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.

Assessing the susceptibility of transgenic mice overexpressing deer prion protein to bovine spongiform encephalopathy.

Several transgenic mouse models have been developed which facilitate the transmission of chronic wasting disease (CWD) of cervids and allow prion strain discrimination. The present study was designed to assess the susceptibility of the prototypic mouse line, Tg(CerPrP)1536(+/-), to bovine spongiform encephalopathy (BSE) prions, which have the ability to overcome species barriers. Tg(CerPrP)1536(+/-) mice challenged with red deer-adapted BSE resulted in 90% to 100% attack rates, and BSE from cattle failed to transmit, indicating agent adaptation in the deer.

Incubation of ovine scrapie with environmental matrix results in biological and biochemical changes of PrP(Sc) over time.

Ovine scrapie can be transmitted via environmental reservoirs. A pool of ovine scrapie isolates were incubated on soil for one day or thirteen months and eluted prion was used to challenge tg338 mice transgenic for ovine PrP. After one-day incubation on soil, two PrP(Sc) phenotypes were present: G338 or Apl338ii. Thirteen months later some divergent PrP(Sc) phenotypes were seen: a mixture of Apl338ii with either G338 or P338, and a completely novel PrP(Sc) deposition, designated Cag338. The data show that prolonged ageing of scrapie prions within an environmental matrix may result in changes in the dominant PrP(Sc) biological/biochemical properties.

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.

Overexpression of chimaeric murine/ovine PrP (A136H154Q171) in transgenic mice facilitates transmission and differentiation of ruminant prions.

Development of transgenic mouse models expressing heterologous prion protein (PrP) has facilitated and advanced in vivo studies of prion diseases affecting humans and animals. Here, novel transgenic mouse lines expressing a chimaeric murine/ovine (Mu/Ov) PrP transgene, including amino acid residues alanine, histidine and glutamine at ovine polymorphic codons 136, 154 and 171 (A136H154Q171), were generated to provide a means of assessing the susceptibility of the ovine AHQ allele to ruminant prion diseases in an in vivo model. Transmission studies showed that the highest level of transgene overexpression, in Tg(Mu/OvPrP(AHQ))EM16 (EM16) mice, conferred high susceptibility to ruminant prions. Highly efficient primary transmission of atypical scrapie from sheep was shown, irrespective of donor sheep PrP genotype, with mean incubation periods (IPs) of 154­178 days post-inoculation (p.i.), 100% disease penetrance and early Western blot detection of protease-resistant fragments (PrP(res)) of the disease-associated isoform, PrP(Sc), in EM16 brain from 110 days p.i. onwards. EM16 mice were also highly susceptible to classical scrapie and bovine spongiform encephalopathy (BSE), with mean IPs 320 and 246 days faster, respectively, than WT mice. Primary passage of atypical scrapie, classical scrapie and BSE showed that the PrP(res) profiles associated with disease in the natural host were faithfully maintained in EM16 mice, and were distinguishable based on molecular masses, antibody reactivities and glycoform percentages. Immunohistochemistry was used to confirm PrP(Sc) deposition in brain sections from terminal phase transmissible spongiform encephalopathy-challenged EM16 mice. The findings indicate that EM16 mice represent a suitable bioassay model for detection of atypical scrapie infectivity and offer the prospect of differentiation of ruminant prions.

Disease characteristics of bovine spongiform encephalopathy following inoculation into mice via three different routes.

Mouse-adapted transmissible spongiform encephalopathy (TSE) strains are routinely distinguished based on reproducible disease characteristics in a given mouse line following inoculation via a consistent route. We investigated whether different administration routes (oral, intragastric (i.g.) and intracerebral (i.c.)) can alter the disease characteristics in IM mice after serial dilution of a stabilized mouse-adapted bovine spongiform encephalopathy (BSE) strain (301V). In addition, the infectivity of distal ileum and mesenteric lymph nodes (ln) sampled at three time points (35 days postinoculation (dpi), 70 dpi and terminal disease) after i.g. inoculation of 301V strain was assessed in mice by i.c. challenge. Strain characteristics were assessed according to standard methodology and PrP(Sc) immunohistochemistry deposition patterns. Mean incubation periods were prolonged following oral or i.g. inoculations compared to the i.c. route. Lesion profiles following i.c. challenges were elevated compared to i.g. and oral routes although vacuolation in the dorsal medulla was consistently high irrespective of the route of administration. Nevertheless, the same PrP(Sc) deposition pattern was associated with each route of administration. Distal and mesenteric ln infectivity was detected as early as 35 dpi and displayed consistent lesion profiles and PrP(Sc) deposition patterns. Our data suggest that although 301V retained its properties, some phenotypic parameters were affected by the route of inoculation. We conclude that bioassay data should be interpreted carefully and should be standardized for route of inoculation.

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.

Infectious titres of sheep scrapie and bovine spongiform encephalopathy agents cannot be accurately predicted from quantitative laboratory test results.

It is widely accepted that abnormal forms of the prion protein (PrP) are the best surrogate marker for the infectious agent of prion diseases and, in practice, the detection of such disease-associated (PrP(d)) and/or protease-resistant (PrP(res)) forms of PrP is the cornerstone of diagnosis and surveillance of the transmissible spongiform encephalopathies (TSEs). Nevertheless, some studies question the consistent association between infectivity and abnormal PrP detection. To address this discrepancy, 11 brain samples of sheep affected with natural scrapie or experimental bovine spongiform encephalopathy were selected on the basis of the magnitude and predominant types of PrP(d) accumulation, as shown by immunohistochemical (IHC) examination; contra-lateral hemi-brain samples were inoculated at three different dilutions into transgenic mice overexpressing ovine PrP and were also subjected to quantitative analysis by three biochemical tests (BCTs). Six samples gave 'low' infectious titres (106·5 to 106·7 LD50 g⁻¹) and five gave 'high titres' (108·¹ to ≥ 108·7 LD50 g⁻¹) and, with the exception of the Western blot analysis, those two groups tended to correspond with samples with lower PrP(d)/PrP(res) results by IHC/BCTs. However, no statistical association could be confirmed due to high individual sample variability. It is concluded that although detection of abnormal forms of PrP by laboratory methods remains useful to confirm TSE infection, infectivity titres cannot be predicted from quantitative test results, at least for the TSE sources and host PRNP genotypes used in this study. Furthermore, the near inverse correlation between infectious titres and Western blot results (high protease pre-treatment) argues for a dissociation between infectivity and PrP(res).

The interpretation of disease phenotypes to identify TSE strains following murine bioassay: characterisation of classical scrapie.

Mouse bioassay can be readily employed for strain typing of naturally occurring transmissible spongiform encephalopathy cases. Classical scrapie strains have been characterised historically based on the established methodology of assessing incubation period of disease and the distribution of disease-specific vacuolation across the brain following strain stabilisation in a given mouse line. More recent research has shown that additional methods could be used to characterise strains and thereby expand the definition of strain "phenotype". Here we present the phenotypic characteristics of classical scrapie strains isolated from 24 UK ovine field cases through the wild-type mouse bioassay. PrPSc immunohistochemistry (IHC), paraffin embedded tissue blots (PET-blot) and Western blotting approaches were used to determine the neuroanatomical distribution and molecular profile of PrPSc associated with each strain, in conjunction with traditional methodologies. Results revealed three strains isolated through each mouse line, including a previously unidentified strain. Moreover IHC and PET-blot methodologies were effective in characterising the strain-associated types and neuroanatomical locations of PrPSc. The use of Western blotting as a parameter to define classical scrapie strains was limited. These data provide a comprehensive description of classical scrapie strain phenotypes on isolation through the mouse bioassay that can provide a reference for further scrapie strain identification.

The interpretation of disease phenotypes to identify TSE strains in mice: characterisation of BSE using PrPSc distribution patterns in the brain.

In individual animals affected by transmissible spongiform encephalopathies, different disease phenotypes can be identified which are attributed to different strains of the agent. In the absence of reliable technology to fully characterise the agent, classification of disease phenotype has been used as a strain typing tool which can be applied in any host. This approach uses standardised data on biological parameters, established for a single host, to allow comparison of different prion sources. Traditionally prion strain characterisation in wild type mice is based on incubation periods and lesion profiles after the stabilisation of the agent into the new host which requires serial passages. Such analysis can take many years, due to prolonged incubation periods. The current study demonstrates that the PrPSc patterns produced by one serial passage in wild type mice of bovine or ovine BSE were consistent, stable and showed minimal and predictable differences from mouse-stabilised reference strains. This biological property makes PrPSc deposition pattern mapping a powerful tool in the identification and definition of TSE strains on primary isolation, making the process of characterisation faster and cheaper than a serial passage protocol. It can be applied to individual mice and therefore it is better suited to identify strain diversity within single inocula in case of co-infections or identify strains in cases where insufficient mice succumb to disease for robust lesion profiles to be constructed. The detailed description presented in this study provides a reference document for identifying BSE in wild type mice.

Safety of a Sustainably Produced, Bioengineered, Nature-Identical Salidroside Compound.

Bioactive phytochemicals such as salidroside have been studied to understand the beneficial effects of Rhodiola rosea, an herbaceous plant used in traditional medicine to increase energy and treat a variety of health issues. However, Rhodiola plants are often slow-growing, and many are endangered in their native habitats. Thus, there is a need for safe, alternative supplies of key phytochemicals from Rhodiola. The salidroside subject of this safety study is a synthetic biology product from fermentation of a bioengineered E. coli that produces salidroside. Here, we present comprehensive test results that support the safety of salidroside manufactured via a patented sustainable bioengineering manufacturing process. In vitro bacterial reverse mutation assays with the bioengineered salidroside show no mutagenicity in any of the concentrations tested. In vivo toxicity studies in rats show no adverse effects from the salidroside product. Based on the results of these studies, we conclude that the bioengineered salidroside discussed here is not genotoxic and demonstrates a no-observed-adverse-effect level (NOAEL) at least 2000 mg/kg bw/day in male and female Sprague-Dawley rats. This study supports that the salidroside compound produced using bioengineered E. coli is a viable alternative to salidroside produced from harvested Rhodiola plants for use as a dietary supplement, food ingredient, or potentially as a pharmaceutical product.

Synthetic Biology Category Wins the 350th Anniversary Merck Innovation Cup.

Over the past 350 years, Merck has developed science and technology especially in health care, life sciences, and performance materials. To celebrate so many productive years, Merck conducted a special expanded anniversary edition of the Innovation Cup in combination with the scientific conference Curious2018 - Future Insight in Darmstadt, Germany.

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.

Phenotype shift from atypical scrapie to CH1641 following experimental transmission in sheep.

The interactions of host and infecting strain in ovine transmissible spongiform encephalopathies are known to be complex, and have a profound effect on the resulting phenotype of disease. In contrast to classical scrapie, the pathology in naturally-occurring cases of atypical scrapie appears more consistent, regardless of genotype, and is preserved on transmission within sheep homologous for the prion protein (PRNP) gene. However, the stability of transmissible spongiform encephalopathy phenotypes on passage across and within species is not absolute, and there are reports in the literature where experimental transmissions of particular isolates have resulted in a phenotype consistent with a different strain. In this study, intracerebral inoculation of atypical scrapie between two genotypes both associated with susceptibility to atypical forms of disease resulted in one sheep displaying an altered phenotype with clinical, pathological, biochemical and murine bioassay characteristics all consistent with the classical scrapie strain CH1641, and distinct from the atypical scrapie donor, while the second sheep did not succumb to challenge. One of two sheep orally challenged with the same inoculum developed atypical scrapie indistinguishable from the donor. This study adds to the range of transmissible spongiform encephalopathy phenotype changes that have been reported following various different experimental donor-recipient combinations. While these circumstances may not arise through natural exposure to disease in the field, there is the potential for iatrogenic exposure should current disease surveillance and feed controls be relaxed. Future sheep to sheep transmission of atypical scrapie might lead to instances of disease with an alternative phenotype and onward transmission potential which may have adverse implications for both public health and animal disease control policies.