GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

The sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A Streptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium Salmonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gacA in a S. mutans rmlD knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gacA as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.     

The structure of glucose‐1‐phosphate thymidylyltransferase from Mycobacterium tuberculosis reveals the location of an essential magnesium ion in the RmlA‐type enzymes

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, continues to be a major threat to populations worldwide. Whereas the disease is treatable, the drug regimen is arduous at best with the use of four antimicrobials over a six‐month period. There is clearly a pressing need for the development of new therapeutics. One potential target for structure‐based drug design is the enzyme RmlA, a glucose‐1‐phosphate thymidylyltransferase. This enzyme catalyzes the first step in the biosynthesis of l ‐rhamnose, which is a deoxysugar critical for the integrity of the bacterium's cell wall. Here, we report the X‐ray structures of M. tuberculosis RmlA in complex with either dTTP or dTDP‐glucose to 1.6 Å and 1.85 Å resolution, respectively. In the RmlA/dTTP complex, two magnesium ions were observed binding to the nucleotide, both ligated in octahedral coordination spheres. In the RmlA/dTDP‐glucose complex, only a single magnesium ion was observed. Importantly, for RmlA‐type enzymes with known three‐dimensional structures, not one model shows the position of the magnesium ion bound to the nucleotide‐linked sugar. As such, this investigation represents the first direct observation of the manner in which a magnesium ion is coordinated to the RmlA product and thus has important ramifications for structure‐based drug design. In the past, molecular modeling procedures have been employed to derive a three‐dimensional model of the M. tuberculosis RmlA for drug design. The X‐ray structures presented herein provide a superior molecular scaffold for such endeavors in the treatment of one of the world's deadliest diseases.     

Computational evaluation of phytocompounds for combating drug resistant tuberculosis by multi-targeted therapy

The cell wall of Mycobacterium tuberculosis interacts with the host counterpart during the pathogenesis of tuberculosis. L-rhamnosyl (L-Rha) residue, a linker connects the arabinogalactan and peptidoglycan moieties in the bacterial cell wall. The biosynthesis of L-rhamnose utilizes four successive enzymes RmlA, RmlB, RmlC and RmlD. Neither rhamnose nor the genes responsible for its synthesis are observed in humans. Thus, drugs inhibiting enzymes of this pathway are unlikely to interfere with metabolic pathways in humans. The adverse drug effects of first and second line drugs along with the development of multi-drug resistance tuberculosis have stimulated the research in search of new therapeutic drugs. Thus, it is attractive to hypothesize that inhibition of the biosynthesis of L-Rha would be lethal to the mycobacteria. Nature provides innumerable secondary metabolites with novel structural architectures with reported activity against M. tuberculosis. Combination of structure based virtual screening with physicochemical and pharmacokinetic studies against rhamnose pathway enzymes identified potential leads. The crucial screening studies recognized four phytocompounds butein, diospyrin, indicanine, and rumexneposide A with good binding affinity towards the rhamnose pathway proteins. Furthermore, the high throughput screening methods recognized butein, a secondary metabolite from Butea monosperma with strong anti-tubercular bioactive spectrum. Butein displayed promising anti-mycobacterial activity which is validated by Microplate alamar blue assay (MABA). The focus on novel agents like these phytocompounds which exhibit preference toward the successive enzymes of a single pathway can prevent the development of bacterial resistance.     

A 96-well microtiter plate assay for high-throughput screening of Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase inhibitors

Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase (RmlB) is the second enzyme for the biosynthesis of dTDP-l-rhamnose, which is a sugar donor to the synthesis of the cell wall linker, d-N-acetylglucosamine-l-rhamnose. RmlB is essential to mycobacterial growth and is not found in humans; therefore, it is a potential target for developing new anti-tuberculosis drugs. So far, there has been no suitable method for high-throughput screening of RmlB inhibitors. Here, the recombinant M. tuberculosis RmlB was purified and an absorbance-based microtiter plate assay was developed for RmlB activity. It could be used for high-throughput screening of RmlB inhibitors. The kinetic properties of M. tuberculosis RmlB, including optimal pH, optimal temperature, the effect of metal ions, and the kinetic parameters, were determined with this assay. The inhibitory effects of dTTP and dTDP on M. tuberculosis RmlB were also studied with the assay.     

The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4‐formamido‐4,6‐dideoxy‐d‐glucose

Recent studies have demonstrated that the O‐antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N‐formylated sugars (3‐formamido‐3,6‐dideoxy‐d ‐glucose or 4‐formamido‐4,6‐dideoxy‐d ‐glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6‐dehydratase, a pyridoxal 5'‐phosphate or PLP‐dependent aminotransferase, and an N‐formyltransferase. To date, there have been no published reports of N‐formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N‐formyltransferase. Given that M. tuberculosis produces l ‐rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6‐dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N‐formylated sugar in M. tuberculosis, namely a PLP‐dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined.     

High-resolution structures of RmlC from Streptococcus suis in complex with substrate analogs locate the active site of this class of enzyme

Nature achieves the epimerization of carbohydrates by a variety of chemical routes. One common route is that performed by the class of enzyme defined by dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC) from the rhamnose pathway. Earlier studies failed to identify the key residues in catalysis. We report the 1.3 A structure of RmlC from Streptococcus suis type 2 and its complexes with dTDP-D-glucose and dTDP-D-xylose. The streptococcal RmlC enzymes belong to a separate subgroup, sharing only 25% identity with RmlC from other bacteria, yet the S. suis enzyme has similar kinetic properties and structure to other RmlC enzymes. Structure, sequence alignment, and mutational analysis have now allowed reliable identification of the catalytic residues and their roles.     

Synthesis and antimicrobial activity of α‐aminoboronic‐containing peptidomimetics

A library of 175 dipeptidomimetics and tripeptidomimetics containing an α‐amino boronic acid or boronate has been synthesized, and the activity toward Mycobacterium tuberculosis, Candida albicans, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa has been screened. Although there is no clear structure–activity relationship, several compounds exhibit promising activity against different pathogens. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Streptococcus mutans requires mature rhamnose‐glucose polysaccharides for proper pathophysiology,morphogenesis and cellular division

The cell wall of Gram‐positive bacteria has been shown to mediate environmental stress tolerance, antibiotic susceptibility, host immune evasion and overall virulence. The majority of these traits have been demonstrated for the well‐studied system of wall teichoic acid (WTA) synthesis, a common cell wall polysaccharide among Gram‐positive organisms. Streptococcus mutans, a Gram‐positive odontopathogen that contributes to the enamel‐destructive disease dental caries, lacks the capabilities to generate WTA. Instead, the cell wall of S. mutans is highly decorated with rhamnose‐glucose polysaccharides (RGP), for which functional roles are poorly defined. Here, we demonstrate that the RGP has a distinct role in protecting S. mutans from a variety of stress conditions pertinent to pathogenic capability. Mutant strains with disrupted RGP synthesis failed to properly localize cell division complexes, suffered from aberrant septum formation and exhibited enhanced cellular autolysis. Surprisingly, mutant strains of S. mutans with impairment in RGP side chain modification grew into elongated chains and also failed to properly localize the presumed cell wall hydrolase, GbpB. Our results indicate that fully mature RGP has distinct protective and morphogenic roles for S. mutans, and these structures are functionally homologous to the WTA of other Gram‐positive bacteria.     

Probiotic Lactobacillus sp. inhibit growth,biofilm formation and gene expression of caries‐inducing Streptococcus mutans

Streptococcus mutans contributes significantly to dental caries, which arises from homoeostasic imbalance between host and microbiota. We hypothesized that Lactobacillus sp. inhibits growth, biofilm formation and gene expression of Streptococcus mutans. Antibacterial (agar diffusion method) and antibiofilm (crystal violet assay) characteristics of probiotic Lactobacillus sp. against Streptococcus mutans (ATCC 25175) were evaluated. We investigated whether Lactobacillus casei (ATCC 393), Lactobacillus reuteri (ATCC 23272), Lactobacillus plantarum (ATCC 14917) or Lactobacillus salivarius (ATCC 11741) inhibit expression of Streptococcus mutans genes involved in biofilm formation, quorum sensing or stress survival using quantitative real‐time polymerase chain reaction (qPCR). Growth changes (OD600) in the presence of pH‐neutralized, catalase‐treated or trypsin‐treated Lactobacillus sp. supernatants were assessed to identify roles of organic acids, peroxides and bacteriocin. Susceptibility testing indicated antibacterial (pH‐dependent) and antibiofilm activities of Lactobacillus sp. against Streptococcus mutans. Scanning electron microscopy revealed reduction in microcolony formation and exopolysaccharide structural changes. Of the oral normal flora, L. salivarius exhibited the highest antibiofilm and peroxide‐dependent antimicrobial activities. All biofilm‐forming cells treated with Lactobacillus sp. supernatants showed reduced expression of genes involved in exopolysaccharide production, acid tolerance and quorum sensing. Thus, Lactobacillus sp. can inhibit tooth decay by limiting growth and virulence properties of Streptococcus mutans.     

Homologs of the Rml enzymes from Salmonella enterica are responsible for dTDP-beta-L-rhamnose biosynthesis in the gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155

The glycan chains of the surface layer (S-layer) glycoprotein from the gram-positive, thermophilic bacterium Aneurinibacillus (formerly Bacillus) thermoaerophilus strain DSM 10155 are composed of L-rhamnose- and D-glycero-D-manno-heptose-containing disaccharide repeating units which are linked to the S-layer polypeptide via core structures that have variable lengths and novel O-glycosidic linkages. In this work we investigated the enzymes involved in the biosynthesis of thymidine diphospho-L-rhamnose (dTDP-L-rhamnose) and their specific properties. Comparable to lipopolysaccharide O-antigen biosynthesis in gram-negative bacteria, dTDP-L-rhamnose is synthesized in a four-step reaction sequence from dTTP and glucose 1-phosphate by the enzymes glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC), and dTDP-4-dehydrorhamnose reductase (RmlD). The rhamnose biosynthesis operon from A. thermoaerophilus DSM 10155 was sequenced, and the genes were overexpressed in Escherichia coli. Compared to purified enterobacterial Rml enzymes, the enzymes from the gram-positive strain show remarkably increased thermostability, a property which is particularly interesting for high-throughput screening and enzymatic synthesis. The closely related strain A. thermoaerophilus L420-91(T) produces D-rhamnose- and 3-acetamido-3,6-dideoxy-D-galactose-containing S-layer glycan chains. Comparison of the enzyme activity patterns in A. thermoaerophilus strains DSM 10155 and L420-91(T) for L-rhamnose and D-rhamnose biosynthesis indicated that the enzymes are differentially expressed during S-layer glycan biosynthesis and that A. thermoaerophilus L420-91(T) is not able to synthesize dTDP-L-rhamnose. These findings confirm that in each strain the enzymes act specifically on S-layer glycoprotein glycan formation.     

Severe invasive streptococcal infection by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

Streptococcus pyogenes, a group A Streptococcus (GAS), has been recognized as the causative pathogen in patients with severe invasive streptococcal infection with or without necrotizing fasciitis. In recent epidemiological studies, Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been isolated from severe invasive streptococcal infection. Complete genome sequence showed that SDSE is the closest bacterial species to GAS, with approximately 70% of genome coverage. SDSE, however, lacks several key virulence factors present in GAS, such as SPE‐B, the hyaluronan synthesis operon and active superantigen against human immune cells. A key event in the ability of GAS to cause severe invasive streptococcal infection was shown to be the acquisition of novel genetic traits such as phages. Strikingly, however, during severe invasive infection, GAS destroys its own covRS two‐component system, which negatively regulates many virulence factor genes, resulting in a hyper‐virulent phenotype. In contrast, this phenomenon has not been observed in SDSE. The present review describes the epidemiology of severe invasive streptococcal infection and the detailed pathogenic mechanisms of GAS and SDSE, emphasizing findings from their genome sequences and analyses of gene expression.     

Two‐dimensional gel‐based proteomic of the caries causative bacterium Streptococcus mutans UA159 and insight into the inhibitory effect of carolacton

Streptococcus mutans is considered to be the most cariogenic organism. Carolacton, isolated from the myxobacterium Sorangium cellulosum, shows the ability to disturb S. mutans biofilm viability that makes it a potential anti‐biofilm drug. However, the molecular mechanism of carolacton remains to be elucidated. In order to use proteomics to characterize the effect of carolacton, we constructed a 2DE‐based proteome reference map of the cytoplasmic and extracellular proteins for S. mutans in the present study. In total, 239 protein spots representing 192 different cytoplasmic proteins were identified by MALDI‐TOF MS and PMF. This represents the highest number of identified proteins so far for S. mutans UA159 in the pI range of 4–7 and would benefit further research on the physiology and pathogenicity of this strain. Based on the constructed reference map, the inhibitory effects of carolacton on S. mutans biofilm and planktonic‐growing cells were investigated. The results of the comparative proteome analysis indicate that carolacton exerts its inhibitory effects by disturbing the peptidoglycan biosynthesis and degradation and thereby causes damages to the integrity of the cell envelope, leading ultimately to cell death.     

Anti‐viral and anti‐bacterial activities of an extract of blackcurrants (Ribes nigrum L.)

The inhibitory effects of an extract of the blackcurrant (Ribes nigrum L.) against pathogens associated with oral, nasopharyngeal and upper respiratory infectious diseases; namely respiratory syncytial virus (RSV), influenza virus A and B (IFV‐A and IFV‐B), adenovirus (AdV), herpes simplex virus type 1, Haemophilus influenzae type B, Streptococcus pneumoniae and Streptococcus mutans, were investigated. Less than 1% concentration of extract of blackcurrant inhibited replication of RSV, IFV‐A and ‐B and HSV‐1 by over 50% and a 10% extract inhibited adsorption of these viruses onto the cell surface by over 95%. The effects on AdV were much less pronounced; the half minimal inhibitory concentration of AdV replication was 2.54 ± 0.26, and a 10% concentration of the extract inhibited AdV adsorption on the cell surface by 72.9 ± 3.4%. The antibacterial activities of the blackcurrant were evaluated based on its efficacy as a disinfectant. A 10% extract disinfected 99.8% of H. Influenzae type B and 78.9% of S. pneumoniae in 10 min, but had no demonstrable effect against S. mutans. The blackcurrant extract still showed antiviral and antibacterial activities after the pH had been made neutral with sodium hydroxide, suggesting that these activities are not the result of acidic reactions or of components precipitated at a neutral pH. These findings demonstrate the potential of blackcurrant extract as a functional food for oral care.     

Rhamnose synthase activity is required for pathogenicity of the vascular wilt fungus Verticillium dahliae

The initial interaction of a pathogenic fungus with its host is complex and involves numerous metabolic pathways and regulatory proteins. Considerable attention has been devoted to proteins that play a crucial role in these interactions, with an emphasis on so‐called effector molecules that are secreted by the invading microbe to establish the symbiosis. However, the contribution of other types of molecules, such as glycans, is less well appreciated. Here, we present a random genetic screen that enabled us to identify 58 novel candidate genes that are involved in the pathogenic potential of the fungal pathogen Verticillium dahliae, which causes vascular wilt diseases in over 200 dicotyledonous plant species, including economically important crops. One of the candidate genes that was identified concerns a putative biosynthetic gene involved in nucleotide sugar precursor formation, as it encodes a putative nucleotide‐rhamnose synthase/epimerase‐reductase (NRS/ER). This enzyme has homology to bacterial enzymes involved in the biosynthesis of the nucleotide sugar deoxy‐thymidine diphosphate (dTDP)‐rhamnose, a precursor of L‐rhamnose, which has been shown to be required for virulence in several human pathogenic bacteria. Rhamnose is known to be a minor cell wall glycan in fungi and has therefore not been suspected as a crucial molecule in fungal–host interactions. Nevertheless, our study shows that deletion of the VdNRS/ER gene from the V. dahliae genome results in complete loss of pathogenicity on tomato and Nicotiana benthamiana plants, whereas vegetative growth and sporulation are not affected. We demonstrate that VdNRS/ER is a functional enzyme in the biosynthesis of uridine diphosphate (UDP)‐rhamnose, and further analysis has revealed that VdNRS/ER deletion strains are impaired in the colonization of tomato roots. Collectively, our results demonstrate that rhamnose, although only a minor cell wall component, is essential for the pathogenicity of V. dahliae.     

RmlC, the third enzyme of dTDP-L-rhamnose pathway, is a new class of epimerase

Deoxythymidine diphosphate (dTDP)-L-rhamnose is the precursor of L-rhamnose, a saccharide required for the virulence of some pathogenic bacteria. dTDP-L-rhamnose is synthesized from glucose-1-phosphate and deoxythymidine triphosphate (dTTP) via a pathway involving four distinct enzymes. This pathway does not exist in humans and the enzymes involved in dTDP-L-rhamnose synthesis are potential targets for the design of new therapeutic agents. Here, the crystal structure of dTDP-6-deoxy-D-xylo-4-hexulose 3,5 epimerase (RmlC, EC5.1.3.13) from Salmonella enterica serovar Typhimurium was determined. The third enzyme of the rhamnose biosynthetic pathway, RmlC epimerizes at two carbon centers, the 3 and 5 positions of the sugar ring. The structure was determined by multiwavelength anomalous diffraction to a resolution of 2.17 A. RmlC is a dimer and each monomer is formed mainly from two beta-sheets arranged in a beta-sandwich. The structure of a dTDP-phenol-RmlC complex shows the substrate-binding site to be located between the two beta-sheets; this site is formed from residues of both monomers. Sequence alignments of other RmlC enzymes confirm that this region is very highly conserved. The enzyme is distinct structurally from other epimerases known and thus, is the first example of a new class of carbohydrate epimerase.     

A new route to dTDP-6-deoxy-l-talose and dTDP-L-rhamnose: dTDP-L-rhamnose 4-epimerase in Burkholderia thailandensis

dTDP-l-rhamnose (dTDP-Rha)-synthesizing dTDP-6-deoxy-l-lyxo-4-hexulose reductase (4-KR) and dTDP-Rha 4-epimerase were characterized from Burkholderia thailandensis E264 by utilizing rmlD_Bth (BTH_I1472) and wbiB_Bth (BTH_I1476), respectively. Incubation of the recombinant WbiB_Bth with RmlA/RmlB/RmlC/Tal, which has previously been shown to generate dTDP-6-deoxy-l-talose (dTDP-6dTal) from α-d-glucose-1-phosphate, dTTP, and NADPH, produced dTDP-Rha. ¹H NMR measurements confirmed that both RmlA/RmlB/RmlC/Tal/WbiB_Bth and RmlA/RmlB/RmlC/RmlD produced dTDP-Rha. WbiB_Bth alone produced dTDP-Rha when incubated with dTDP-6dTal. This is the first report to demonstrate epimerase activity interconverting between dTDP-Rha and dTDP-6dTal.     

Structural studies on KijD1, a sugar C‐3′‐methyltransferase

Kijanimicin is an antitumor antibiotic isolated from Actinomadura kijaniata. It is composed of three distinct moieties: a pentacyclic core, a monosaccharide referred to as d ‐kijanose, and a tetrasaccharide chain composed of l ‐digitoxose units. d ‐Kijanose is a highly unusual nitro‐containing tetradeoxysugar, which requires at least ten enzymes for its production. Here we describe a structural analysis of one of these enzymes, namely KijD1, which functions as a C‐3′‐methyltransferase using S‐adenosylmethionine as its cofactor. For this investigation, two ternary complexes of KijD1, determined in the presence of S‐adenosylhomocysteine (SAH) and dTDP or SAH and dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐3‐methyl‐d ‐glucose, were solved to 1.7 or 1.6 Å resolution, respectively. Unexpectedly, these structures, as well as additional biochemical analyses, demonstrated that the quaternary structure of KijD1 is a dimer. Indeed, this is in sharp contrast to that previously observed for the sugar C‐3′‐methyltransferase isolated from Micromonospora chalcea. By the judicious use of site‐directed mutagenesis, it was possible to convert the dimeric form of KijD1 into a monomeric version. The quaternary structure of KijD1 could not have been deduced based solely on bioinformatics approaches, and thus this investigation highlights the continuing need for experimental validation.     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

Biosynthesis of the leucine derived α‐, β‐ and γ‐hydroxynitrile glucosides in barley (Hordeum vulgare L.)

Barley (Hordeum vulgare L.) produces five leucine‐derived hydroxynitrile glucosides (HNGs), of which only epiheterodendrin is a cyanogenic glucoside. The four non‐cyanogenic HNGs are the β‐HNG epidermin and the γ‐HNGs osmaronin, dihydroosmaronin and sutherlandin. By analyzing 247 spring barley lines including landraces and old and modern cultivars, we demonstrated that the HNG level varies notably between lines whereas the overall ratio between the compounds is constant. Based on sequence similarity to the sorghum (Sorghum bicolor) genes involved in dhurrin biosynthesis, we identified a gene cluster on barley chromosome 1 putatively harboring genes that encode enzymes in HNG biosynthesis. Candidate genes were functionally characterized by transient expression in Nicotiana benthamiana. Five multifunctional P450s, including two CYP79 family enzymes and three CYP71 family enzymes, and a single UDP‐glucosyltransferase were found to catalyze the reactions required for biosynthesis of all five barley HNGs. Two of the CYP71 enzymes needed to be co‐expressed for the last hydroxylation step in sutherlandin synthesis to proceed. This observation, together with the constant ratio between the different HNGs, suggested that HNG synthesis in barley is organized within a single multi‐enzyme complex.     

Mutations in the essential FAS II β‐hydroxyacyl ACP dehydratase complex confer resistance to thiacetazone in Mycobacterium tuberculosis and Mycobacterium kansasii

It has recently been shown that the anti‐mycobacterial pro‐drug thiacetazone (TAC) inhibits the conversion of double bonds of mycolic acid precursors into cyclopropyl rings in Mycobacterium bovis var BCG, M. marimum and M. chelonae by affecting the cyclopropyl mycolic acid synthases (CMASs) as judged by the build‐up of unsaturated mycolate precursors. In our hands, TAC inhibits mycolic acid biosynthesis in Mycobacterium tuberculosis and M. kansasii with almost negligible accumulation of those precursors. Our observations that ‘de novo’ biosynthesis of all the mycolic acid families decreased upon TAC treatment prompted us to analyse the role of each one of the Type II Fatty Acid Synthase (FASII) enzymes. Overexpression of the hadABC operon, encoding the essential FASII dehydratase complex, but not of any of the remaining FASII genes acting on the elongation of fatty acyl chains leading to the synthesis of meromycolic acids, resulted in high level of resistance to TAC in M. tuberculosis. Spontaneous M. tuberculosis and M. kansasii TAC‐resistant mutants isolated during this work revealed mutations in the hadABC genes strongly supporting our proposal that these enzymes are new players in the resistance to this anti‐mycobacterial compound.