nucleotide sequence of the genes encoding the three components of 4-chlorobenzoate (4-CBA) dehalogenase from Pseudomonas sp. CBS-3 and examined the origin of these proteins by homology analysis. Open reading frame 1 (ORF1) encodes a 30-kDa 4-CBA-coenzyme A dehalogenase related to enoyl-coenzyme A hydratases functioning in fatty acid beta-oxidation. ORF2 encodes a 57-kDa protein which activates 4-CBA by acyl adenylation/thioesterification. This 4-CBA:coenzyme A ligase shares significant sequence similarity with a large group of proteins, many of which catalyze similar chemistry in beta-oxidation pathways or in siderophore and antibiotic synthetic pathways. These proteins have in common a short stretch of sequence, (T,S)(S,G)G(T,S)(T,E)G(L,X)PK(G,-), which is particularly highly conserved and which may represent an important new class of "signature" sequence. We were unable to find any proteins homologous in sequence to the 16-kDa 4-hydroxybenzoate-coenzyme A thioesterase encoded by ORF3. Analysis of the chemistry and function of the proteins found to be structurally related to the 4-CBA:coenzyme A ligase and the 4-CBA-coenzyme A dehalogenase supports the proposal that they evolved from a beta-oxidation pathway.     

Zwittermicin A-producing strains of Bacillus cereus from diverse soils.

Bacillus cereus UW85 produces a novel aminopolyol antibiotic, zwittermicin A, that contributes to the ability of UW85 to suppress damping-off of alfalfa caused by Phytophthora medicaginis. UW85 produces a second antibiotic, provisionally designated antibiotic B, which also contributes to suppression of damping-off but has not been structurally defined yet and is less potent than zwittermicin A. The purpose of this study was to isolate genetically diverse strains of B. cereus that produce zwittermicin A and suppress disease. We found that most isolates of B. cereus that were sensitive to phage P7 or inhibited the growth of Erwinia herbicola produced zwittermicin A; therefore, phage typing and E. herbicola inhibition provided indirect, but rapid screening tests for identification of zwittermicin A-producing isolates. We used these tests to screen a collection of 4,307 B. cereus and Bacillus thuringiensis isolates obtained from bacterial stock collections and from diverse soils collected in Honduras, Panama, Australia, The Netherlands, and the United States. A subset of the isolates screened by the P7 sensitivity and E. herbicola inhibition tests were assayed directly for production of zwittermicin A, leading to the identification of 57 isolates that produced zwittermicin A; 41 of these isolates also produced antibiotic B. Eight isolates produced antibiotic B but not zwittermicin A. The assay for phage P7 sensitivity was particularly useful because of its simplicity and rapidity and because 22 of the 23 P7-sensitive isolates tested produced zwittermicin A. However, not all zwittermicin A-producing isolates were sensitive to P7, and the more labor-intensive E. herbicola inhibition assay identified a larger proportion of the zwittermicin A producers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Culture conditions that influence accumulation of zwittermicin A by Bacillus cereus UW85

Bacillus cereus strain UW85 produces an antibiotic, designated zwittermicin A, that is associated with the ability of UW85 to suppress damping-off disease of alfalfa (Medicago sativa) caused by the oomycete pathogen, Phytophthora medicaginis, in a laboratory bioassay. We have identified certain culture conditions that promote or suppress zwittermicin A accumulation by UW85. Maximum accumulation was detected in supernatants of trypticase soy broth cultures after sporulation, which is when cultures of UW85 provide the greatest suppression of damping-off on alfalfa. Inorganic amendments to trypticase soy broth cultures had the following effects on zwittermicin A accumulation and disease suppression: phosphate (50 mM or more) reduced zwittermicin A accumulation and disease suppression; ferric iron (0.25–1.0 mM) enhanced zwittermicin A accumulaiton and disease suppression; micronutrients (manganese, boron, copper, molybdenum, zinc) had no effect on zwittermicin A accumulation or disease suppression. Cultures of UW85 grown in chemically defined minimal medium supplemented with casein hydrolysate or grown in defined medium containing the minimal requirements for growth supplemented with five amino acids (Gln, Arg, Met, Phe, Ile) accumulated zwittermicin A. In minimal medium, alfalfa seed exudate inhibited growth of UW85, whereas alfalfa sprout exudate enhanced zwittermicin A accumulation by 40%. These data indicate that the accumulation of zwittermicin A can be modulated by specific nutrients, inorganic compounds, and plant-derived factors. These results will facilitate the improvement of large-scale purification of zwittermicin A, suggest appropriate conditions under which to conduct further genetic and biochemical analyses, and further substantiate the association between antibiotic accumulation and disease suppression by UW85.     

Functional identification of conjugation and replication regions of the tetracycline resistance plasmid pCW3 from Clostridium perfringens

Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes' genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.     

Structure-based functional classification of hypothetical protein MTH538 from Methanobacterium thermoautotrophicum

The structure of MTH538, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum, has been determined by NMR spectroscopy. MTH538 is one of numerous structural genomics targets selected in a genome-wide survey of uncharacterized sequences from this organism. MTH538 is a so-called singleton, a sequence not closely related to any other (known) sequences. The structure of MTH538 closely resembles the known structures of receiver domains from two component response regulator systems, such as CheY, and is similar to the structures of flavodoxins and GTP-binding proteins. Tests on MTH538 for characteristic activities of CheY and flavodoxin were negative. MTH538 did not become phosphorylated in the presence of acetyl phosphate and Mg(2+), although it appeared to bind Mg(2+). MTH538 also did not bind flavin mononucleotide (FMN) or coenzyme F(420). Nevertheless, sequence and structure parallels between MTH538/CheY and two families of ATPase/phosphatase proteins suggest that MTH538 may have a role in a phosphorylation-independent two-component response regulator system.     

The acrAB locus is involved in modulating intracellular acetyl coenzyme A levels in a strain of Escherichia coli CM2555 expressing the chloramphenicol acetyltransferase (cat) gene

Recently, an Escherichia coli CM2555 strain was described as sensitive to chloramphenicol when expressing the chloramphenicol resistance gene (cat) from a multicopy plasmid. This sensitivity was linked to dysfunction of the acrA gene, which encodes a component of the AcrAB-TolC multidrug efflux pump. Preliminary data indicate that the sensitivity phenotype might be due to a decline in intracellular acetyl coenzyme A concentration accompanying the reaction catalyzed by chloramphenicol acetyltransferase, the cat-encoded resistance protein. Here, we demonstrate that the acrA dysfunction is the factor impairing the intracellular acetyl coenzyme A levels in the cat-expressing CM2555 strain. This effect might be alleviated by the interplay of proteins constituting two homologous efflux systems: AcrAB-TolC and AcrEF-TolC. However, our results show also that this is a genetic background-specific phenomenon, as the decrease in acetyl coenzyme A level is not evident in a cat-bearing acrAB derivative of the commonly used strain C600.     

Structural and Functional Similarity between the Bacterial Type III Secretion System Needle Protein PrgI and the Eukaryotic Apoptosis Bcl-2 Proteins

Background

Functional similarity is challenging to identify when global sequence and structure similarity is low. Active-sites or functionally relevant regions are evolutionarily more stable relative to the remainder of a protein structure and provide an alternative means to identify potential functional similarity between proteins. We recently developed the FAST-NMR methodology to discover biochemical functions or functional hypotheses of proteins of unknown function by experimentally identifying ligand binding sites. FAST-NMR utilizes our CPASS software and database to assign a function based on a similarity in the structure and sequence of ligand binding sites between proteins of known and unknown function.

Methodology/Principal Findings

The PrgI protein from Salmonella typhimurium forms the needle complex in the type III secretion system (T3SS). A FAST-NMR screen identified a similarity between the ligand binding sites of PrgI and the Bcl-2 apoptosis protein Bcl-xL. These ligand binding sites correlate with known protein-protein binding interfaces required for oligomerization. Both proteins form membrane pores through this oligomerization to release effector proteins to stimulate cell death. Structural analysis indicates an overlap between the PrgI structure and the pore forming motif of Bcl-xL. A sequence alignment indicates conservation between the PrgI and Bcl-xL ligand binding sites and pore formation regions. This active-site similarity was then used to verify that chelerythrine, a known Bcl-xL inhibitor, also binds PrgI.

Conclusions/Significance

A structural and functional relationship between the bacterial T3SS and eukaryotic apoptosis was identified using our FAST-NMR ligand affinity screen in combination with a bioinformatic analysis based on our CPASS program. A similarity between PrgI and Bcl-xL is not readily apparent using traditional global sequence and structure analysis, but was only identified because of conservation in ligand binding sites. These results demonstrate the unique opportunity that ligand-binding sites provide for the identification of functional relationships when global sequence and structural information is limited.     

Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant.

Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis.     

Specific interactions of metal ions with Cys-Xaa-Cys unit inserted into the peptide sequence

In this work five peptides with Cys-Xaa-Cys motif were studied including Ac-Cys-Gly-Cys-NH(2), Ac-Cys-Pro-Cys-Pro-NH(2), their N-unprotected analogues and the N-terminal fragment of metallothionein-3, Met-Asp-Pro-Glu-Thr-Cys-Pro-Cys-Pro-NH(2). All these peptides were found to be very effective ligands for Ni(2+), Zn(2+) and Cd(2+) ions. Potentiometric and spectroscopic (UV-Vis, CD and MCD) studies have proved that sulfur atoms are critical donors for the metal ions coordination. The amide nitrogen may participate in the metal ion binding only in the case when Gly is adjacent to Cys residues. Ac-Cys-Gly-Cys-NH(2) may serve as a low molecular weight model for cluster A, which is a binding unit of nickel ion in acetyl coenzyme A synthase. This bifunctional enzyme from anaerobic microorganisms catalyzes the formation of acetyl coenzyme A from CO, a methyl group donated by the corrinoid-iron-sulfur protein and coenzyme A. Other peptides studied in this work were Ac-Cys-Pro-Cys-Pro-NH(2) and Met-Asp-Pro-Glu-Thr-Cys-Pro-Cys-NH(2) originating from metallothionein sequence. These motifs are characteristic for the sequence of cysteine rich metallothionein-3 (MT-3) called also neuronal growth inhibitory factor (GIF). Cys-Pro-Cys-Pro fragment of protein was demonstrated to be crucial for the inhibitory activity of the protein.     

Enzyme classification by ligand binding

The problem of assigning a biochemical function to newly discovered proteins has been traditionally approached by expert enzymological analysis, sequence analysis, and structural modeling. In recent years, the appearance of databases containing protein-ligand interaction data for large numbers of protein classes and chemical compounds have provided new ways of investigating proteins for which the biochemical function is not completely understood. In this work, we introduce a method that utilizes ligand-binding data for functional classification of enzymes. The method makes use of the existing Enzyme Commission (EC) classification scheme and the data on interactions of small molecules with enzymes from the BRENDA database. A set of ligands that binds to an enzyme with unknown biochemical function serves as a query to search a protein-ligand interaction database for enzyme classes that are known to interact with a similar set of ligands. These classes provide hypotheses of the query enzyme's function and complement other computational annotations that take advantage of sequence and structural information. Similarity between sets of ligands is computed using point set similarity measures based upon similarity between individual compounds. We present the statistics of classification of the enzymes in the database by a cross-validation procedure and illustrate the application of the method on several examples.     

Biochemical and mutational analyses of AcuA, the acetyltransferase enzyme that controls the activity of the acetyl coenzyme a synthetase (AcsA) in Bacillus subtilis

The acuABC genes of Bacillus subtilis comprise a putative posttranslational modification system. The AcuA protein is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, the AcuC protein is a class I histone deacetylase, and the role of the AcuB protein is not known. AcuA controls the activity of acetyl coenzyme A synthetase (AcsA; EC 6.2.1.1) in this bacterium by acetylating residue Lys549. Here we report the kinetic analysis of wild-type and variant AcuA proteins. We contrived a genetic scheme for the identification of AcuA residues critical for activity. Changes at residues H177 and G187 completely inactivated AcuA and led to its rapid turnover. Changes at residues R42 and T169 were less severe. In vitro assay conditions were optimized, and an effective means of inactivating the enzyme was found. The basic kinetic parameters of wild-type and variant AcuA proteins were obtained and compared to those of eukaryotic GNATs. Insights into how the isolated mutations may exert their deleterious effect were investigated by using the crystal structure of an AcuA homolog.     

Interaction of the fluorescent analogue stearoyl-(1,N6)-etheno coenzyme A with chicken liver acetyl coenzyme A carboxylase

The interaction of stearoyl-(1,N6)-etheno coenzyme A (stearoyl-epsilon-CoA) with acetyl coenzyme A carboxylase was investigated by using fluorescence spectroscopy. The fluorescence emission of stearoyl-epsilon-CoA was partially quenched by acetyl coenzyme A carboxylase. Analysis of the data for dissociation constant (KD) and the stoichiometry of the interaction (n) gave values of 5.06 nM and 1.2, respectively, at pH 7.6 in 50 mM Tris-HCl and 25 degrees C. The KD value is comparable to the inhibition constant (Ki) obtained previously by others for the inhibition of rat liver acetyl coenzyme A carboxylase by long chain fatty acyl-CoAs. Citrate (which is known to polymerize and thus activate carboxylase) caused a partial quenching of the protein fluorescence of carboxylase, presumably due to polymerization of the enzyme. The quenching of the stearoyl-epsilon-CoA fluorescence caused by carboxylase as well as the inhibition of carboxylase activity by stearoyl-epsilon-CoA was reversed by citrate, but only in the presence of 6-O-methylglucose polysaccharide which forms a stable complex with fatty acyl-CoA. This shows that the stearoyl-epsilon-CoA bound to the enzyme is displaced by citrate only in the presence of an acceptor of fatty acyl-CoA. These results support the reciprocal relationship of citrate and fatty acyl-CoA in the regulation of acetyl coenzyme A carboxylase.     

Comparison of sequence-based and structure-based phylogenetic trees of homologous proteins: Inferences on protein evolution

Several studies based on the known three-dimensional (3-D) structures of proteins show that two homologous proteins with insignificant sequence similarity could adopt a common fold and may perform same or similar biochemical functions. Hence, it is appropriate to use similarities in 3-D structure of proteins rather than the amino acid sequence similarities in modelling evolution of distantly related proteins. Here we present an assessment of using 3-D structures in modelling evolution of homologous proteins. Using a dataset of 108 protein domain families of known structures with at least 10 members per family we present a comparison of extent of structural and sequence dissimilarities among pairs of proteins which are inputs into the construction of phylogenetic trees. We find that correlation between the structure-based dissimilarity measures and the sequence-based dissimilarity measures is usually good if the sequence similarity among the homologues is about 30% or more. For protein families with low sequence similarity among the members, the correlation coefficient between the sequence-based and the structure-based dissimilarities are poor. In these cases the structure-based dendrogram clusters proteins with most similar biochemical functional properties better than the sequence-similarity based dendrogram. In multi-domain protein families and disulphide-rich protein families the correlation coefficient for the match of sequence-based and structure-based dissimilarity (SDM) measures can be poor though the sequence identity could be higher than 30%. Hence it is suggested that protein evolution is best modelled using 3-D structures if the sequence similarities (SSM) of the homologues are very low.