An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor.

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein-protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. KEY POINTS: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED.

The Best of Both Worlds-Streptomyces coelicolor and Streptomyces venezuelae as Model Species for Studying Antibiotic Production and Bacterial Multicellular Development.

Streptomyces bacteria have been studied for more than 80 years thanks to their ability to produce an incredible array of antibiotics and other specialized metabolites and their unusual fungal-like development. Their antibiotic production capabilities have ensured continual interest from both academic and industrial sectors, while their developmental life cycle has provided investigators with unique opportunities to address fundamental questions relating to bacterial multicellular growth. Much of our understanding of the biology and metabolism of these fascinating bacteria, and many of the tools we use to manipulate these organisms, have stemmed from investigations using the model species Streptomyces coelicolor and Streptomyces venezuelae. Here, we explore the pioneering work in S. coelicolor that established foundational genetic principles relating to specialized metabolism and development, alongside the genomic and cell biology developments that led to the emergence of S. venezuelae as a new model system. We highlight key discoveries that have stemmed from studies of these two systems and discuss opportunities for future investigations that leverage the power and understanding provided by S. coelicolor and S. venezuelae.

mRNA levels of tricarboxylic acid cycle genes in Streptomyces coelicolor M145 cultured on glucose.

BACKGROUND: Streptomyces strains degrade many complex organic compounds and produce secondary metabolites. In aerobic organisms such as Streptomyces species, the tricarboxylic acid (TCA) cycle represents an indispensable central carbon metabolic pathway for energy generation and metabolic intermediary replenishment. Although various precursors for antibiotic biosynthesis are derived from this cycle, relatively few studies have focused on determining how a single carbon source can impact this metabolic pathway at different growth phases. In this study, we identified chromosomal genes involved in the TCA cycle in Streptomyces coelicolor and determined their mRNA levels. METHODS AND RESULTS: We searched the genes involved in the TCA cycle in S. coelicolor through bioinformatic analysis. Growth, glucose concentration quantification and RNA isolation were made from cultures of S. coelicolor grown on minimal medium with glucose along 72 h. mRNA levels of all identified genes were obtained by RT-qPCR. Five enzymes encoded by a single gene each were found, while for the rest at least two genes were found. The results showed that all the genes corresponding to the TCA enzymes were transcribed at very different levels and some of them displayed growth-phase dependent expression. CONCLUSION: All TCA cycle-associated genes, including paralog genes, were differentially transcribed in S. coelicolor grown in minimal medium with glucose as carbon source. Some of them, such as succinyl-CoA synthetase and succinate dehydrogenase, have low mRNA levels, which could limit the carbon flux through the TCA cycle. Our findings suggest that the genetic expansion of TCA cycle genes could confer to S. coelicolor the ability to adapt to diverse nutritional conditions and metabolic changes through different paralog genes expression.

Heterologous expression of 8-demethyl-tetracenomycin (8-dmtc) affected Streptomyces coelicolor life cycle.

Heterologous hosts are highly important to detect the expression of biosynthetic gene clusters that are cryptic or poorly expressed in their natural hosts. To investigate whether actinorhodin-overproducer Streptomyces coelicolor ∆ppk mutant strain could be a possible prototype as a heterologous expression host, a cosmid containing most of the elm gene cluster of Streptomyces olivaceus Tü2353 was integrated into chromosomes of both S. coelicolor A3(2) and ∆ppk strains. Interestingly, it was found that the production of tetracyclic polyketide 8-demethyl-tetracenomycin (8-DMTC) by recombinant strains caused significant changes in the morphology of cells. All the pellets and clumps were disentangled and mycelia were fragmented in the recombinant strains. Moreover, they produce neither pigmented antibiotics nor agarase and did not sporulate. By eliminating the elm biosynthesis genes from the cosmid, we showed that the morphological properties of recombinants were caused by the production of 8-DMTC. Extracellular application of 8-DMTC on S. coelicolor wild-type cells caused a similar phenotype with the 8-DMTC-producing recombinant strains. The results of this study may contribute to the understanding of the effect of 8-DMTC in Streptomyces since the morphological changes that we have observed have not been reported before. It is also valuable in that it provides useful information about the use of Streptomyces as hosts for the heterologous expression of 8-DMTC.

The phosphoenolpyruvate-pyruvate-oxaloacetate node genes and enzymes in Streptomyces coelicolor M-145.

The phosphoenolpyruvate-pyruvate-oxaloacetate node is a major branch within the central carbon metabolism and acts as a connection point between glycolysis, gluconeogenesis, and the TCA cycle. Phosphoenolpyruvate carboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malic enzymes, and pyruvate kinase, among others, are enzymes included in this node. We determined the mRNA levels and specific activity profiles of some of these genes and enzymes in Streptomyces coelicolor M-145. The results obtained in the presence of glucose demonstrated that all genes studied of the phosphoenolpyruvate-pyruvate-oxaloacetate node were expressed, although at different levels, with 10- to 100-fold differences. SCO3127 (phosphoenolpyruvate carboxylase gene) and SCO5261 (NADP+-dependent malic enzyme gene) showed the highest expression in the rapid growth phase, and the mRNA levels corresponding to SCO5896 (phosphoenolpyruvate-utilizing enzyme gene), and SCO0546 (pyruvate carboxylase gene) increased 5- to 10-fold towards the stationary phase. In casamino acids, in general mRNA levels of S. coelicolor were lower than in glucose, however, results showed greater mRNA expression of SCO4979 (PEP carboxykinase), SCO0208 (pyruvate phosphate dikinase gene), and SCO5261 (NADP+-dependent malic enzyme). These results suggest that PEP carboxylase (SCO3127) is an important enzyme during glucose catabolism and oxaloacetate replenishment. On the other hand, phosphoenolpyruvate carboxykinase, pyruvate phosphate dikinase, and NADP+-malic enzyme could have an important role in gluconeogenesis in S. coelicolor.

Deletion of the hypothetical protein SCO2127 of Streptomyces coelicolor allowed identification of a new regulator of actinorhodin production.

Although the specific function of SCO2127 remains elusive, it has been assumed that this hypothetical protein plays an important role in carbon catabolite regulation and therefore in antibiotic biosynthesis in Streptomyces coelicolor. To shed light on the functional relationship of SCO2127 to the biosynthesis of actinorhodin, a detailed analysis of the proteins differentially produced between the strain M145 and the Δsco2127 mutant of S. coelicolor was performed. The delayed morphological differentiation and impaired production of actinorhodin showed by the deletion strain were accompanied by increased abundance of gluconeogenic enzymes, as well as downregulation of both glycolysis and acetyl-CoA carboxylase. Repression of mycothiol biosynthetic enzymes was further observed in the absence of SCO2127, in addition to upregulation of hydroxyectoine biosynthetic enzymes and SCO0204, which controls nitrite formation. The data generated in this study reveal that the response regulator SCO0204 greatly contributes to prevent the formation of actinorhodin in the ∆sco2127 mutant, likely through the activation of some proteins associated with oxidative stress that include the nitrite producer SCO0216.

Carbon Catabolite Regulation of Secondary Metabolite Formation and Morphological Differentiation in Streptomyces coelicolor.

In the genus Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. However, there is little information about the mechanism involved in these effects. In the present work, it was found that glucose exerted a suppressive effect on the Streptomyces coelicolor actinorhodin (Act) and undecylprodigiosin (Red) production, as well as in its morphological differentiation. Accordingly, using a high-density microarray approach in S. coelicolor grown under glucose repression, at early growth stages, a negative effect was exerted on the transcription of genes involved in Act and Red production, when compared with non-repressive conditions. Seven genes of Act and at least ten genes of Red production were down-regulated by glucose. Stronger repression was observed on the initial steps of antibiotics formation. On the contrary, the coelimycin P1 cluster was up-regulated by glucose. Regarding differentiation, no sporulation was observed in the presence of glucose and expression of a set of genes of the bld cascade was repressed as well as chaplins and rodlins genes. Finally, a series of transcriptional regulators involved in both processes were up- or down-regulated by glucose. This is the first global transcriptomic approach performed to understand the molecular basis of the glucose effect on the synthesis of secondary metabolism and differentiation in the genus Streptomyces. The results of this study are opening new avenues for further exploration.

Transcriptomic analysis of a classical model of carbon catabolite regulation in Streptomyces coelicolor.

BACKGROUND: In the genus Streptomyces, one of the most remarkable control mechanisms of physiological processes is carbon catabolite repression (CCR). This mechanism regulates the expression of genes involved in the uptake and utilization of alternative carbon sources. CCR also affects the synthesis of secondary metabolites and morphological differentiation. Even when the outcome effect of CCR in different bacteria is the same, their essential mechanisms can be quite different. In several streptomycetes glucose kinase (Glk) represents the main glucose phosphorylating enzyme and has been regarded as a regulatory protein in CCR. To evaluate the paradigmatic model proposed for CCR in Streptomyces, a high-density microarray approach was applied to Streptomyces coelicolor M145, under repressed and non-repressed conditions. The transcriptomic study was extended to assess the ScGlk role in this model by comparing the transcriptomic profile of S. coelicolor M145 with that of a ∆glk mutant derived from the wild-type strain, complemented with a heterologous glk gene from Zymomonas mobilis (Zmglk), insensitive to CCR but able to grow in glucose (ScoZm strain). RESULTS: Microarray experiments revealed that glucose influenced the expression of 651 genes. Interestingly, even when the ScGlk protein does not have DNA binding domains and the glycolytic flux was restored by a heterologous glucokinase, the ScGlk replacement modified the expression of 134 genes. From these, 91 were also affected by glucose while 43 appeared to be under the control of ScGlk. This work identified the expression of S. coelicolor genes involved in primary metabolism that were influenced by glucose and/or ScGlk. Aside from describing the metabolic pathways influenced by glucose and/or ScGlk, several unexplored transcriptional regulators involved in the CCR mechanism were disclosed. CONCLUSIONS: The transcriptome of a classical model of CCR was studied in S. coelicolor to differentiate between the effects due to glucose or ScGlk in this regulatory mechanism. Glucose elicited important metabolic and transcriptional changes in this microorganism. While its entry and flow through glycolysis and pentose phosphate pathway were stimulated, the gluconeogenesis was inhibited. Glucose also triggered the CCR by repressing transporter systems and the transcription of enzymes required for secondary carbon sources utilization. Our results confirm and update the agar model of the CCR in Streptomyces and its dependence on the ScGlk per se. Surprisingly, the expected regulatory function of ScGlk was not found to be as global as thought before (only 43 out of 779 genes were affected), although may be accompanied or coordinated by other transcriptional regulators. Aside from describing the metabolic pathways influenced by glucose and/or ScGlk, several unexplored transcriptional regulators involved in the CCR mechanism were disclosed. These findings offer new opportunities to study and understand the CCR in S. coelicolor by increasing the number of known glucose and ScGlk -regulated pathways and a new set of putative regulatory proteins possibly involved or controlling the CCR.

AllR Controls the Expression of Streptomyces coelicolor Allantoin Pathway Genes.

Streptomyces species are native inhabitants of soil, a natural environment where nutrients can be scarce and competition fierce. They have evolved ways to metabolize unusual nutrients, such as purines and its derivatives, which are highly abundant in soil. Catabolism of these uncommon carbon and nitrogen sources needs to be tightly regulated in response to nutrient availability and environmental stimulus. Recently, the allantoin degradation pathway was characterized in Streptomyces coelicolor. However, there are questions that remained unanswered, particularly regarding pathway regulation. Here, using a combination of proteomics and genetic approaches, we identified the negative regulator of the allantoin pathway, AllR. In vitro studies confirmed that AllR binds to the promoter regions of allantoin catabolic genes and determined the AllR DNA binding motif. In addition, effector studies showed that allantoic acid, and glyoxylate, to a lesser extent, inhibit the binding of AllR to the DNA. Inactivation of AllR repressor leads to the constitutive expression of the AllR regulated genes and intriguingly impairs actinorhodin and undecylprodigiosin production. Genetics and proteomics analysis revealed that among all genes from the allantoin pathway that are upregulated in the allR mutant, the hyi gene encoding a hydroxypyruvate isomerase (Hyi) is responsible of the impairment of antibiotic production.

Alteration of coenzyme specificity of malate dehydrogenase from Streptomyces coelicolor A3(2) by site-directed mutagenesis.

We describe here for the first time the alteration of coenzyme specificity of malate dehydrogenase (MDH) from Streptomyces coelicolor A3(2) (ScMDH). In the present study, we replaced four amino acid residues in the Rossmann fold (ßB-αC) region of NADH-dependent ScMDH by site-directed mutagenesis with those of NADPH-dependent MDH (Glu42Gly, Ile43Ser, Pro45Arg, and Ala46Ser). The coenzyme specificity of the mutant enzyme (ScMDH-T4) was examined. Coenzyme specificity of ScMDH-T4 was shifted 2231.3-fold toward NADPH using kcat/Km(coenzyme) as the measurement of coenzyme specificity. Accordingly, the effect of the replacements on coenzyme specificity is discussed. Our work provides further insight into the coenzyme specificity of ScMDH.

Allantoin catabolism influences the production of antibiotics in Streptomyces coelicolor.

Purines are a primary source of carbon and nitrogen in soil; however, their metabolism is poorly understood in Streptomyces. Using a combination of proteomics, metabolomics, and metabolic engineering, we characterized the allantoin pathway in Streptomyces coelicolor. When cells grew in glucose minimal medium with allantoin as the sole nitrogen source, quantitative proteomics identified 38 enzymes upregulated and 28 downregulated. This allowed identifying six new functional enzymes involved in allantoin metabolism in S. coelicolor. From those, using a combination of biochemical and genetic engineering tools, it was found that allantoinase (EC 3.5.2.5) and allantoicase (EC 3.5.3.4) are essential for allantoin metabolism in S. coelicolor. Metabolomics showed that under these growth conditions, there is a significant intracellular accumulation of urea and amino acids, which eventually results in urea and ammonium release into the culture medium. Antibiotic production of a urease mutant strain showed that the catabolism of allantoin, and the subsequent release of ammonium, inhibits antibiotic production. These observations link the antibiotic production impairment with an imbalance in nitrogen metabolism and provide the first evidence of an interaction between purine metabolism and antibiotic biosynthesis.

Crystal structures and mutational analyses of acyl-CoA carboxylase beta subunit of Streptomyces coelicolor.

The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are homologue multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities toward these substrates is only partially understood. To further understand the molecular basis of how the active site residues can modulate the substrate recognition, we mutated D422, N80, R456, and R457 of PccB, the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal structure were compared systematically to establish the sequence-structure-function relationship that correlates the observed substrate specificity toward acetyl-, propionyl-, and butyryl-CoA with active site geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, influencing not only the active site properties but further altering protein stability and causing long-range conformational changes. Mutations of N80, R456, and R457 lead to variations in the quaternary structure of the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in maintaining the active protein conformation as well as a critical role in substrate binding.

FasR, a novel class of transcriptional regulator, governs the activation of fatty acid biosynthesis genes in Streptomyces coelicolor.

Membrane lipid homeostasis is essential for bacterial survival and adaptation to different environments. The regulation of fatty acid biosynthesis is therefore crucial for maintaining the correct composition and biophysical properties of cell membranes. This regulation implicates a biochemical control of key enzymes and a transcriptional regulation of genes involved in lipid metabolism. In Streptomyces coelicolor we found that control of lipid homeostasis is accomplished, at least in part, through the transcriptional regulation of fatty acid biosynthetic genes. A novel transcription factor, FasR (SCO2386), controls expression of fabDHPF operon and lies immediately upstream of fabD, in a cluster of genes that is highly conserved within actinomycetes. Disruption of fasR resulted in a mutant strain, with severe growth defects and a delay in the timing of morphological and physiological differentiation. Expression of fab genes was downregulated in the fasR mutant, indicating a role for this transcription factor as an activator. Consequently, the mutant showed a significant drop in fatty acid synthase activity and triacylglyceride accumulation. FasR binds specifically to a DNA sequence containing fabDHPF promoter region, both in vivo and in vitro. These data provide the first example of positive regulation of genes encoding core proteins of saturated fatty acid synthase complex.

The Streptomyces coelicolor genome encodes a type I ribosome-inactivating protein.

Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in numerous plants, but also constitute a subunit of the bacterial Shiga toxin. Classification of plant RIPs is based on the absence (type I) or presence (type II) of an additional lectin module. In Shiga toxin, sugar binding is mediated by a distinct RIP-associated homopentamer. In the genome of two actinomycetes, we identified RIP-like proteins that resemble plant type I RIPs rather than the RIP subunit (StxA) of Shiga toxin. Some representatives of ß- and γ-proteobacteria also contain genes encoding RIP-like proteins, but these are homologous to StxA. Here, we describe the isolation and initial characterization of the RIP-like gene product SCO7092 (RIPsc) from the Gram-positive soil bacterium Streptomyces coelicolor. The ripsc gene was expressed in Escherichia coli as a recombinant protein of about 30 kDa, and displayed the characteristic N-glycosidase activity causing specific rRNA depurination. In Streptomyces lividans and E. coli, RIPsc overproduction resulted in a dramatic decrease in the growth rate. In addition, intracellular production was deleterious for Saccharomyces cerevisiae. However, when applied externally to microbial cells, purified RIPsc did not display antibacterial or antifungal activity, suggesting that it cannot enter these cells. In a cell-free system, however, purified S. coelicolor RIPsc protein displayed strong inhibitory activity towards protein translation.

Characterization of the methyl-specific restriction system of Streptomyces coelicolor A3(2) and of the role played by laterally acquired nucleases.

The methyl-specific restriction system of Streptomyces coelicolor A3(2) was analyzed by carrying out transformations with unmethylated and methylated pSET152 DNA. Streptomyces coelicolor was found to strongly restrict DNA methylated in vivo by the Dam, Dcm and Hsd modification systems of Escherichia coli. Hsd-modified DNA was restricted as strongly as Dam-modified DNA, even though there are significantly fewer sites on the plasmid; Dcm-modified plasmid was restricted more strongly then either Dam- or Hsd-modified DNA. Restriction of plasmid DNA modified in vitro by different methylases also showed a greater dependence on the methylated sequence than on the number of methylated sites. Streptomyces coelicolor mutants were constructed that lacked genes identified as the likely candidates for encoding methyl-specific restriction nucleases (the products of the SCO4213, SCO4631 and SCO2863 genes, as well as the SCO3261-SCO3262 operon) that are located in the laterally acquired genomic islands of the S. coelicolor chromosome; these mutants showed partial alleviation of methylated DNA restriction. Cloning of these genes in the close relative Streptomyces lividans increased the restriction of methylated DNA by this species, confirming their role as part of the methyl-specific restriction system of S. coelicolor.

A eukaryote-like cardiolipin synthase is present in Streptomyces coelicolor and in most actinobacteria.

Cardiolipin (CL) is an anionic membrane lipid present in bacteria, plants, and animals, but absent from archaea. It is generally thought that bacteria use an enzyme belonging to the phospholipase D superfamily as cardiolipin synthase (Cls) catalyzing a reversible phosphatidyl group transfer from one phosphatidylglycerol (PG) molecule to another PG to form CL and glycerol. In contrast, in eukaryotes a Cls of the CDP-alcohol phosphatidyltransferase superfamily uses cytidine diphosphate-diacylglycerol (CDP-DAG) as the donor of the phosphatidyl group, which is transferred to a molecule of PG to form CL. Searching the genome of the actinomycete Streptomyces coelicolor A3(2) we identified a gene coding for a putative Cls of the CDP-alcohol phosphatidyltransferase superfamily (Sco1389). Here we show that expression of Sco1389 in a CL-deficient Rhizobium etli mutant restores CL formation. In an in vitro assay Sco1389 condenses CDP-DAG with PG to form CL and therefore catalyzes the same reaction as eukaryotic cardiolipin synthases. This is the first time that a CDP-alcohol phosphatidyltransferase from bacteria is shown to be responsible for CL formation. The broad occurrence of putative orthologues of Sco1389 among the actinobacteria suggests that CL synthesis involving a eukaryotic type Cls is common in actinobacteria.

Heterologous production of polyketides in bacteria.

Polyketide natural products are among the most important microbial metabolites in human medicine and are widely used to treat both acute and degenerative diseases. The need to develop new drugs has prompted the idea of using heterologous systems for the expression of polyketide biosynthetic pathways. The basic idea behind this approach is to use heterologous bacterial systems with better growth and genetic characteristics that could support better production of a certain compound than the original host or that could allow the generation of novel analogues through combinatorial biosynthesis. Moreover, these hosts could be used to express "cryptic" secondary metabolic pathways or serve as surrogate hosts in metagenomics experiments in order to find potential new bioactive compounds. In this chapter we discuss recent advances in the heterologous production of polyketides in bacteria and describe some methodological improvements of the systems.

Cloning and expression of the sco2127 gene from Streptomyces coelicolor M145.

It is known that Streptomyces peucetius var. caesius mutants resistant to 2-deoxyglucose (Dog(R)) exhibit glucose transport deficiency, low glucose kinase (Glk) activity and insensitivity to carbon catabolite repression (CCR). This phenotype can be pleiotropically complemented by a 576-bp gene encoding SCO2127 from Streptomyces coelicolor, suggesting the participation of this protein in the CCR process. In the present work, the sco2127 region was subcloned into pQE30 and its transcription product (SCO2127-His(6)) overexpressed. This procedure allowed purification of SCO2127 (with a Ni-sepharose resin) and production of polyclonal antibodies. In western blot assays, the antibodies gave a positive reaction against protein extracts from both S. coelicolor and S. peucetius var. caesius, appearing as a single band of 34 kDa. No protein was detected using extracts from a S. coelicolor mutant lacking the sco2127 gene (Deltasco2127). In agreement with its possible involvement in the CCR process, SCO2127 was detected during the logarithmic growth phase of S. coelicolor grown in minimal medium supplemented with 50 and 100 mM glucose. In addition, when 50 mM glucose was utilized, SCO2127 and residual glucose concentration simultaneously decreased at later stages of the microbial growth.

Multiple pathways for triacylglycerol biosynthesis in Streptomyces coelicolor.

The terminal reaction in triacylglyceride (TAG) biosynthesis is the esterification of diacylglycerol (DAG) with a fatty acid molecule. To study this reaction in Streptomyces coelicolor, we analyzed three candidate genes (sco0958, sco1280, and sco0123) whose products significantly resemble the recently identified wax ester synthase/acyl-coenzyme A (CoA):DAG acyltransferase (DGAT) from Acinetobacter baylyi. The deletion of either sco0123 or sco1280 resulted in no detectable decrease in TAG accumulation. In contrast, the deletion of sco0958 produced a dramatic reduction in neutral lipid production, whereas the overexpression of this gene yielded a significant increase in de novo TAG biosynthesis. In vitro activity assays showed that Sco0958 mediates the esterification of DAG using long-chain acyl-CoAs (C(14) to C(18)) as acyl donors. The K(m) and V(max) values of this enzyme for myristoyl-CoA were 45 muM and 822 nmol mg(-1) min(-1), respectively. Significantly, the triple mutant strain was not completely devoid of storage lipids, indicating the existence of alternative TAG-biosynthetic routes. We present strong evidence demonstrating that the residual production of TAG in this mutant strain is mediated, at least in part, by an acyl-CoA-dependent pathway, since the triple mutant still exhibited DGAT activity. More importantly, there was substantial phospholipid:DGAT (PDAT) activity in the wild type and in the triple mutant. This is the first time that a PDAT activity has been reported for bacteria, highlighting the extreme metabolic diversity of this industrially important soil microorganism.