Crystallization and rhenium MAD phasing of the acyl-homoserinelactone synthase EsaI.

Acyl-homoserine-L-lactones (AHLs) are diffusible chemical signals that are required for virulence of many Gram-negative bacteria. AHLs are produced by AHL synthases from two substrates, S-adenosyl-L-methionine and acyl-acyl carrier protein. The AHL synthase EsaI, which is homologous to the AHL synthases from other pathogenic bacterial species, has been crystallized in the primitive tetragonal space group P4(3), with unit-cell parameters a = b = 66.40, c = 47.33 A. The structure was solved by multiple-wavelength anomalous diffraction with a novel use of the rhenium anomalous signal. The rhenium-containing structure has been refined to a resolution of 2.5 A and the perrhenate ion binding sites and liganding residues have been identified.

Acylhomoserine lactone synthase activity of the Vibrio fischeri AinS protein.

Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone from an acyl-acyl carrier protein and S-adenosylmethionine. Another V. fischeri gene, ainS, directs the synthesis of N-octanoylhomoserine lactone. The AinS protein shows no significant sequence similarity with LuxI family members, but it does show sequence similarity with the Vibrio harveyi LuxM protein. The luxM gene is required for the synthesis of N-(3-hydroxybutyryl)-L-homoserine lactone. To gain insights about whether AinS and LuxM represent a second family of acylhomoserine lactone synthases, we have purified AinS as a maltose-binding protein (MBP) fusion protein. The purified MBP-AinS fusion protein catalyzed the synthesis of N-octanoylhomoserine lactone from S-adenosylmethionine and either octanoyl-acyl carrier protein or, to a lesser extent, octanoyl coenzyme A. With the exception that octanoyl coenzyme A served as an acyl substrate for the MBP-AinS fusion protein, the substrates for and reaction kinetics of the MBP-AinS fusion protein were similar to those of the several LuxI family members previously studied. We conclude that AinS is an acylhomoserine lactone synthase and that it represents a second family of such enzymes.

Acyl homoserine-lactone quorum-sensing signal generation.

Acyl homoserine lactones (acyl-HSLs) are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression. These signals are synthesized by members of the LuxI family of proteins. To understand the mechanism of acyl-HSL synthesis we have purified the Pseudomonas aeruginosa RhlI protein and analyzed the kinetics of acyl-HSL synthesis by this enzyme. Purified RhlI catalyzes the synthesis of acyl-HSLs from acyl-acyl carrier proteins and S-adenosylmethionine. An analysis of the patterns of product inhibition indicated that RhlI catalyzes signal synthesis by a sequential, ordered reaction mechanism in which S-adenosylmethionine binds to RhlI as the initial step in the enzymatic mechanism. Because pathogenic bacteria such as P. aeruginosa use acyl-HSL signals to regulate virulence genes, an understanding of the mechanism of signal synthesis and identification of inhibitors of signal synthesis has implications for development of quorum sensing-targeted antivirulence molecules.

In vivo evidence that S-adenosylmethionine and fatty acid synthesis intermediates are the substrates for the LuxI family of autoinducer synthases.

Many gram-negative bacteria synthesize N-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In Escherichia coli, synthesis of N-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a fadD mutant blocked in beta-oxidative fatty acid degradation. Also, conditions known to induce the fad regulon did not increase autoinducer synthesis. In contrast, cerulenin and diazoborine, specific inhibitors of fatty acid synthesis, both blocked autoinducer synthesis even in a strain dependent on beta-oxidative fatty acid degradation for growth. These data provide the first in vivo evidence that the acyl chains in autoinducers synthesized by LuxI-family synthases are derived from acyl-acyl carrier protein substrates rather than acyl coenzyme A substrates. Also, we show that decreased levels of intracellular S-adenosylmethionine caused by expression of bacteriophage T3 S-adenosylmethionine hydrolase result in a marked reduction in autoinducer synthesis, thus providing direct in vivo evidence that the homoserine lactone ring of LuxI-family autoinducers is derived from S-adenosylmethionine.

Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

Polymorphism of the yeast pyruvate carboxylase 2 gene and protein: effects on protein biotinylation.

In Saccharomyces cerevisiae there are two isoenzymes of pyruvate carboxylase (Pyc) encoded by separate genes designated PYC1 and PYC2. We report the isolation and sequencing of a PYC2 gene, and the localization of both genes on the physical map of S. cerevisiae. Comparison with the previously reported sequence [Stucka, Dequin, Salmon and Gancedo (1991) Mol. Gen. Genet. 229, 307-315] revealed significant differences within the open reading frame. The most notable difference was near the 3' end, where we found a single base deletion reducing the open reading frame by 15 bases. We have confirmed the C-terminus of Pyc2 encoded by the gene isolated here by expressing and purifying an 86-amino-acid biotin-domain peptide. In addition, we investigated the effects of the two changes in the Pyc2 biotin domain (K1155R substitution and Q1178P/five-amino-acid extension) on the extent of biotinylation in vivo by Escherichia coli biotin ligase, and compared the biotinylation of peptides containing these changes with that of two different-length Pyc1 biotin-domain peptides. The K1155R substitution had very little effect on biotinylation, but the five-amino-acid C-terminal extension to Pyc2 and the N-terminal extension to Pycl both improved biotinylation in vivo.

Regulation of pyruvate carboxylase isozyme (PYC1, PYC2) gene expression in Saccharomyces cerevisiae during fermentative and nonfermentative growth.

In Saccharomyces cerevisiae there are two isoenzymes of pyruvate carboxylase (Pyc) encoded by separate genes, designated PYC1 and PYC2. In the wild type yeast, the expression of both genes is influenced by both the growth phase and the type of carbon source, indicating discrete regulatory mechanisms and metabolic roles for PYC1 and PYC2. On glucose minimal medium PYC1 and PYC2 are differentially regulated as shown by a constant level of PYC1 expression throughout the main growth phase compared to a high level of PYC2 expression only in the early growth phase. On ethanol minimal medium, the growth-related pattern of PYC1 and PYC2 expression was similar as shown by a 3.6-fold decline from early to mid log phase. PYC1 expression, however, was activated 10-fold above PYC2 mRNA levels during this period of growth. To further investigate the roles of the two PYC genes we determined the growth phenotypes and expression levels of PYC in pyc1 and pyc2 single null mutants. During fermentative growth, the lack of either PYC gene had little effect on the level and pattern of expression of the other PYC gene, indicating further their separate regulation. In comparison to the pyc2 null, the pyc1 null strain showed a 3- to 4-fold lower level of Pyc activity and Pyc protein concentration. Moreover, the pyc1 null showed a strong requirement for L-aspartate for efficient growth, indicating the importance of PYC1 expression for the synthesis of C4 intermediates. DV6.2 (PYC1, pyc2 delta) showed a 3.2-fold higher level of activity on ethanol minimal medium when compared to growth on glucose minimal medium, and supported growth in the absence of L-aspartate. The pyc1 null, MW21.3 (pyc1 delta, PYC2), on the other hand, did not support growth on ethanol in the absence of aspartate. This study represents the first report on the characterisation of expression of the PYC genes in yeast throughout growth. Their metabolic roles for both fermentative and gluconeogenic growth are considered.

Yeast pyruvate carboxylase: identification of two genes encoding isoenzymes.

In Saccharomyces cerevisiae, pyruvate carboxylase [EC 6.4.1.1] has an important anaplerotic role in the production of oxaloacetate from pyruvate. We report here the existence of two pyruvate carboxylase isozymes, which are encoded by separate genes within the yeast genome. Null mutants were constructed by one step gene disruption of the characterised PYC gene in the yeast genome. The mutants were found to have 10-20% residual pyruvate carboxylase activity, which was attributable to a protein of identical size and immunogenically related to pyruvate carboxylase. Immunocytochemical labelling studies on ultrathin sections of embedded whole cells from the null mutants showed the isozyme to be located exclusively in the cytoplasm. We have mapped the genes encoding both enzymes and shown the previously characterised gene, designated PYC1, to be on chromosome VII whilst PYC2 is on chromosome II.