The Mycobacterium tuberculosis Ag85A is a novel diacylglycerol acyltransferase involved in lipid body formation

Mycobacterium tuberculosis accumulates large amounts of triacylglycerol (TAG) which acts as storage compounds for energy and carbon. The mycobacterial triacylglycerols stored in the form of intracellular lipid droplets are essential for long-term survival of M. tuberculosis during a dormant state. We report here that when the M. tuberculosis mycolytransferase Ag85A is overexpressed in Mycobacterium smegmatis mc(2)155, cell morphology was changed and the cells became grossly enlarged. A massive formation of lipid bodies and a change in lipid pattern was observed simultaneously. We suspected a possible role of Ag85A in the acyl lipid metabolism and discovered that the enzyme possesses acyl-CoA:diacylglycerol acyltransferase (DGAT) activity in addition to its well-known function as mycolyltransferase. Ag85A mediates the transesterification of diacylglycerol using long-chain acyl-CoA as acyl donors. The K(m) and K(cat) values for palmitoleoyl-coenzyme A were 390 μM and 55.54 min(-1) respectively. A docking model suggests that palmitoleoyl-coenzyme A and 1,2-dipalmitin occupy the same active site as trehalose 6,6'-dimycolate and trehalose 6'-monomycolate. The site-directed Ser126Ala mutation of the active site proved that this residue is involved in the catalytic activity of this enzyme. Although not proven conclusively for dormant stage of M. tuberculosis, our novel finding about the synthesis of TAGs by Ag85A strongly suggests that Ag85A may play a significant role in the formation of lipid storage bodies and thus also in the establishment and maintenance of a persistent tuberculosis infection.     

Synthesis and biological evaluation of trehalose analogs as potential inhibitors of mycobacterial cell wall biosynthesis

Analogs of trehalose are reported that were designed to interfere with mycolylation pathways in the mycobacterial cell wall. Several derivatives of 6,6'-dideoxytrehalose, including N,N'-dialkylamino and 6,6'-bis(sulfonamido) analogs, were prepared and evaluated for antimycobacterial activity against Mycobacterium tuberculosis H(37)Ra and a panel of clinical isolates of Mycobacterium avium. 6,6'-Diaminotrehalose and its diazido precursor were both inactive, but significant activity apparently related to aliphatic chain length was found among the sulfonamides, N-alkylamines, and one of the amidines.     

Production and partial characterization of anti-cord factor (trehalose-6,6'-dimycolate) IgG antibody in rabbits recognizing mycolic acid subclasses of Mycobacterium tuberculosis or Mycobacterium avium

An ELISA with cord factor (trehalose-6,6'-dimycolate) is useful for the serodiagnosis of tuberculosis. To clarify the exact antigenic epitope in cord factor, recognized by a rabbit anti-cord factor IgG antibody, and to ascertain the most sensitive and specific diagnostic test antigen, rabbits were immunized with two kinds of cord factors isolated from Mycobacterium tuberculosis or Mycobacterium avium and the reactivities of the sera were tested against cord factors or the component mycolic acid methyl esters by ELISA. The serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against M. tuberculosis cord factor, but less reactive against M. avium cord factor. In contrast, the serum from rabbits immunized with M. avium cord factor was highly reactive against M. avium cord factor but less reactive against M. tuberculosis cord factor. Moreover, the serum from rabbits immunized with M. tuberculosis cord factor reacted against mycolic acid methyl esters, especially methoxy mycolic acid methyl ester. On the other hand, the serum from rabbits immunized with M. tuberculosis cord factor was less reactive against trehalose-6-monomycolate and not reactive against sulfolipid (2,3,6,6'-tetraacyl trehalose 2'-sulfate). From these results, it was concluded that the anti-cord factor IgG antibody, produced experimentally in rabbits, recognized the differences in the cord factor structures, i.e. the hydrophobic moiety rather than the carbohydrate moiety. It was also noted that the serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against methoxy mycolic acid as an epitope. This paper is the first to describe how the anti-cord factor IgG antibody can recognize the mycolic acid subclasses, which differ according to the species of mycobacteria.     

Characterization of sulfolipids of Mycobacterium tuberculosis H37Rv by multiple-stage linear ion-trap high-resolution mass spectrometry with electrospray ionization reveals that the family of sulfolipid II predominates

Mycobacterium tuberculosis, the causative agent of tuberculosis, is unique among bacterial pathogens in that it contains a wide array of complex lipids and lipoglycans on its cell wall. Among them, the sulfated glycolipid, termed the sulfolipid, is thought to mediate specific host-pathogen interactions during infection. Sulfolipids (SLs), including sulfolipid I (SL-I) and sulfolipid II (SL-II), are 2,3,6,6'-tetraacyltrehalose 2'-sulfates. SL-I was identified as a family of homologous 2-palmitoyl(stearoyl)-3-phthioceranoyl-6,6'-bis(hydroxyphthioceranoy1)trehalose 2'-sulfates and was believed to be the principal sulfolipid of M. tuberculosis strain H37Rv. We cultured and extracted sulfolipids using various conditions, including those originally described, and employed high-resolution multiple-stage linear ion-trap mass spectrometry with electrospray ionization to characterize the structure of the principal SL. We revealed that SL-II, a family of homologous 2-stearoyl(palmitoyl)-3,6,6'-tris(hydroxyphthioceranoy1)trehalose 2'-sulfates, rather than SL-I is the principal sulfolipid class. We identified a great number of isomers resulting from permutation of the various hydroxyphthioceranoyl substituents at positions 6 and 6' of the trehalose backbone for each of the SL-II species in the entire family. We redefined the structure of this important lipid family that was misassigned using the traditional methods 40 years ago.     

Rapid serodiagnosis of human mycobacteriosis by ELISA using cord factor (trehalose-6,6'-dimycolate) purified from Mycobacterium tuberculosis as antigen

IgG antibodies against purified cord factor (trehalose-6,6'-dimycolate, TDM) in sera of 99 patients infected with mycobacteria (42 patients with tuberculosis excreting tubercle bacilli in the sputum, 11 patients with non-tuberculous mycobacteriosis excreting acid-fast bacilli in the sputum, and 46 patients without bacilli in the sputum but diagnosed as having pulmonary tuberculosis by chest X-ray films and physical examination), five patients with lung cancer, and 100 healthy controls which included subjects positive and negative for the tuberculin test were tested by the ELISA with TDM purified from Mycobacterium tuberculosis H37Rv as the antigen. Of the 99 cases of mycobacteriosis, 83 patients (83.8%) had positive results (48 samples from 53 patients, or 90.5%, with bacilli in the sputum, and 35 samples from 46 patients (76%) with tuberculosis diagnosed clinically). The sera of the five patients with lung cancer and the 100 controls all gave negative results. Thus, the sensitivity and specificity were 83.8% and 100%, respectively. ELISA with TDM as the antigen is simple, reproducible, and useful for the rapid serodiagnosis of general mycobacterial infections including tuberculosis, because it does not involve the cultivation of bacteria.     

Anti-cord factor (trehalose 6,6'dimycolate) IgG antibody in tuberculosis patients recognizes mycolic acid subclasses.

The detection of anti-cord factor (trehalose 6,6'-dimycolate) IgG antibody in active (smear-and/or culture-positive) and inactive (smear-and culture-negative) tuberculosis patients is a useful serodiagnostic tool that can be used for early clinical diagnosis of the disease. We estimated the titers of anticord factor IgG antibody in the sera of tuberculosis patients, and compared them with those of Mycobacterium avium-infected patients. Most of the serum samples obtained from the tuberculosis patients were highly reactive against M. tuberculosis (MTB) cord factor isolated from M. tuberculosis H37Rv, a human-type mycobacterial strain, whereas they were less reactive against M. avium (MAC) cord factor. Similarly, most of the serum samples of the MAC-infected patients were highly reactive against MAC cord factor and less reactive against MTB cord factor. These results suggest that anti-cord factor IgG antibody recognizes the mycolic acid subclasses as an epitope which comprises cord factor, since MTB and MAC cord factor differ in mycolic acid subclasses and molecular species composition. To clarify the exact antigenic epitope in cord factor and to find out a more sensitive and specific diagnostic test antigen, we examined the reactivity of patients' sera to glycolipids containing trehalose (cord factor and sulfolipid) obtained from various mycobacterial species. Furthermore, the reactivity of human antisera to various mycolic acid subclasses (alpha-, methoxy and keto mycolic acids) of MTB cord factor was compared. We found that anti-cord factor IgG antibody in the sera of human tuberculosis patients most strikingly recognized methoxy mycolic acid in the cord factor of M. tuberculosis, whereas it recognized alpha- and keto mycolic acids weakly. Pre-absorption studies of antibody with MTB cord factor or methoxy mycolic acid methyl ester showed that anti-cord factor antibody was absorbed partially, but consistently. This is the first report describing that the specific subclass of mycolic acid from mycobacteria is antigenic in the humoral immune system of human tuberculosis infection.     

Serodiagnosis of extrapulmonary tuberculosis by enzyme-linked immunosorbent assay (ELISA)

Antibodies against Mycobacterium tuberculosis antigenic glycolipids were determined by enzyme-linked immunosorbent assay (ELISA). The 720 sera were collected from adult patients under investigation, suspected with extrapulmonary tuberculosis. The test performance was estimated according to definitive diagnosis in terms of specificity, sensitivity, positive predictive value and negative predictive value. These parameters calculated on 142 sera from patients with extrapulmonary tuberculosis and on 578 sera from patients with different nontuberculosis diseases were 92%, 81.6%, 70.9% and 95.1%, respectively. The specificity decreased to 85% when tuberculosis was associated with cancer or hepatic cirrhosis. In reactivated tuberculosis the sensitivity and the positive predictive value were 86.9% and 83.3%, respectively. Our results showed that ELISA was conclusive for patients with active tuberculosis, before the initiation of the treatment. The sensitivity decreased to 30% in inactive forms. It was demonstrated that ELISA was positive in cases with negative microscopy genitourinary tuberculosis. ELISA could be used as a supporting test in the laboratory diagnosis of active extrapulmonary tuberculosis in adults, disregarding the site involved.     

Cord factor (trehalose-6-6'-dimycolate) of in vivo-derived Mycobacterium lepraemurium.

Harvests of Mycobacterium lepraemurium obtained from livers of moribund infected mice yielded M. lepraemurium cell walls that were extracted with solvent to provide crude M. lepraemurium cell wall lipids. By solvent fractionation and chromatography on DEAE cellulose and cellulose, a cord factor-like glycolipids contaminated with mycoside C was obtained. Additional solvent treatment provided the purified glycolipid, which was identified as 6,6'-trehalose dimycolate, by infrared and chromatographic comparison with authentic samples from M. tuberculosis, by identification of trehalose and specific mycolates of M. lepraemurium, and by permethylation analysis. This constitutes the first unequivocal identification of cord factor as a product of in vivo-derived mycobacteria.     

Rapid Serodiagnosis of Mycobacterium avium-intracellulare Complex Infection by ELISA with Cord Factor (Trehalose 6, 6′-Dimycolate), and Serotyping Using the Glycopeptidolipid Antigen

Mycobacterium avium-intracellulare complex (MAC) is one of the most important opportunistic pathogens, particularly in patients with acquired immunodeficiency syndrome (AIDS). The aim of this study was to determine whether an enzyme-linked immunosorbent assay (ELISA) using trehalose 6,6′-dimycolate (TDM) as an antigen can be used for the rapid serodiagnosis of MAC infection. We also identified MAC serotypes by ELISA using serotype-specific glycopeptidolipid (GPL) antigen. To confirm our findings, the thin-layer chromatographic (TLC) behavior of serotype-specific GPL of the strains isolated from MAC-infected patients was also tested. Forty patients infected with MAC and 30 healthy controls were tested. Thirty-two of the 40 MAC-infected patients had higher titers of serum antibodies against MAC TDM than against MTB TDM, while all 30 healthy control sera were unreactive to MAC TDM and MTB TDM. Results of the GPL ELISA indicated that 20 of the 40 MAC-infected patients' sera were reactive against serotype 4 GPL, 3 against serotype 8 GPL, and 1 against serotype 16 GPL. A TLC analysis of the GPL of the 40 MAC isolates showed that 16 strains were of serotype 4, 5 of serotype 8, and 2 of serotype 16. Results of the GPL ELISA were in good accord with those of the TLC analysis for most patients. Our findings suggest that ELISA using TDM is useful for rapid serodiagnosis of MAC infection, and that complementary ELISA testing using serotype-specific GPL gives additional detailed information concerning MAC serotypes.     

Detection of early secretory antigenic target-6 antibody for diagnosis of tuberculosis in non-human primates

Tuberculosis is one of the most economically devastating, zoonotic infections of captive non-human primates. The limitations of the tuberculin skin test, which is currently used to diagnose tuberculosis in living non-human primates, make it necessary to find new, simple, and economical diagnostic methods. We describe use of an enzyme-linked immunoassay to detect IgG antibodies against early secretory antigenic target (ESAT)-6, a small protein secreted by virulent tubercle bacilli, in paired (pre- and post-outbreak) sera from 57 non-human primates involved in an outbreak of Mycobacterium bovis infection in a research colony. Of 25 animals with tuberculosis lesions at necropsy, 22 (88%) had high serum levels of the ESAT-6 antibody. The ESAT-6 antibody was found in 16% (5/32) of post-outbreak sera from animals in which tuberculosis could not be confirmed at necropsy. The strong association between the ESAT-6 antibody and tuberculosis in non-human primates documented in this study, together with the robustness of the serologic assay, make the ESAT-6 ELISA a valuable tool for diagnosis of tuberculosis in captive non-human primates.     

Antigens in culture supernatant of Mycobacterium tuberculosis: epitopes defined by monoclonal and human antibodies.

Antigens of Mycobacterium tuberculosis found in the supernatant of heat-treated cultures were characterized in order to explore whether antigens from this source could be used for the development of a serological test. Culture supernatants and sonicates of 12, 25 and 39 d cultures were analysed by SDS-PAGE. In culture supernatant, major protein bands of 65, 24, and 12 kDa were visible after Coomassie brilliant blue staining. Using murine monoclonal antibodies in Western blots, a pattern of protein bands distinct from that of the corresponding M. tuberculosis sonicates was found in all the culture supernatants. Gel permeation chromatography, in the presence of SDS, was used to separate the major protein bands in the culture supernatant. In ELISA, sera from 20 of 26 patients with tuberculosis reacted with fractions containing mainly 24 kDa or 12 kDa proteins, whereas none of the control sera reacted. In Western blots, each patient serum had its own characteristic banding pattern with culture supernatant, but all the sera from tuberculosis patients and control subjects reacted with protein bands of 65, 61, 58, 30 and 24 kDa. The 12 kDa protein was recognized only by sera from patients with tuberculosis in both Western blots and ELISA. This suggests that different kinds of epitopes on proteins of M. tuberculosis are detected by human antibodies in Western blots and ELISA. We assume that epitopes recognized in Western blots by patients with tuberculosis and control subjects are ubiquitous and are also present on normal commensal bacteria. Epitopes recognized by only some patients with tuberculosis in Western blots may be linear and M. tuberculosis specific. Epitopes recognized by tuberculosis patients but by none of the control subjects in ELISA may be conformation related and M. tuberculosis specific. The major protein bands found in supernatants of heat-treated cultures, 24 and 12 kDa, possess epitopes that may be M. tuberculosis specific and are potentially valuable for the development of a serological test.     

血清学诊断中结核杆菌磷酸烯醇型丙酮酸羧激酶的应用

[目的]在原核系统中表达结核杆菌磷酸烯醇型丙酮酸羧激酶(phosphoenolpyruvate car-boxykinase PEPCK),并研究该蛋白在诊断结核病人血清抗体中的应用价值.[方法]应用基因重组技术表达重组蛋白结核杆菌磷酸烯醇型丙酮酸羧激酶,经亲和层析法纯化表达产物.用表达的重组蛋白免疫小鼠,研究其免疫学特性.间接酶联免疫吸附试验(Enzyme link immunosorbent assay,ELISA)检测结核病人血清中特异性IgG抗体,并与结核杆菌抗体胶体金法诊断试剂盒检测结果对比.[结果]试验表明转化入大肠杆菌中的重组质粒能够表达并纯化出相对分子量为72 kDa的重组蛋白;Western blot证实重组蛋白能够与小鼠抗BCG血清发生特异性反应;重组蛋白免疫小鼠后,小鼠血清中的抗体滴度可达1∶1280以上;重组蛋白用作ELISA包被抗原检测病人血清阳性率为17.3%(30/173),其中排菌病人的阳性率为32.5%(13/42),不排菌病人的阳性率为12.9%.该方法结果与结核杆菌抗体胶体金法诊断试剂盒的检测结果相比,敏感性为51.0%,特异性为96.7%.[结论]结核杆菌PEPCK具有较好的免疫原性和抗原性,有可能作为结核病血清学诊断的一组抗原之一.     

Revised structure of a trehalose-containing immunoreactive glycolipid of Mycobacterium tuberculosis

Nuclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry as well as various chemical degradations and chromatographic techniques were used to re-examine the structure of a highly immunoreactive glycolipid previously described in Mycobacterium tuberculosis (strain Canetti) as a 2,3-diacyl trehalose 2'-sulfate (labelled SL-IV). Ion exchange chromatography allowed the recognition of a neutral and an acidic glycolipid, indistinguishable on conventional silica gel. The neutral glycolipid was shown to be serologically identical to SL-IV and its structure was established as 2,3-diacyl trehalose. It corresponded to the non-chemically defined highly observed immunoreactive lipid previously recognized by others in M. tuberculosis (H37Rv).     

Production and characterization of monoclonal antibodies specific for Mycobacterium bovis

A series of monoclonal antibodies (MAbs), specific for Mycobacterium bovis and BCG strains, were tested extensively for cross-reactivity to a wide range of mycobacterial species using ELISA, Western blotting and dot-blot analysis. The MAbs bound specifically to M. bovis and BCG and showed limited cross-reactivity with some strains of M. tuberculosis. All these MAbs recognized a 22 kDa protein previously termed MPB70, and by competitive ELISA analysis appeared to detect at least three M. bovis-specific determinants on the MPB70 molecule.     

Rapid serodiagnostic test for pulmonary tuberculosis

Immune complexes (ICs) were isolated from sera of patients with pulmonary tuberculosis (PT), non-tuberculous pulmonary diseases and healthy control subjects by polyethylene glycol (PEG) precipitation method. Specific mycobacterial antibody in ICs was tested against an affinity purified mycobacterial antigen by counter-current immunoelectrophoresis (CIE). The results show that specific mycobacterial antibodies are present only in ICs of patients with PT (70%). Therefore CIE could be used as a simple and rapid serodiagnostic test in patients with PT, particularly when bacteriological methods in sputum specimens are negative for Mycobacterium tuberculosis. CIE has several operational advantages over ELISA and best suited to laboratories with limited resources.     

Nucleotide sequence analysis and serologic characterization of the Mycobacterium intracellulare homologue of the Mycobacterium tuberculosis 19 kDa antigen

Disseminated Mycobacterium avium/Mycobacterium intracellulare complex (MAC) disease is a frequent complication in patients with the acquired immune deficiency syndrome (AIDS). In this report, we present the nucleotide sequence of the M. intracellulare MI22 gene. Computer sequence comparisons reveal that the MI22 gene, which encodes a serologically active protein, has 78% DNA sequence identity and 77% protein sequence identity with the seroreactive 19 kDa Mycobacterium tuberculosis lipoprotein antigen. Southern blot hybridizations indicate that an MI22 gene probe binds similar-sized restriction fragments in M. tuberculosis and M. intracellular genomic DNA. In addition, immunoblot analyses demonstrate that MI22 is recognized by sera from tuberculosis patients. These data further support the existence of 19 kDa MAC and M. tuberculosis protein homologues. Phase partitioning experiments and the presence of a consensus lipid modification site in the deduced MI22 protein sequence strongly suggest that M122 is also a lipoprotein. Comparative analyses of these mycobacterial antigenic homologues may provide the basis for the design of species-specific diagnostic reagents.     

Trehalose synthase converts glycogen to trehalose

Trehalose (alpha,alpha-1,1-glucosyl-glucose) is essential for the growth of mycobacteria, and these organisms have three different pathways that can produce trehalose. One pathway involves the enzyme described in the present study, trehalose synthase (TreS), which interconverts trehalose and maltose. We show that TreS from Mycobacterium smegmatis, as well as recombinant TreS produced in Escherichia coli, has amylase activity in addition to the maltose <--> trehalose interconverting activity (referred to as MTase). Both activities were present in the enzyme purified to apparent homogeneity from extracts of Mycobacterium smegmatis, and also in the recombinant enzyme produced in E. coli from either the M. smegmatis or the Mycobacterium tuberculosis gene. Furthermore, when either purified or recombinant TreS was chromatographed on a Sephacryl S-200 column, both MTase and amylase activities were present in the same fractions across the peak, and the ratio of these two activities remained constant in these fractions. In addition, crystals of TreS also contained both amylase and MTase activities. TreS produced both radioactive maltose and radioactive trehalose when incubated with [(3)H]glycogen, and also converted maltooligosaccharides, such as maltoheptaose, to both maltose and trehalose. The amylase activity was stimulated by addition of Ca(2+), but this cation inhibited the MTase activity. In addition, MTase activity, but not amylase activity, was strongly inhibited, and in a competitive manner, by validoxylamine. On the other hand, amylase, but not MTase activity, was inhibited by the known transition-state amylase inhibitor, acarbose, suggesting the possibility of two different active sites. Our data suggest that TreS represents another pathway for the production of trehalose from glycogen, involving maltose as an intermediate. In addition, the wild-type organism or mutants blocked in other trehalose biosynthetic pathways, but still having active TreS, accumulate 10- to 20-fold more glycogen when grown in high concentrations (> or = 2% or more) of trehalose, but not in glucose or other sugars. Furthermore, trehalose mutants that are missing TreS do not accumulate glycogen in high concentrations of trehalose or other sugars. These data indicate that trehalose and TreS are both involved in the production of glycogen, and that the metabolism of trehalose and glycogen is interconnected.     

A new synthesis of cord factors and analogs

Treatment of 6,6′-di-O-trityl-trehalose (1) [2] with benzyl chloride in dioxane followed by acid hydrolysis and chromatography gave the chromatographically pure 2,3,4,2′,3′,4′-hexa-O-benzyl trehalose (2). Compound 2 was converted into the corresponding 6,6′-di-O-methane-sulphonyl derivative 3 in quantitative yield. Treatment of the latter compound with the potassium salts of 4-[p-(hexadecyloxy)-phenyl]butyric acid, corynomycolic acid and mycolic acid from Mycobacterium bovis afforded the corresponding benzylated-6,6′-di-O-acyl esters 4, 5 and 6 respectively. Catalytic hydrogenolysis of 4, 5, and 6 yielded 6,6′-di-O-4-[p-(hexadecyloxy)-phenyl] butyryl-trehalose 7; 6,6′-di-O-corynomycolyl-trehalose 8; and 6,6′-di-O-bovi-mycolyl-trehalose 9 respectively.     

Identification, function and structure of the mycobacterial sulfotransferase that initiates sulfolipid-1 biosynthesis

Sulfolipid-1 (SL-1) is an abundant sulfated glycolipid and potential virulence factor found in Mycobacterium tuberculosis. SL-1 consists of a trehalose-2-sulfate (T2S) disaccharide elaborated with four lipids. We identified and characterized a conserved mycobacterial sulfotransferase, Stf0, which generates the T2S moiety of SL-1. Biochemical studies demonstrated that the enzyme requires unmodified trehalose as substrate and is sensitive to small structural perturbations of the disaccharide. Disruption of stf0 in Mycobacterium smegmatis and M. tuberculosis resulted in the loss of T2S and SL-1 formation, respectively. The structure of Stf0 at a resolution of 2.6 A reveals the molecular basis of trehalose recognition and a unique dimer configuration that encloses the substrate into a bipartite active site. These data provide strong evidence that Stf0 carries out the first committed step in the biosynthesis of SL-1 and establish a system for probing the role of SL-1 in M. tuberculosis infection.     

Trehalose is required for growth of Mycobacterium smegmatis

Mycobacteria contain high levels of the disaccharide trehalose in free form as well as within various immunologically relevant glycolipids such as cord factor and sulfolipid-1. By contrast, most bacteria use trehalose solely as a general osmoprotectant or thermoprotectant. Mycobacterium tuberculosis and Mycobacterium smegmatis possess three pathways for the synthesis of trehalose. Most bacteria possess only one trehalose biosynthesis pathway and do not elaborate the disaccharide into more complex metabolites, suggesting a distinct role for trehalose in mycobacteria. We disabled key enzymes required for each of the three pathways in M. smegmatis by allelic replacement. The resulting trehalose biosynthesis mutant was unable to proliferate and enter stationary phase unless supplemented with trehalose. At elevated temperatures, however, the mutant was unable to proliferate even in the presence of trehalose. Genetic complementation experiments showed that each of the three pathways was able to recover the mutant in the absence of trehalose, even at elevated temperatures. From a panel of trehalose analogs, only those with the native alpha,alpha-(1,1) anomeric stereochemistry rescued the mutant, whereas alternate stereoisomers and general osmo- and thermoprotectants were inactive. These findings suggest a dual role for trehalose as both a thermoprotectant and a precursor of critical cell wall metabolites.