Edward DeMoll

Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis

BACKGROUND Virulence in the pathogenic bacterium Yersinia pestis, causative agent of bubonic plague, has been correlated with the biosynthesis and transport of an iron-chelating siderophore, yersiniabactin, which is induced under iron-starvation conditions. Initial DNA sequencing suggested that this system is highly conserved among the pathogenic Yersinia. Yersiniabactin contains a phenolic group and three five-membered thiazole heterocycles that serve as iron ligands. RESULTS The entire Y. pestis yersiniabactin region has been sequenced. Sequence analysis of yersiniabactin biosynthetic regions (irp2-ybtE and ybtS) reveals a strategy for siderophore production using a mixed polyketide synthase/nonribosomal peptide synthetase complex formed between HMWP1 and HMWP2 (encoded by irp1 and irp2). The complex contains 16 domains, five of them variants of phosphopantetheine-modified peptidyl carrier protein or acyl carrier protein domains. HMWP1 and HMWP2 also contain methyltransferase and heterocyclization domains. Mutating ybtS revealed that this gene encodes a protein essential for yersiniabactin synthesis. CONCLUSIONS The HMWP1 and HMWP2 domain organization suggests that the yersiniabactin siderophore is assembled in a modular fashion, in which a series of covalent intermediates are passed from the amino terminus of HMWP2 to the carboxyl terminus of HMWP1. Biosynthetic labeling studies indicate that the three yersiniabactin methyl moieties are donated by S-adenosylmethionine and that the linker between the thiazoline and thiazolidine rings is derived from malonyl-CoA. The salicylate moiety is probably synthesized using the aromatic amino-acid biosynthetic pathway, the final step of which converts chorismate to salicylate. YbtS might be necessary for converting chorismate to salicylate.

Yersiniabactin from Yersinia pestis: biochemical characterization of the siderophore and its role in iron transport and regulation

A siderophore-dependent iron transport system of the pathogenic yersiniae plays a role in the pathogenesis of these organisms. The structure of the yersiniabactin (Ybt) siderophore produced by Yersinia enterocolitica has been elucidated. This paper reports the purification of Ybt from Yersinia pestis and demonstrates that it has the same structure as Ybt from Y. enterocolitica. Purified Ybt had a formation constant for Fe3+ of approximately 4x10(-36). Addition of purified Ybt from Y. pestis enhanced iron uptake by a siderophore-negative (irp2) strain of Y. pestis. Maximal expression of the Ybt outer-membrane receptor, Psn, in this strain was dependent upon exogenously supplied Ybt. Regulation of Psn expression by Ybt occurred at the transcriptional level. Y. pestis DNA was used to construct irp2 and psn mutations in Yersinia pseudotuberculosis. The irp2 mutant strain no longer synthesized Ybt and the psn mutant strain could not use exogenously supplied Ybt. As in Y. pestis, Ybt was required for maximal expression of Psn. Regulation by Ybt occurred at the transcriptional level. In contrast to Y. pestis, in which a psn mutation does not repress synthesis of Ybt siderophore or expression of the iron-regulated HMWP1 and HMWP2 proteins, the same mutation in Y. pseudotuberculosis partially repressed these products.

Characterization of human T-cell responses to Yersinia enterocolitica superantigen

We reported that antigenic preparations from Yersinia enterocolitica stimulate murine T cells in a manner consistent with that of superantigens. As a consequence we examined whether Y. enterocolitica antigenic preparations stimulate human T-cell cultures. Human T cells, enriched from peripheral blood lymphocytes, were stimulated to proliferate in the presence of Y. enterocolitica cytoplasmic and membrane preparations. This activity has also been shown to be sensitive to protease treatment, indicating the presence of a protein, and when separated by ion-exchange chromatography a single peak of activity is resolved. Furthermore, this proliferation was inhibited, in a dose-dependent manner, by the presence of antibodies directed against MHC class II antigens, indicating a requirement for these molecules. When these cells were stained with a panel of V beta-specific antibodies to determine if there was an enrichment of a particular V beta-bearing T-cell subset after stimulation, results indicate a significant enrichment of T cells bearing V beta 3, V beta 12, V beta 14, and V beta 17 over controls. Taken together, these data are consistent with a Y. enterocolitica product acting as a superantigen for human T cells.

Borrelia burgdorferi EbfC Defines a Newly-Identified, Widespread Family of Bacterial DNA-Binding Proteins

The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where ‘n’ can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5′ of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel α-helical ‘tweezer’-like structure.

Acetyl-CoA decarbonylase/synthase complex from Archaeoglobus fulgidus

Abstract The acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex catalyzes the reversible cleavage and synthesis of acetyl-CoA in methanogens. This report of the enzyme complex in Archaeoglobus fulgidus demonstrates the existence of a functional ACDS complex in an organism that is not a methanogen. The A. fulgidus enzyme complex contained five subunits of 89, 72, 50, 49.5, and 18.5 kDa, and it catalyzed the overall synthesis of acetyl-CoA according to the following reaction:w CO2 + 2 Fdred(Fe2+) + 2 H+ + CH3– H4SPt + CoA ⇌ acetyl-CoA + H4SPt + 2 Fdox(Fe3+) + H2Owhere Fd is ferredoxin, and CH3–H4SPt and H4SPt denote N5-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively.

Reduced synthesis of the Ybt siderophore or production of aberrant Ybt-like molecules activates transcription of yersiniabactin genes in Yersinia pestis

Synthesis of the siderophore yersiniabactin (Ybt) proceeds by a mixed nonribosomal peptide synthetase/polyketide synthase mechanism. Transcription of ybt genes encoding biosynthetic and transport functions is repressed under excess iron conditions by Fur, but is also activated by Ybt via the transcriptional regulator YbtA. While mutations in most biosynthetic genes and ybtA negate transcription activation from the regulated promoters, three biosynthetic mutations do not reduce this transcriptional activation. Here we show that two of these mutants, one lacking the putative type II thioesterase (TE) YbtT and the other with a mutation in the TE domain of HMWP1, produce reduced levels of authentic Ybt that are capable of signalling activity. Alanine substitutions in two residues of YbtT that are essential for catalytic activity in other type II TEs reduced the ability of Yersinia pestis to grow under iron-chelated conditions. The third mutant, which lacks the salicylate synthase YbtS, did not make authentic Ybt but did produce a signalling molecule. Finally, a Δpgm strain of Y. pestis, which lacks essential Ybt biosynthetic genes, also produced a signalling molecule that can activate transcription of ybt genes. The non-Ybt signal molecules from these two mutants are likely separate compounds. While these compounds are not biologically relevant to normal Ybt regulation, a comparison of the structures of Ybt and other signalling molecules will help in determining the chemical structures recognized as a Ybt signal.

A single operon-encoded form of the acetyl-CoA decarbonylase/synthase multienzyme complex responsible for synthesis and cleavage of acetyl-CoA in Methanosarcina thermophila

Methanogens growing on C-1 substrates synthesize 2-carbon acetyl groups in the form of acetyl-CoA for carbon assimilation using the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) which contains five different subunits encoded within an operon. In species growing on acetate ACDS also functions to cleave the acetate C-C bond for energy production by methanogenesis. A number of species of Methanosarcina that are capable of growth on either C-1 compounds or acetate contain two separate ACDS operons, and questions have been raised about whether or not these operons play separate roles in acetate synthesis and cleavage. Methanosarcina thermophila genomic DNA was analyzed for the presence of two ACDS operons by PCR amplifications with different primer pairs, restriction enzyme analyses, DNA sequencing and Southern blot analyses. A single ACDS operon was identified and characterized, with no evidence for more than one. MALDI mass spectrometric analyses were carried out on ACDS preparations from methanol- and acetate-grown cells. Peptide fragmentation patterns showed that the same ACDS subunits were present regardless of growth conditions. The evidence indicates that a single form of ACDS is used both for acetate cleavage during growth on acetate and for acetate synthesis during growth on C-1 substrates.

Extraction, Purification, and Identification of Yersiniabactin, the Siderophore of Yersinia pestis

This unit describes in detail the extraction, purification, and identification of Yersiniabactin the siderophore of Yersinia pestis. Iron is essential for bacterial growth. Although relatively abundant, access to iron is limited in nature by low solubility. This problem is exacerbated for pathogenic bacteria, which must also defeat the host organisms innate defenses, including mechanisms to sequester iron. One solution to these problems is production of water soluble, small molecules with high affinities for iron called siderophores. This protocol has been fine tuned for Yersiniabactin purification but may be easily modified for use in isolating other siderophores or similar molecules. Curr. Protoc. Microbiol. 23:5B.3.1‐5B.3.22.

Observed Resistance to Pyrimidine Analogs and Sensitivity to Uracil in Drosophila is attributed to Deregulation of Pyrimidine Metabolism

Pyrimidine nucleotides play a central role in cellular metabolism and regulation. In most organisms two pathways provide pyrimidines: the de novo biosynthetic pathway and the salvage pathway. Drosophila melanogaster, the fruit fly, is an ideal organism for study of the genetic basis and regulatory mechanisms of various metabolic pathways. De novo pyrimidine biosynthesis in the fruit fly is a six-step pathway, which is catalyzed by enzymes encoded by three separate genes (Freund and Jarry, 1987; Rawls et al., 1993; Eisenberg et al., 1993). The gene rudimentary (r) is Drosophila’s equivalent of the mammalian gene for CAD (Freund and Jarry, 1987). De novo pyrimidine biosynthesis is important for the proper development of flies. However, the salvage pathway can suffice when the external supply of pyrimidines is very high (Falk and Nash, 1974).