The length of lipoteichoic acid polymers controls Staphylococcus aureus cell size and envelope integrity.

The opportunistic pathogen Staphylococcus aureus is protected by a cell envelope that is crucial for viability. In addition to peptidoglycan, lipoteichoic acid (LTA) is an especially important component of the S. aureus cell envelope. LTA is an anionic polymer anchored to a glycolipid in the outer leaflet of the cell membrane. It was known that deleting the gene for UgtP, the enzyme that makes this glycolipid anchor, causes cell growth and division defects. In Bacillus subtilis, growth abnormalities from the loss of ugtP have been attributed to both the absence of the encoded protein and to the loss of its products. Here, we show that growth defects in S. aureus ugtP deletion mutants are due to the long, abnormal LTA polymer that is produced when the glycolipid anchor is missing from the outer leaflet of the membrane. Dysregulated cell growth leads to defective cell division, and these phenotypes are corrected by mutations in the LTA polymerase, ltaS, that reduce polymer length. We also show that S. aureus mutants with long LTA are sensitized to cell wall hydrolases, beta-lactam antibiotics, and compounds that target other cell envelope pathways. We conclude that control of LTA polymer length is important for S. aureus physiology and promotes survival under stressful conditions, including antibiotic stress.IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of community- and hospital-acquired infections and is responsible for a large fraction of deaths caused by antibiotic-resistant bacteria. S. aureus is surrounded by a complex cell envelope that protects it from antimicrobial compounds and other stresses. Here we show that controlling the length of an essential cell envelope polymer, lipoteichoic acid, is critical for controlling S. aureus cell size and cell envelope integrity. We also show that genes involved in LTA length regulation are required for resistance to beta-lactam antibiotics in MRSA. The proteins encoded by these genes may be targets for combination therapy with an appropriate beta-lactam.

Dissecting modular synthases through inhibition: A complementary chemical and genetic approach.

Modular synthases, such as fatty acid, polyketide, and non-ribosomal peptide synthases (NRPSs), are sophisticated machineries essential in both primary and secondary metabolism. Various techniques have been developed to understand their genetic background and enzymatic abilities. However, uncovering the actual biosynthetic pathways remains challenging. Herein, we demonstrate a pipeline to study an assembly line synthase by interrogating the enzymatic function of each individual enzymatic domain of BpsA, a NRPS that produces the blue 3,3'-bipyridyl pigment indigoidine. Specific inhibitors for each biosynthetic domain of BpsA were obtained or synthesized, and the enzymatic performance of BpsA upon addition of each inhibitor was monitored by pigment development in vitro and in living bacteria. The results were verified using genetic mutants to inactivate each domain. Finally, the results complemented the currently proposed biosynthetic pathway of BpsA.

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation.

Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the d-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. The d-alanylation machinery includes two membrane proteins of unclear function, DltB and DltD, which are somehow involved in transfer of d-alanine from a carrier protein inside the cell to teichoic acids on the cell surface. Here, we probed the role of DltD in the human pathogen Staphylococcus aureus using both cell-based and biochemical assays. We first exploited a known synthetic lethal interaction to establish the essentiality of each gene in the DLT pathway for d-alanylation of lipoteichoic acid (LTA) and confirmed this by directly detecting radiolabeled d-Ala-LTA both in cells and in vesicles prepared from mutant strains of S. aureus We developed a partial reconstitution of the pathway by using cell-derived vesicles containing DltB, but no other components of the d-alanylation pathway, and showed that d-alanylation of previously formed lipoteichoic acid in the DltB vesicles requires the presence of purified and reconstituted DltA, DltC, and DltD, but not of the LTA synthase LtaS. Finally, based on the activity of DltD mutants in cells and in our reconstituted system, we determined that Ser-70 and His-361 are essential for d-alanylation activity, and we propose that DltD uses a catalytic dyad to transfer d-alanine to LTA. In summary, we have developed a suite of assays for investigating the bacterial DLT pathway and uncovered a role for DltD in LTA d-alanylation.

Reconstitution of Staphylococcus aureus Lipoteichoic Acid Synthase Activity Identifies Congo Red as a Selective Inhibitor.

Lipoteichoic acid (LTA) is an anionic surface polymer that is essential for normal growth of Staphylococcus aureus, making the LTA polymerase, LTA synthase (LtaS), a proposed drug target for combating Staphylococcal infections. LtaS is a polytopic membrane protein with five membrane-spanning helices and an extracellular domain, and it uses phosphatidylglycerol to assemble a glycerol phosphate chain on a glycosylated diacylglycerol membrane anchor. We report here the first reconstitution of LtaS polymerization activity and show that the azo dye Congo red inhibits this enzyme both in vitro and in cells. Related azo dyes and the previously reported LtaS inhibitor 1771 have weak or no in vitro inhibitory activity. Synthetic lethality with mutant strains known to be nonviable in the absence of LTA confirms selective inhibition by Congo red. As the only validated LtaS inhibitor, Congo red can serve as a probe to understand how inhibiting lipoteichoic acid biosynthesis affects cell physiology and may also guide the discovery of more potent inhibitors for use in treating S. aureus infections.

A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles toward their use in non-native microbial hosts.

The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life.

Covering: up to 2013. Although holo-acyl carrier protein synthase, AcpS, a phosphopantetheinyl transferase (PPTase), was characterized in the 1960s, it was not until the publication of the landmark paper by Lambalot et al. in 1996 that PPTases garnered wide-spread attention being classified as a distinct enzyme superfamily. In the past two decades an increasing number of papers have been published on PPTases ranging from identification, characterization, structure determination, mutagenesis, inhibition, and engineering in synthetic biology. In this review, we comprehensively discuss all current knowledge on this class of enzymes that post-translationally install a 4'-phosphopantetheine arm on various carrier proteins.

Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology.

Many pharmaceuticals on the market today belong to a large class of natural products called nonribosomal peptides (NRPs). Originating from bacteria and fungi, these peptide-based natural products consist not only of the 20 canonical L-amino acids, but also non-proteinogenic amino acids, heterocyclic rings, sugars, and fatty acids, generating tremendous chemical diversity. As a result, these secondary metabolites exhibit a broad array of bioactivity, ranging from antimicrobial to anticancer. The biosynthesis of these complex compounds is carried out by large multimodular megaenzymes called nonribosomal peptide synthetases (NRPSs). Each module is responsible for incorporation of a monomeric unit into the natural product peptide and is composed of individual domains that perform different catalytic reactions. Biochemical and bioinformatic investigations of these enzymes have uncovered the key principles of NRP synthesis, expanding the pharmaceutical potential of their enzymatic processes. Progress has been made in the manipulation of this biosynthetic machinery to develop new chemoenzymatic approaches for synthesizing novel pharmaceutical agents with increased potency. This review focuses on the recent discoveries and breakthroughs in the structural elucidation, molecular mechanism, and chemical biology underlying the discrete domains within NRPSs.

Harvesting the biosynthetic machineries that cultivate a variety of indispensable plant natural products.

Plants are a sustainable resource for valuable natural chemicals best illustrated by large-scale farming centered on specific products. Here, we review recent discoveries of plant metabolic pathways producing natural products with unconventional biomolecular structures. Prenylation of polyketides by aromatic prenyltransferases (aPTases) ties together two of the major groups of plant specialized chemicals, terpenoids and polyketides, providing a core modification leading to new bioactivities and downstream metabolic processing. Moreover, PTases that biosynthesize Z-terpenoid precursors for small molecules such as lycosantalene have recently been found in the tomato family. Gaps in our understanding of how economically important compounds such as cannabinoids are produced are being identified using next-generation 'omics' to rapidly advance biochemical breakthroughs at an unprecedented rate. For instance, olivetolic acid cyclase, a polyketide synthase (PKS) co-factor from Cannabis sativa, directs the proper cyclization of a polyketide intermediate. Elucidations of spatial and temporal arrangements of biosynthetic enzymes into metabolons, such as those used to control the efficient production of natural polymers such as rubber and defensive small molecules such as linamarin and lotaustralin, provide blueprints for engineering streamlined production of plant products.

Biosynthetic potential of sesquiterpene synthases: product profiles of Egyptian Henbane premnaspirodiene synthase and related mutants.

The plant terpene synthase (TPS) family is responsible for the biosynthesis of a variety of terpenoid natural products possessing diverse biological functions. TPSs catalyze the ionization and, most commonly, rearrangement and cyclization of prenyl diphosphate substrates, forming linear and cyclic hydrocarbons. Moreover, a single TPS often produces several minor products in addition to a dominant product. We characterized the catalytic profiles of Hyoscyamus muticus premnaspirodiene synthase (HPS) and compared it with the profile of a closely related TPS, Nicotiana tabacum 5-epi-aristolochene synthase (TEAS). The profiles of two previously studied HPS and TEAS mutants, each containing nine interconverting mutations, dubbed HPS-M9 and TEAS-M9, were also characterized. All four TPSs were compared under varying temperature and pH conditions. In addition, we solved the X-ray crystal structures of TEAS and a TEAS quadruple mutant complexed with substrate and products to gain insight into the enzymatic features modulating product formation. These informative structures, along with product profiles, provide new insight into plant TPS catalytic promiscuity.

Structure, biochemistry, and inhibition of essential 4'-phosphopantetheinyl transferases from two species of Mycobacteria.

4'-Phosphopantetheinyl transferases (PPTase) post-translationally modify carrier proteins with a phosphopantetheine moiety, an essential reaction in all three domains of life. In the bacterial genus Mycobacteria, the Sfp-type PPTase activates pathways necessary for the biosynthesis of cell wall components and small molecule virulence factors. We solved the X-ray crystal structures and biochemically characterized the Sfp-type PPTases from two of the most prevalent Mycobacterial pathogens, PptT of M. tuberculosis and MuPPT of M. ulcerans. Structural analyses reveal significant differences in cofactor binding and active site composition when compared to previously characterized Sfp-type PPTases. Functional analyses including the efficacy of Sfp-type PPTase-specific inhibitors also suggest that the Mycobacterial Sfp-type PPTases can serve as therapeutic targets against Mycobacterial infections.

Confluence of structural and chemical biology: plant polyketide synthases as biocatalysts for a bio-based future.

Type III plant polyketide synthases (PKSs) biosynthesize a dazzling array of polyphenolic products that serve important roles in both plant and human health. Recent advances in structural characterization of these enzymes and new tools from the field of chemical biology have facilitated exquisite probing of plant PKS iterative catalysis. These tools have also been used to exploit type III PKSs as biocatalysts to generate new chemicals. Going forward, chemical, structural and biochemical analyses will provide an atomic resolution understanding of plant PKSs and will serve as a springboard for bioengineering and scalable production of valuable molecules in vitro, by fermentation and in planta.