Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria.

The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority. meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor. The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the construction of in vivo selection systems. We have isolated the asd, dapA, dapB, dapD, and dapE genes involved in the DAP biosynthetic pathway of Mycobacterium bovis BCG. These genes were isolated by complementation of Escherichia coli mutations with an expression library of BCG DNA. Our analysis of these genes suggests that BCG may use more than one pathway for biosynthesis of DAP. The nucleotide sequence of the BCG dapB gene was determined. The activity of the product of this gene in Escherichia coli provided evidence that the gene may encode a novel bifunctional dihydrodipicolinate reductase and DAP dehydrogenase.     

Analogs of diaminopimelic acid as inhibitors of meso-diaminopimelate dehydrogenase and LL-diaminopimelate epimerase

Analogs 1-8 of diaminopimelic acid (DAP) were synthesized and tested for inhibition of purified meso-DAP D-dehydrogenase from Bacillus sphaericus and of LL-DAP epimerase from Escherichia coli. The dehydrogenase was assayed by monitoring NADPH formation spectrophotometrically at 340 nm. N-Hydroxy DAP 4, N-amino DAP 5, and 4-methylene DAP 6 are substrates of the dehydrogenase with relative rates exceeding those of the meso isomers of the thia analogs 1ab, 2ab, and 3ab. DAP epimerase was assayed by coupling the epimerization of LL-DAP to DL-DAP (Km = 0.26 mM) with the dehydrogenase-catalyzed oxidation of DL-DAP by NADP. Lanthionine isomers 1ab and 1c were stronger inhibitors of the epimerase (Ki = 0.18 mM, Ki' = 0.67 mM, and Ki = 0.42 mM, respectively) than the corresponding meso-sulfoxide 2ab or the meso-sulfone 3ab. Other isomers of 2 and 3, as well as compounds 7 and 8, showed no epimerase inhibition. N-Hydroxy DAP 4 was the most potent competitive inhibitor (Ki = 0.0056 mM) of the epimerase, whereas N-amino DAP 5 is weaker (Ki = 2.9 mM) and 4-methylene DAP 6 is a noncompetitive inhibitor (Ki' = 0.95 mM). Although none of the analogs tested showed time-dependent inactivation of either enzyme, compounds 4, 5, 6, and 7 display substantial antibacterial activities. Possible mechanisms of epimerase inhibition and significance of the DAP pathway as a target for antibiotics are discussed.     

Pseudomonas aeruginosa diaminopimelate decarboxylase: evolutionary relationship with other amino acid decarboxylases

The lysA gene encodes meso-diaminopimelate (DAP) decarboxylase (E.C.4.1.1.20), the last enzyme of the lysine biosynthetic pathway in bacteria. We have determined the nucleotide sequence of the lysA gene from Pseudomonas aeruginosa. Comparison of the deduced amino acid sequence of the lysA gene product revealed extensive similarity with the sequences of the functionally equivalent enzymes from Escherichia coli and Corynebacterium glutamicum. Even though both P. aeruginosa and E. coli are Gram-negative bacteria, sequence comparisons indicate a greater similarity between enzymes of P. aeruginosa and the Gram- positive bacterium C. glutamicum than between those of P. aeruginosa and E. coli enzymes. Comparison of DAP decarboxylase with protein sequences present in data bases revealed that bacterial DAP decarboxylases are homologous to mouse (Mus musculus) ornithine decarboxylase (E.C.4.1.1.17), the key enzyme in polyamine biosynthesis in mammals. On the other hand, no similarity was detected between DAP decarboxylases and other bacterial amino acid decarboxylases.      

Synthesis of Desthiobiotin from 7,8-Diaminopel-argonic Acid in Biotin Auxotrophs of Escherichia coli K-12

The synthesis of desthiobiotin from 7,8-diaminopelargonic acid (DAP) was demonstrated in resting cell suspensions of Escherichia coli K-12 bioA mutants under conditions in which the biotin locus was derepressed. The biosynthetically formed desthiobiotin was identified by chromatography, electrophoresis, and by its ability to support the growth of yeast and those E. coli biotin auxotrophs that are blocked earlier in the biotin pathway. Optimal conditions for desthiobiotin synthesis were determined. Desthiobiotin synthetase activity was repressed 67% when partially derepressed resting cells were incubated in the presence of 3 ng of biotin per ml. Serine, bicarbonate, and glucose stimulated desthiobiotin synthesis apparently by acting as sources of CO(2). The results of this study are consistent with an earlier postulated pathway for biotin biosynthesis in E. coli: pimelic acid --> 7-oxo-8-aminopelargonic acid --> DAP --> desthiobiotin --> biotin.     

Cloning and sequence analysis of the meso-diaminopimelate decarboxylase gene from Bacillus methanolicus MGA3 and comparison to other decarboxylase genes.

The lysA gene of Bacillus methanolicus MGA3 was cloned by complementation of an auxotrophic Escherichia coli lysA22 mutant with a genomic library of B. methanolicus MGA3 chromosomal DNA. Subcloning localized the B. methanolicus MGA3 lysA gene into a 2.3-kb SmaI-SstI fragment. Sequence analysis of the 2.3-kb fragment indicated an open reading frame encoding a protein of 48,223 Da, which was similar to the meso-diaminopimelate (DAP) decarboxylase amino acid sequences of Bacillus subtilis (62%) and Corynebacterium glutamicum (40%). Amino acid sequence analysis indicated several regions of conservation among bacterial DAP decarboxylases, eukaryotic ornithine decarboxylases, and arginine decarboxylases, suggesting a common structural arrangement for positioning of substrate and the cofactor pyridoxal 5'-phosphate. The B. methanolicus MGA3 DAP decarboxylase was shown to be a dimer (M(r) 86,000) with a subunit molecular mass of approximately 50,000 Da. This decarboxylase is inhibited by lysine (Ki = 0.93 mM) with a Km of 0.8 mM for DAP. The inhibition pattern suggests that the activity of this enzyme in lysine-overproducing strains of B. methanolicus MGA3 may limit lysine synthesis.     

Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity

In innate immunity, pattern recognition molecules recognize cell wall components of microorganisms and activate subsequent immune responses, such as the induction of antimicrobial peptides and melanization in Drosophila. The diaminopimelic acid (DAP)-type peptidoglycan potently activates imd-dependent induction of antibacterial peptides. Peptidoglycan recognition protein (PGRP) family members act as pattern recognition molecules. PGRP-LC loss-of-function mutations affect the imd-dependent induction of antibacterial peptides and resistance to Gram-negative bacteria, whereas PGRP-LE binds to the DAP-type peptidoglycan, and a gain-of-function mutation induces constitutive activation of both the imd pathway and melanization. Here, we generated PGRP-LE null mutants and report that PGRP-LE functions synergistically with PGRP-LC in producing resistance to Escherichia coli and Bacillus megaterium infections, which have the DAP-type peptidoglycan. Consistent with this, PGRP-LE acts both upstream and in parallel with PGRP-LC in the imd pathway, and is required for infection-dependent activation of melanization in Drosophila. A role for PGRP-LE in the epithelial induction of antimicrobial peptides is also suggested.     

Kinetics of uptake and incorporation of meso-diaminopimelic acid in different Escherichia coli strains.

The rate at which the peptidoglycan precursor meso-diaminopimelic acid (DAP) is incorporated into the cell wall of Escherichia coli cells was determined by pulse-label experiments. For different E. coli strains, the incorporation rate was compared with the rate of uptake of DAP into the cell. With E. coli W7, a dap lys mutant generally used in this kind of studies, steady-state incorporation was reached only after about 0.75 of the doubling time. This lag period can be ascribed to the presence of a large internal DAP pool in the cells. An E. coli K-12 lysA strain was constructed which could be grown without DAP in its medium. Consequently, due to the higher specific activity of the added [3H]DAP, faster incorporation and higher levels of radioactivity in the peptidoglycan layer were observed in the K-12 lysA strain than in the W7 strain. In addition, uptake and incorporation were faster in steady state (within about 0.2 of the doubling time), indicating a smaller DAP pool. The lag period could be further diminished and the incorporation rate could be increased by feedback inhibition of the biosynthetic pathway to DAP with threonine and methionine. These results make MC4100 lysA a suitable strain for studies on peptidoglycan synthesis. To explain our observations, we suggest the existence of an expandable pool of DAP in E. coli which varies with the DAP concentration in the growth medium. With 2 microgram of DAP per ml, the size of the pool is severalfold the amount of DAP contained in the cell wall. This pool can be partly washed out of the cells. Grown without DAP, MC4100 lysA still has a small pool caused by endogenous synthesis, which accounts for the fact that steady-state [3H]DAP incorporation in the lysA strain still shows a lag period.     

Preparation, characterization, and optimization of an in vitro C30 carotenoid pathway

The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4'-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives.     

Efficient and quantitative incorporation of diaminopimelic acid into the peptidoglycan of Salmonella typhimurium

Abstract Diaminopimelic acid is incorporated into the peptidoglycan of Salmonella typhimurium in an efficient and quantitative manner. The amount of DAP incorporated is similar to the number of molecules estimated to exist in the Salmonella cell wall. In contrast, strains of E. coli , including those most used for studies of cell wall synthesis, are much less efficient in the incorporation of diaminopimelic acid. The lysine-requiring strains of E. coli appear to excrete diaminopimelic acid related material during growth and this accounts, in part, for the inefficient incorporation of radioactive diaminopimelic acid into Escherichia strains. In addition, the Escherichia strains are much less permeable to DAP than Salmonella strains. Cysteine and cystine inhibit the incorporation of DAP into the cell and this result suggests that Salmonella uses the cystine uptake system to allow DAP into the cell.     

In vivo metabolism of 2,2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal microorganisms and ruminants and its use as a marker of bacterial biomass.

Cells of Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino[G-3H]pimelic acid ([3H]DAP) as models of gram-positive and gram-negative bacteria, respectively. Two experiments were conducted to study the in vivo metabolism of 2,2'-diaminopimelic acid (DAP) in sheep. In experiment 1, cells of [3H]DAP-labeled B. megaterium GW1 were infused into the rumen of one sheep and the radiolabel was traced within microbial samples, digesta, and the whole animal. Bacterially bound [3H]DAP was extensively metabolized, primarily (up to 70% after 8 h) via decarboxylation to [3H]lysine by both ruminal protozoa and ruminal bacteria. Recovery of infused radiolabel in urine and feces was low (42% after 96 h) and perhaps indicative of further metabolism by the host animal. In experiment 2, [3H]DAP-labeled B. megaterium GW1 was infused into the rumens of three sheep and [3H]DAP-labeled E. coli W7-M5 was infused into the rumen of another sheep. The radioactivity contents of these mutant bacteria were insufficient to use as tracers, but the metabolism of DAP was monitored in the total, free, and peptidyl forms. Free DAP, as a proportion of total DAP in duodenal digesta, varied from 0 to 9.5%, whereas peptidyl DAP accounted for 8.3 to 99.2%. These data reflect the extensive metabolism of bacterially bound DAP within the gastrointestinal tracts of ruminant animals and serve as a serious caution to the uncritical use of DAP as a marker of bacterial biomass in the digesta of these animals.     

Genetic control of Escherichia coli K-12 strains' assimilation of 2,6-diaminopurine as a purine source]

Mutations of the resistance to 2,6-diaminopurine (apt), which affect adenine phosphoribosyltransferase, fail to permit the growth of Escherichia coli pur mutants (purine auxotrophs which cannot make inosine monophosphate de novo) on the medium with 2,6-diaminopurine (DAP) as the sole source of purines. Addition of a small amount of hypoxantine, but not guanine, stimulated the growth of mutants of pur apt and pur apt+ genotypes on the medium with DAP. The utilization of DAP as purine source in the presence of hypoxantine is blocked by mutations guaC (guanosine monophosphate reductase), add (adenosine deaminase) and pup (purine necleoside phosphorylase), suggesting that DAP are utilized via purine nucleoside phosphorylase and adenosine deaminase. The drm mutation (that increases the level of pentose-1-phosphate in the cell) does not activate the utilization of DAP. The results indicate that a step, that limits the utilization of DAP as the sole source of purines by pur mutants of E. coli, is the deamination of DAP nucleoside.     

Genetic Analysis of Diaminopimelic Acid- and Lysine-Requiring Mutants of Escherichia coli

Several diaminopimelic acid (DAP)- and lysine-requiring mutants of Escherichia coli were isolated and studied by genetic, physiological, and biochemical means. The genes concerned with DAP-lysine synthesis map at several different sites on the E. coli chromosome and, therefore, do not constitute a single operon. Three separate loci affecting DAP synthesis are located in the 0 to 2.5 min region of the genetic map. The order of the loci in this region is thr-dapB-pyrA-ara-leu-pan-dapC-tonA-dapD. Two additional DAP genes map in the region between min 47 and 48, with the gene order being gua-dapA-dapE-ctr. The lys locus at min 55 determines the synthesis of the enzyme DAP decarboxylase, which catalyzes the conversion of DAP into lysine. The order of the genes in this region is serA-lysA-thyA.     

A bacterium that has three pathways to regulate membrane lipid fluidity

Well-studied bacteria such as Bacillus subtilis and Escherichia coli each have only a single pathway for synthesis of the unsaturated fatty acids required to make functional membrane lipids. In marked contrast, unsaturated fatty acid synthesis in Pseudomonas aeruginosa proceeds by three distinct pathways.     

Biochemical and phylogenetic characterization of a novel diaminopimelate biosynthesis pathway in prokaryotes identifies a diverged form of LL-diaminopimelate aminotransferase

A variant of the diaminopimelate (DAP)-lysine biosynthesis pathway uses an LL-DAP aminotransferase (DapL, EC 2.6.1.83) to catalyze the direct conversion of L-2,3,4,5-tetrahydrodipicolinate to LL-DAP. Comparative genomic analysis and experimental verification of DapL candidates revealed the existence of two diverged forms of DapL (DapL1 and DapL2). DapL orthologs were identified in eubacteria and archaea. In some species the corresponding dapL gene was found to lie in genomic contiguity with other dap genes, suggestive of a polycistronic structure. The DapL candidate enzymes were found to cluster into two classes sharing approximately 30% amino acid identity. The function of selected enzymes from each class was studied. Both classes were able to functionally complement Escherichia coli dapD and dapE mutants and to catalyze LL-DAP transamination, providing functional evidence for a role in DAP/lysine biosynthesis. In all cases the occurrence of dapL in a species correlated with the absence of genes for dapD and dapE representing the acyl DAP pathway variants, and only in a few cases was dapL coincident with ddh encoding meso-DAP dehydrogenase. The results indicate that the DapL pathway is restricted to specific lineages of eubacteria including the Cyanobacteria, Desulfuromonadales, Firmicutes, Bacteroidetes, Chlamydiae, Spirochaeta, and Chloroflexi and two archaeal groups, the Methanobacteriaceae and Archaeoglobaceae.     

Development of stable, genetically well-defined conditionally viableEscherichia coli strains

Summary The diaminopimelate (DAP) pathway provides the cell with lysine and with DAP, a vital cell wall constituent. Mutations in the DAP pathway of lysine biosynthesis are lethal for cells exposed to lysine in the absence of DAP. In this paper, the substitution of thedapD gene ofEscherichia coli with the kanamycin resistance gene from Tn903 is described and its possible uses are discussed.     

Functional genomic analysis of the Bacillus subtilis Tat pathway for protein secretion

Protein secretion from Bacillus species is a major industrial production tool with a market of over $1 billion per year. However, standard export technologies, based on the well-characterised general secretory (Sec) pathway, are frequently inapplicable for the production of proteins. The recently discovered twin-arginine translocation (Tat) pathway offers additional potential to transport proteins. Here we review the use of functional genomic and proteomic approaches to explore the Tat pathway of Bacillus subtilis. The properties of Tat pathway components and the twin-arginine signal peptides that direct proteins into this pathway are discussed. Where appropriate, a comparison is made with Tat systems from other organism, such as Escherichia coli. Recent findings with the latter organism in particular provide proof-of-principle that the Tat pathway can be exploited for the production of Sec-incompatible proteins.     

The three adaptation systems of Bacillus subtilis chemotaxis

Adaptation has a crucial role in the gradient-sensing mechanism that underlies bacterial chemotaxis. The Escherichia coli chemotaxis pathway uses a single adaptation system involving reversible receptor methylation. In Bacillus subtilis, the chemotaxis pathway seems to use three adaptation systems. One involves reversible receptor methylation, although quite differently than in E. coli. The other two involve CheC, CheD and CheV, which are chemotaxis proteins not found in E. coli. Remarkably, no one system is absolutely required for adaptation or is independently capable of generating adaptation. In this review, we discuss these three novel adaptation systems in B. subtilis and propose a model for their integration.     

地衣芽胞杆菌对白色念珠菌等的拮抗作用

目的了解地衣芽胞杆菌在试管内与阴道正常菌群共生关系的情况。方法将地衣芽胞杆菌菌液分别与葡萄球菌、大肠埃希菌、白色念珠菌、德氏乳杆菌混合培养,定量计数各菌在不同时间内单独培养和混合培养时各菌的活菌数。结果地衣芽胞杆菌生长不受金黄色葡萄球菌、白色念珠菌和大肠埃希菌的影响,金黄色葡萄球菌和白色念珠菌在有地衣芽胞杆菌存在的情况下,其生长受到明显的抑制（P〈0.05）;乳杆菌在12-48 h内,有显著的抑制地衣芽胞杆菌生长的作用,而乳杆菌的生长不受地衣芽胞杆菌的存在与否而正常生长。结论地衣芽胞杆菌对金黄色葡萄球菌及白色念珠菌在体外具有明显的拮抗作用,地衣芽胞杆菌对大肠埃希菌、乳杆菌无明显的体外拮抗作用。     

An LL-diaminopimelate aminotransferase defines a novel variant of the lysine biosynthesis pathway in plants

Although lysine (Lys) biosynthesis in plants is known to occur by way of a pathway that utilizes diaminopimelic acid (DAP) as a central intermediate, the available evidence suggests that none of the known DAP-pathway variants found in nature occur in plants. A new Lys biosynthesis pathway has been identified in Arabidopsis (Arabidopsis thaliana) that utilizes a novel transaminase that specifically catalyzes the interconversion of tetrahydrodipicolinate and LL-diaminopimelate, a reaction requiring three enzymes in the DAP-pathway variant found in Escherichia coli. The LL-DAP aminotransferase encoded by locus At4g33680 was able to complement the dapD and dapE mutants of E. coli. This result, in conjunction with the kinetic properties and substrate specificity of the enzyme, indicated that LL-DAP aminotransferase functions in the Lys biosynthetic direction under in vivo conditions. Orthologs of At4g33680 were identified in all the cyanobacterial species whose genomes have been sequenced. The Synechocystis sp. ortholog encoded by locus sll0480 showed the same functional properties as At4g33680. These results demonstrate that the Lys biosynthesis pathway in plants and cyanobacteria is distinct from the pathways that have so far been defined in microorganisms.