No abstract

Diacyl phthiocerol esters and their congeners are mycobacterial virulence factors. The biosynthesis of these complex lipids remains poorly understood. Insight into their biosynthesis will aid the development of rationally designed drugs that inhibit their production. In this study, we investigate a biosynthetic step required for diacyl (phenol)phthiocerol ester production, i.e., the reduction of the keto group of (phenol)phthiodiolones. We utilized comparative genomics to identify phthiodiolone ketoreductase gene candidates and provide a genetic analysis demonstrating gene function for two of these candidates. Moreover, we present data confirming the existence of a diacyl phthiotriol intermediate in diacyl phthiocerol biosynthesis. We also elucidate the mechanism underlying diacyl phthiocerol deficiency in some mycobacteria, such as Mycobacterium ulcerans and Mycobacterium kansasii. Overall, our findings shed additional light on the biosynthesis of an important group of mycobacterial lipids involved in virulence.A unique feature of the mycobacteria is their outer cell wall layer. The unusual lipid composition of this layer contributes to the characteristic pathogenicity and resiliency of mycobacterial pathogens (4,7,8,15,22,24). Among the lipids of the mycobacterial outer cell wall layer are diesters of long-chain multimethyl-branched fatty acids and ␤ glycol-containing longchain polyketides (PKs) (14,15,20,24). These diesters, collectively referred to herein as diacylated PKs (DPs), are produced by Mycobacterium tuberculosis, M. leprae, M. bovis, M. microti, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. haemophilum, and M. gastri. With the exception of M. gastri, all these mycobacteria are pathogenic. Depending on the species, the fatty acid component of the diesters is called either mycocerosic acid or phthioceranic acid. The ␤ glycol-containing PKs are phthiocerols (POLs), phthiodiolones (PONEs), phenolphthiocerols (pPOLs), or phenolphthiodiolones (pPONEs). The diesters of pPOLs and pPONEs are glycosylated on the aromatic ring. Finally, the difference between (p)POLs and (p)PONEs is that the former lipid family has a 3-methoxy group [or 2-methoxy in (p)POLs of the B series], whereas the latter family has a 3-keto group [or 2-keto in (p)PONEs of the B series] (Fig. 1).Discovered over 70 years ago (39, 40), DPs have recently become the focus of intense attention after several lines of evidence indicated their role as virulence factors. Most importantly, abrogation of the production of M. tuberculosis nonglycosylated DPs (often referred to as phthiocerol dimycocerosates) was recently shown to correlate with attenuation of virulence in mouse infection models (6,13,33,34,36,37). Furthermore, production of glycosylated DPs (usually referred to as phenolglycolipids [PGLs]) and not production of only nonglycosylated DPs (32) correlates with M. tuberculosis hypervirulence in the mouse model, and the M. tuberculosis PGLs were demonstrated to down-regulate macrophage immune function in vitro (32), hinting at ...

Since the work of Jorgenson and Lukacs in the early 1980s, 1,2 capillary electrophoresis (CE) has experienced rapid growth and been applied to a wide array of analytes. The dominant area of application of CE has been for biomolecules. However, there has been a steady and increasing interest in using CE for the determination of metal ions. 3The attractive features of CE for metal ion determination are the same as those for other separations: its exceptional separation efficiency; its rapid separations; and its low mass detection limits. However, as with other types of analytes, the primary limitation of CE is its poor concentration detection limits. Most (85%) publications of CE for metal ions have used absorbance detection. 3 Unfortunately, few hydrated metal ions absorb significantly above 185 nm. Greater sensitivity is achieved by complexing the metal ions with a metallochromic dye. Examples include PAR for transition metals, 4 Arsenazo I for alkaline earth metals 5 and Arsenate III for lanthanides and uranium. 6,7 Alternatively, up to 26 metal cations have been separated with parts-per-billion detection limits using indirect absorbance detection. [8][9][10][11] Regardless, the most sensitive detection scheme in CE is laser induced fluorescence (LIF). 12-14 CE-LIF determinations of metals typically use the 8-hydroxyquinoline-5-sulfonic acid (HQS) fluorescent ligand shown in Fig. 1 . Optimum excitation of the metal-HQS is at 393 nm with maximum emission in the 500 -520 nm range. 17 Fluorescence excitation of metal-HQS complexes separated by CE has been performed using the 325 nm HeCd laser line, 15,18,19 the 351.1 -364.8 nm lines of an argon ion laser 20 and with a broad band mercury-xenon lamp. 16 However, none of these excitation sources is ideal. They all are expensive and have limited lifetimes (∼5000 h). They are also relatively bulky, making them unattractive for miniaturized instruments. 19 Semiconductor or diode lasers are promising alternatives to conventional lasers. Diode lasers are cheap, require no maintenance, and have lifetimes approaching 50000 h. 21 These lasers are also extremely compact and can run off battery power.Further, the output stability of diode lasers is significantly better than that of conventional lasers (0.01% vs. 1% noise). 22 These properties suggest that diode lasers are ideally suited for LIF detection. In October of 1999, Nichia Corporation (Japan) launched the commercial sale of their InGaN-based violet laser diode.23-27 * The diode operates at 405 nm with a power output of 5 mW and has an estimated lifetime of 2000 to 5000 h. Such short wavelength semiconductor lasers have long been sought for applications such as full-color electroluminescent displays, laser printers, undersea optical communications and optical data storage. For instance, a violet diode laser would quadruple the data capacity of current DVD players.In addition to its applications in electronics and telecommunications, the violet diode laser offers tremendous potential for analytical chemistry. Recently, ...