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Branched-chain fatty acids (BCFA) are encountered in Gram-positive bacteria, but less so in other organisms. The bacterial BCFA in membranes are typically saturated, with both odd- and even-numbered carbon chain lengths, and with methyl branches at either the ω-1 (iso) or ω-2 (anteiso) positions. The acylation with BCFA also contributes to the structural diversity of microbial natural products and potentially modulates biological activity. For the tunicamycin (TUN) family of natural products, the toxicity toward eukaryotes is highly dependent upon N-acylation with trans-2,3-unsaturated BCFA. The loss of the 2,3-unsaturation gives modified TUN with reduced eukaryotic toxicity but crucially with retention of the synergistic enhancement of the β-lactam group of antibiotics. Here, we infer from genomics, mass spectrometry, and deuterium labeling that the trans-2,3-unsaturated TUN variants and the saturated cellular lipids found in TUN-producing Streptomyces are derived from the same pool of BCFA metabolites. Moreover, non-natural primers of BCFA metabolism are selectively incorporated into the cellular lipids of TUN-producing Streptomyces and concomitantly produce structurally novel neo-branched TUN N-acyl variants.

Angustmycin A has anti-mycobacterial and cytokinin activities, and contains an intriguing structure in which an unusual sugar with C5′-C6′ dehydration is linked to adenine via an N-glycosidic bond. However, the logic underlying the biosynthesis of this molecule has long remained obscure. Here, we address angustmycin A biosynthesis by the full deciphering of its pathway. We demonstrate that AgmD, C, A, E, and B function as d-allulose 6-phosphate 3-epimerase, d-allulose 6-phosphate pyrophosphokinase, adenine phosphoallulosyltransferase, phosphoribohydrolase, and phosphatase, respectively, and that these collaboratively catalyze the relay reactions to biosynthesize angustmycin C. Additionally, we provide evidence that AgmF is a noncanonical dehydratase for the final step to angustmycin A via a self-sufficient strategy for cofactor recycling. Finally, we have reconstituted the entire six-enzyme pathway in vitro and in E. coli leading to angustmycin A production. These results expand the enzymatic repertoire regarding natural product biosynthesis, and also open the way for rational and rapid discovery of other angustmycin related antibiotics., Angustmycin A is a nucleoside antibiotic having anti-mycobacterial and cytokinin activities. Here, the authors report the whole pathway leading to angustmycin A biosynthesis in Streptomyces and achieve the heterologous production of angustmycin A in E. coli.

Summary Minimycin (MIN) is a C-nucleoside antibiotic structurally related to pseudouridine, and indigoidine is a naturally occurring blue pigment produced by diverse bacteria. Although MIN and indigoidine have been known for decades, the logic underlying the divergent biosynthesis of these interesting molecules has been obscure. Here, we report the identification of a minimal 5-gene cluster (min) essential for MIN biosynthesis. We demonstrated that a non-ribosomal peptide synthetase (MinA) governs “the switch” for the divergent biosynthesis of MIN and the cryptic indigoidine. We also demonstrated that MinCN (the N-terminal phosphatase domain of MinC), MinD (uracil phosphoribosyltransferase), and MinT (transporter) function together as the safeguard enzymes, which collaboratively constitute an unusual self-resistance system. Finally, we provided evidence that MinD, utilizing an unprecedented substrate-competition strategy for self-resistance of the producer cell, maintains competition advantage over the active molecule MIN-5′-monophosphate by increasing the UMP pool in vivo. These findings greatly expand our knowledge regarding natural product biosynthesis., Graphical Abstract, Highlights • A minimal 5-gene cluster (min) is essential for minimycin biosynthesis • Divergent biosynthesis of minimycin and indigoidine is mediated by an NRPS enzyme • A cascade of three safeguard enzymes constitutes the unusual self-resistance system • MinD functions as the key safeguard enzyme by increasing the UMP pool in vivo, Biotechnology; Microbial Biotechnology; Systems Biology

Stenocarpella maydis is the most common corn ear rot pathogen throughout the world. We previously reported that this fungus secretes a protease, Stm-cmp, which functions as a chitinase-modifying protein (CMP) to truncate corn ChitA chitinase. In the current manuscript we describe the purification of Stm-cmp from fungal cultures and characterize its activity using in vitro protease assays. Hydrophobic interaction chromatography was a key purification step as Stm-cmp bound the column under low salt conditions, allowing its separation from co-extracted proteins. The purified fraction consisted of predominantly a single protein with a mass of 100 kDa. SDS-PAGE-based protease assays in which corn ChitA chitinase was treated with the purified Stm-cmp demonstrated that it functions as an exoprotease, progressively shortening ChitA to create two truncated proteins. MALDI-TOF-based protease assays demonstrated that the protease also functions as an endoprotease to release a large peptide from the amino-terminus that is subsequently trimmed by removal of three amino acids from the amino-terminus. The complex behavior of Stm-cmp is surprising because previously characterized CMPs function as endoproteases. The biophysical characteristics of Stm-cmp observed during purification, its molecular mass, and its unique activity indicate that Stm-cmp is unrelated to the three previously described types of CMPs. The diversity of CMP proteases suggests that ChitA and related plant proteins play an important role in plant defense.

The alarming growth of antibiotic resistance that is currently ongoing is a serious threat to human health. One of the most promising novel antibiotic targets is MraY (phospho-MurNAc-pentapeptide-transferase), an essential enzyme in bacterial cell wall synthesis. Through recent advances in biochemical research, there is now structural information available for MraY, and for its human homologue GPT (GlcNAc-1-P-transferase), that opens up exciting possibilities for structure-based drug design. The antibiotic compound tunicamycin is a natural product inhibitor of MraY that is also toxic to eukaryotes through its binding to GPT. In this work, we have used tunicamycin and modified versions of tunicamycin as tool compounds to explore the active site of MraY and to gain further insight into what determines inhibitor potency. We have investigated tunicamycin variants where the following motifs have been modified: the length and branching of the tunicamycin fatty acyl chain, the saturation of the fatty acyl chain, the 6″-hydroxyl group of the GlcNAc ring, and the ring structure of the uracil motif. The compounds are analyzed in terms of how potently they bind to MraY, inhibit the activity of the enzyme, and affect the protein thermal stability. Finally, we rationalize these results in the context of the protein structures of MraY and GPT.

Streptococcus suis is a major pathogen of swine that is also considered an emerging zoonotic pathogen of humans. Because of increasing antibiotic resistance in S. suis, new antimicrobial agents are being sought. Liamocins are unique polyol lipids produced by certain strains of the fungus Aureobasidium pullulans. Liamocins recently have been found to have antibacterial activity with specificity for Streptococcus species. However, liamocins have not previously been tested for inhibition of S. suis. Ten strains of S. suis isolated from domestic pigs were tested for susceptibility to liamocins by the broth microdilution method. All 10 were susceptible, with MICs ranging from 39 to 78 μg/mL. Viability assays using the LIVE/DEAD BacLight Bacterial Viability Kit indicated that liamocins are bactericidal to S. suis. Cell-leakage assays showed that cells treated with liamocins leaked small molecular mass nucleic acids into the extracellular matrix, suggesting that liamocins act via disruption of the cell membrane. Liamocins thus show potential for development as a specific antibacterial to reduce colonization of S. suis in swine.

Chitinases are thought to play a role in plant resistance to pathogens, but the extent of this role is unknown. The gene for a maize ( Zea mays ) chitinase, “chitinase 2”, previously reported to be induced by two ear rot pathogens in one maize inbred, was cloned from mRNA isolated from milk stage kernels of several different inbreds reported to be susceptible or resistant to ear rot pathogens. The chitinase gene sequence of some of the susceptible inbreds had frame shifts which would result in a nonfunctional protein. Other susceptible inbreds, and a few resistant inbreds, were missing regions that would contribute to chitin binding. A putative functional clone from a resistant inbred was expressed in yeast, and produced a protein with chitinase activity against different oligomers of N -acetyl glucosamine and modified chitin. The yeast-produced chitinase was also resistant to degradation by proteases from maize ear rot fungi, and enhanced antifungal activity of miconazole towards Fusarium graminearum . When introduced in maize callus transgenically, the callus expressing the chitinase 2 gene had significantly less growth of the ear rot pathogen, F. graminearium , than callus which did not express the gene. This information suggests susceptibility and resistance to ear rot pathogens is influenced not only by expression levels, but also by the sequence of putative resistance genes. Previous reports of increased expression of putative resistance genes need to be tempered with the realization that they may play no role in resistance if the genes code for nonfunctional or reduced function proteins.

Liamocins are antimicrobial compounds with specificity for Streptococcus spp., produced by certain strains of the fungus Aureobasidium pullulans. Recent studies have identified strains and culture medium for liamocin production. However, this culture medium has not previously been optimized for liamocin production. In this study, we employ one-factor-at-a-time and statistical methods to develop an optimized medium for liamocin production. Liamocin yields from optimized medium were nearly double those from standard medium, up to 22 g liamocin/L. This represents the highest yields of liamocin reported to date. Furthermore, MALDI-TOF analyses show that liamocins produced from optimized medium are similar to those produced from standard medium.

Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a specific peptide bond but select one of many Gly-Gly bonds within the polyglycine region. Some Gly-Gly bonds are cleaved frequently while others are never cleaved. Moreover, we have previously shown that PGHs from different fungal pathogens prefer to cleave different Gly-Gly peptide bonds. It is not understood how PGHs selectively cleave the ChitA linker, especially because its polyglycine structure lacks peptide sidechains. To gain insights into this process we synthesized several peptide analogs of ChitA to evaluate them as potential substrates and inhibitors of Es-cmp, a PGH from the plant pathogenic fungus Epicoccum sorghi. Our results showed that part of the PGH recognition site for substrate chitinases is adjacent to the polyglycine linker on the carboxy side. More specifically, four amino acid residues were implicated, each spaced four residues apart on an alpha helix. Moreover, analogous peptides with selective Gly->sarcosine (N-methylglycine) mutations or a specific Ser->Thr mutation retained inhibitor activity but were no longer cleaved by PGH. Additonally, our findings suggest that peptide analogs of ChitA that inhibit PGH activity could be used to strengthen plant defenses.

The β-lactams are the most widely used group of antibiotics in human health and agriculture, but this is under threat due to the persistent rise of pathogenic resistance. Several compounds, including tunicamycin (TUN), can enhance the antibacterial activity of the β-lactams to the extent of overcoming resistance, but the mammalian toxicity of TUN has precluded its use in this role. Selective hydrogenation of TUN produces modified compounds (TunR1 and TunR2), which retain the enhancement of β-lactams while having much lower mammalian toxicity. Here we show that TunR1 and TunR2 enhance the antibacterial activity of multiple β-lactam family members, including penems, cephems, and third-generation penicillins, to a similar extent as does the native TUN. Eleven of the β-lactams tested were enhanced from 2 to >256-fold against Bacillus subtilis, with comparable results against a penicillin G-resistant strain. The most significant enhancements were obtained with third-generation aminothiazolidyl cephems, including cefotaxime, ceftazidime, and cefquinome. These results support the potential of low toxicity tunicamycin analogs (TunR1 and TunR2) as clinically valid, synergistic enhancers for a broad group of β-lactam antibiotics.

Liamocins are polyol lipids produced by the fungus Aureobasidium pullulans, and have selective antibacterial activity against Streptococcus species. Liamocins produced by A. pullulans strain NRRL 50380 on sucrose medium have a d-mannitol head group ester-linked to 3,5-dihydroxydecanoate acyl chains, three or four of which are joined together by 1,5-polyester bonds (liamocins Man-A1 and Man-B1), and similar 3'-O-acetylated analogs (Man-A2 and Man-B2). However, other types of liamocins are produced depending on the choice of strain and growth conditions. In the current study, growth on different polyols, but not sugars, resulted in considerable structural variation, including liamocins with d-galactitol (dulcitol), d-sorbitol (glucitol), d- and l-arabitol, d-xylitol, l-threitol and glycerol head groups. The head groups of liamocins produced on arabitol were shown to be entirely composed of d-arabitol. These liamocin variants were structurally characterized by NMR and MS, and tested for antibacterial activity. The new liamocin variants also had selective activity against Streptococcus. Liamocin structural variants are novel antibacterials against Streptococcus sp. that merit further investigation.

Antibiotic resistance poses an increasing threat to global health, and it is urgent to reverse the present trend by accelerating development of new natural product derived drugs. Nucleoside antibiotics, a valuable family of promising natural products with remarkable structural features and diverse biological activities, have played significant roles in healthcare and for plant protection. Understanding the biosynthesis of these intricate molecules has provided a foundation for bioengineering the microbial cell factory towards yield enhancement and structural diversification. In this review, we summarize the recent progresses in employing synthetic biology-based strategies to improve the production of target nucleoside antibiotics. Moreover, we delineate the advances on rationally accessing the chemical diversities of natural nucleoside antibiotics.

Maize is a crop of worldwide importance, but insects and plant pathogens limit sustainable production. The development of maize varieties with improved resistance will be facilitated by identification of relevant resistance alleles through appropriate biological assays. A barwin-like gene (which we call maizewin) located in a QTL for Fusarium resistance annotated such that potential glucanase and chitinase activity could occur in the translated protein, was cloned from an ear rot resistant inbred and transgenically expressed in maize callus. Although limited increases in insect resistance occurred in the transgenic callus material containing the maizewin gene compared to a GUS control gene, resistance to Fusarium pathogens was common and associated with increased glucanase activity. The gene appeared to produce a protein with glucanase and chitosanase activity, consistent with an antifungal role. The allele that produces the functional resistant protein may be relatively uncommon in maize germplasm. Incorporation of a functional maizewin allele into susceptible germplasm should help increase production by contributing to control of both insect pests and plant pathogens.

Subtilases are a large family of serine proteases that occur throughout biology. A small subset contain protease-associated (PA) domains that are structurally separate from but encoded within the active site. In bacteria, subtilase PA domains function to recruit specific protein substrates. Here we demonstrate that a protease secreted by the fungal corn pathogen Stenocarpella maydis, which truncates corn ChitA chitinase, is a PA domain subtilase. Protease was purified from S. maydis cultures and tryptic peptides were analyzed by LC-MS/MS. Ions were mapped to two predicted PA domain subtilases. Yeast strains were engineered to express each protease. One failed to produce recombinant protein while the other secreted protease that truncated ChitA. This protease, that we named kilbournase, was purified and characterized. It cleaved multiple peptide bonds in the amino-terminal chitin binding domain of ChitA while leaving the catalytic domain intact. Kilbournase was more active on the ChitA-B73 alloform compared to ChitA-LH82 and did not cleave AtChitIV3, a homolog from Arabidopsis thaliana, indicating a high level of specificity. Truncation of corn ChitA by kilbournase resembles truncation of human C5a by Streptococcus pyogenes ScpA, arguing that PA domain proteases in bacteria and fungi may commonly target specific host proteins.

A gene annotated as a chitinase-like protein that occurred in a quantitative trait locus for fungal maize ear rot resistance was cloned from a maize inbred with Fusarium proliferatum/verticillioides resistance. The protein sequence was similar to type III chitinases and homologous proteins without chitinase activity generally referred to as narbonins or chitolectins. The gene was expressed in Pichia pastoris, and the resulting protein was purified and examined for different types of activity. The yeast-produced protein did not hydrolyze chitin substrates, or 4-nitrophenyl sugars, but did hydrolyze 4-nitrophenyl acetate and 4-nitrophenyl palmitate. The protein also significantly retarded the growth of first instar fall armyworms (Spodptera fruigiperda) and significantly enhanced the activity of miconazole nitrate toward F. graminearum. The gene was biolistically introduced into maize BMS callus and the transgene was detectible by PCR. Some transformant lines significantly reduced corn earworm (Helicoverpa zea), fall armyworm, F. graminearum, F. proliferaturm and F. verticillioides growth compared to GUS transformed control callus. When transformed callus protein extracts were separated by native polyacrylamide gel electrophoresis, a band of 4-nitrophenyl acetate activity was found in bioactive callus that was not present in GUS control callus. Although the true target of the hydrolase is not known, it does appear to play a role in resistance to pests of maize and appears to be a previously unreported type of resistance mechanism.

A new arabinogalactan is described that is produced in large quantity from the cut stems of the North American grape species Vitis riparia (Frost grape). The sugar composition consists of l-arabinofuranose (l-Araf, 55.2%) and d-galactopyranose (d-Galp 30.1%), with smaller components of d-xylose (11.2%), d-mannose (3.5%), and glucuronic acid (GlcA, ∼2%), the latter linked via a galactosyl residue. Permethylation identified 3-linked Galp residues, some substituted at the 2-position with Galp or Manp, terminal Araf and Xylp, and an internal 3-substituted Araf. NMR (HSQC, TOCSY, HMBC, DOSY) identified βGalp and three αAraf spin systems, in an Araf-α1,3-Araf-α1,2-Araf-α1,2-Galp structural motif. Diffusion-ordered NMR showed that the FGP has a molecular weight of 1-10 MDa. Unlike gum arabic, the FGP does not contain a hydroxyproline-rich protein (HPRP). FGP forms stable gels at15% w/v and at 1-12% solutions are viscous and are excellent emulsifiers of flavoring oils (grapefruit, clove, and lemongrass), giving stable emulsions for ≥72 h. Lower concentrations (0.1% w/v) were less viscous, yet still gave stable grapefruit oil/water emulsions. Hence, FGP is a β1,3-linked arabinogalactan with potential as a gum arabic replacement in the food and beverage industries.

Polyglycine hydrolases are secreted fungal proteases that cleave glycine–glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochliobolus carbonum (Bz-cmp). Here we report the identity of their encoding genes and the primary amino acid sequences of the proteins responsible for these activities. Peptides from a tryptic digest of Es-cmp were analyzed by LC-MS/MS and the spectra obtained were matched to a draft genome sequence of E. sorghi. From this analysis, a 642 amino acid protein containing a predicted β-lactamase catalytic region of 280 amino acids was identified. Heterologous strains of the yeast Pichia pastoris were created to express this protein and its homolog from C. carbonum from their cDNAs. Both strains produced recombinant proteins with polyglycine hydrolase activity as shown by SDS-PAGE and MALDI-MS based assays. Site directed mutagenesis was used to mutate the predicted catalytic serine of Es-cmp to glycine, resulting in loss of catalytic activity. BLAST searching of publicly available fungal genomes identified full-length homologous proteins in 11 other fungi of the class Dothideomycetes, and in three fungi of the related class Sordariomycetes while significant BLAST hits extended into the phylum Basidiomycota. Multiple sequence alignment led to the identification of a network of seven conserved tryptophans that surround the β-lactamase-like region. This is the first report of a predicted β-lactamase that is an endoprotease.

T-2 toxin is a trichothecene mycotoxin produced when Fusarium fungi infect grains, especially oats and wheat. Ingestion of T-2 toxin contaminated grain can cause diarrhea, hemorrhaging, and feed refusal in livestock. Cereal crops infected with mycotoxin-producing fungi form toxin glycosides, sometimes called masked mycotoxins, which are a potential food safety concern because they are not detectable by standard approaches and may be converted back to the parent toxin during digestion or food processing. The work reported here addresses four aspects of T-2 toxin-glucosides: phytotoxicity, stability after ingestion, antibody detection, and the anomericity of the naturally occurring T-2 toxin-glucoside found in cereal plants. T-2 toxin-?-glucoside was chemically synthesized and compared to T-2 toxin-?-glucoside prepared with Blastobotrys muscicola cultures and the T-2 toxin-glucoside found in naturally contaminated oats and wheat. The anomeric forms were separated chromatographically and differ in both NMR and mass spectrometry. Both anomers were significantly degraded to T-2 toxin and HT-2 toxin under conditions that mimic human digestion, but with different kinetics and metabolic end products. The naturally occurring T-2 toxin-glucoside from plants was found to be identical to T-2 toxin-?-glucoside prepared with B. muscicola. An antibody test for the detection of T-2 toxin was not effective for the detection of T-2 toxin-?-glucoside. This anomer was produced in sufficient quantity to assess its animal toxicity.

Corn (Zea mays) and Arabidopsis (Arabidopsis thaliana) produce GH family 19 plant class IV chitinases. These chitinases contain two domains: a small N-terminal hevein region, and a C-terminal chitinase. Numerous structures of GH19 chitinase domains have been reported, including the chitinase domain of corn ChitA. Structural information on the N-terminal domains, however, is lacking. Fusarium pathogens secrete fungalysin proteases that cleave some class IV chitinases at a well-defined Gly-Cys site between the two domains. To study the structure of the peptide domain we used the fungalysin protease Fv-cmp as a tool to release the hevein domain from plant class IV chitinases, allowing their direct study. MALDI-TOF MS analysis of fungalysin-released peptides from plant class IV chitinases from corn and Arabidopsis allowed visualization of multiple isotopomers, resulting in accurate mass determination. When treated with DTT, peptide ions increased in mass by six mass units, suggesting breakage of three disulfide bonds. When reduced peptides were S-alkylated, peptides were converted to a series of evenly spaced ions with various states of alkylation, confirming the presence of six reduced cysteines. This chemical data was complemented by use of molecular modeling to determine the fold of the peptide and location of disulfide bonds. The chemical data and molecular model combine to create a structural model of a hevein domain from a GH19 chitinase.

A novel emulsifier, Frost grape polysaccharide (FGP), isolated from natural exudate of the species Vitis riparia Michx, was physically and rheologically characterized. The determination of the physical, structural, thermodynamic, emulsification, film, and rheological properties of FGP provide essential details for the commercial adoption of this novel plant polysaccharide. FGP is capable of producing exceptionally stable emulsions when compared with the industrially ubiquitous gum arabic (GA). The FGP isolate contained a negligible amount of nitrogen (0.03%), indicating that it does not contain an associated glycoprotein, unlike GA. Solutions of FGP have a high degree of thermostability, displaying no loss in viscosity with temperature cycling and no thermal degradation when held at 90 °C. FGP is an excellent film former, producing high tensile strength films which remain intact at temperatures up to 200 °C. This work identified a number of potential food and pharmaceutical applications where FGP is significantly superior to GA.

Tunicamycin is a Streptomyces-derived inhibitor of eukaryotic protein N-glycosylation and bacterial cell wall biosynthesis, and is a potent and general toxin by these biological mechanisms. The antibacterial activity is dependent in part upon a π-π stacking interaction between the tunicamycin uridyl group and a specific Phe residue within MraY, a tunicamycin-binding protein in bacteria. We have previously shown that reducing the tunicamycin uridyl group to 5,6-dihydrouridyl (DHU) significantly lowers its eukaryotic toxicity, potentially by disrupting the π-stacking with the active site Phe. The present report compares the catalytic hydrogenation of tunicamycin and uridine with various precious metal catalysts, and describe optimum conditions for the selective production of N-acyl reduced tunicamycin or for tunicamycins reduced in both the N-acyl and uridyl double bonds. At room temperature, Pd-based catalysts are selective for the N-acyl reduction, whereas Rh-based catalysts favor the double reduction to provide access to fully reduced tunicamycin. The reduced DHU is highly base-sensitive, leading to amide ring opening under mild alkaline conditions.

Tunicamycins (TUN) are inhibitors of the UDP-HexNAc: polyprenol-P HexNAc-1-P transferase family of enzymes, which initiate the biosynthesis of bacterial peptidoglycan and catalyze the first step in eukaryotic protein N-glycosylation. The TUN are therefore general and potent toxins to both eukaryotes and prokaryotes. Screening a library of synthetic TUN against Bacillus and yeast identified TUN that are antibacterial, but have significantly reduced eukaryotic toxicity. One of these (Tun-15:0) differs from the native TUN control only by the lack of the conjugated double bond in the tunicaminyl N-acyl group. Tun-15:0 also showed reduced inhibition for protein N-glycosylation in a Pichia-based bioassay. Natural TUN was subsequently modified by chemically reducing the N-acyl double bond (TunR1) or both the N-acyl and uridyl double bonds (TunR2). TunR1 and TunR2 retain their antibacterial activity, but with considerably reduced eukaryotic toxicity. In protein N-glycosylation bioassays, TunR1 is a less potent inhibitor than native TUN and TunR2 is entirely inactive. Importantly, the less toxic TunR1 and TunR2 both enhance the antibacterial activity of β-lactams: oxacillin by 32- to 64-fold, comparable with native TUN, and with similar enhancements for methicillin and penicillin G. Hence, the modified TUNs, TunR1 and TunR2, are potentially important as less-toxic synergistic enhancers of the β-lactams.

An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of β-endoglucanase with specificity towards schizophyllan. Concentrated enzyme preparations from these strains showed specific activities of 1.7-4.3 U β-glucanase/mg protein. Using dilutions of these enzymes in time course digestions, schizophyllan was progressively modified to reduced molecular weight species. Glucose and oligosaccharides were found only in the more complete digestions, and thus modified schizophyllan can be produced quantitatively, without loss, to small molecules. Permethylation analysis confirmed that modified schizophyllan retains the fundamental linkage structure of native schizophyllan. Modified schizophyllan species showed progressively reduced viscosity profiles, and all exhibited pseudoplasticity in response to shear thinning.

Graphical abstract, We have sought a universal platform for elucidating and exploiting specificity of glycan-mediated adhesion by potentially uncharacterized microorganisms. Several techniques exist to explore microbial interactions with carbohydrate structures. Many are unsuitable for investigating specific mechanisms or uncharacterized organisms, requiring pure cultures, labeling techniques, expensive equipment, or other limitations such as questionable stability, stereospecificity, or scalability. We have adapted an affinity chromatography resin as a model to overcome these drawbacks, among others. It readily allows for the quantification, selection, and manipulation of target organisms based on interactions with glycan ligands. To maximize its utility as a selective screening method, we have constructed the tool such that it:•Promotes whole-cell interactions using viable, unaltered cells.•Provides robust spatial interactions with target glycans, presented with controlled stereo-specificity, for high affinity/avidity interactions that reflect a complex in vivo matrix.•Has the ability to utilize any reducing glycan, is quick, efficient, safe, and affordable to construct, and is scalable and reusable for multiple applications.

Cmps (chitinase-modifying proteins) are fungal proteases that truncate plant class IV chitinases by cleaving near their N-termini. We previously described Fv-cmp, a fungalysin protease that cleaves a conserved glycine–cysteine bond within the hevein domain. In the present paper we describe a new type of cmp, polyglycine hydrolases, as proteases that selectively cleave glycine–glycine peptide bonds within the polyglycine linker of plant class IV chitinases. Polyglycine hydrolases were purified from Cochliobolus carbonum (syn. Bipolaris zeicola; Bz-cmp) and Epicoccum sorghi (syn. Phoma sorghina; Es-cmp) and were shown to cleave three different maize class IV chitinase substrates. The proteolytic cleavage sites were assessed by SDS/PAGE and MALDI–TOF-MS and indicated the cleavage of multiple peptide bonds within the polyglycine linker regions. Site-directed mutagenesis was used to produce mutants of maize ChitB chitinase in which two serine residues in its linker were systematically modified to glycine. Serine to glycine changes in the ChitB linker resulted in higher susceptibility to truncation by Bz-cmp and altered substrate specificity for Bz-cmp and Es-cmp, such that different glycine–glycine peptide bonds were cleaved. Removal of the hevein domain led to loss of Es-cmp activity, indicating that interactions outside of the active site are important for recognition. Our findings demonstrate that plant class IV chitinases with polyglycine linkers are targeted for truncation by selective polyglycine hydrolases that are secreted by plant pathogenic fungi. This novel proteolysis of polyglycine motifs is previously unreported, but the specificity is similar to that of bacterial lysostaphin proteases, which cleave pentaglycine cross-links from peptidoglycan.

Two related actinomycetes, Glycomyces sp. strain NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338, were identified as potential phosphonic acid producers by screening for the gene encoding phosphoenolpyruvate (PEP) mutase, which is required for the biosynthesis of most phosphonates. Using a variety of analytical techniques, both strains were subsequently shown to produce phosphonate-containing exopolysaccharides (EPS), also known as phosphonoglycans. The phosphonoglycans were purified by sequential organic solvent extractions, methanol precipitation, and ultrafiltration. The EPS from the Glycomyces strain has a mass of 40 to 50 kDa and is composed of galactose, xylose, and five distinct partially O -methylated galactose residues. Per-deutero-methylation analysis indicated that galactosyl residues in the polysaccharide backbone are 3,4-linked Gal, 2,4-linked 3-MeGal, 2,3-linked Gal, 3,6-linked 2-MeGal, and 4,6-linked 2,3-diMeGal. The EPS from the Stackebrandtia strain is comprised of glucose, galactose, xylose, and four partially O -methylated galactose residues. Isotopic labeling indicated that the O -methyl groups in the Stackebrandtia phosphonoglycan arise from S -adenosylmethionine. The phosphonate moiety in both phosphonoglycans was shown to be 2-hydroxyethylphosphonate (2-HEP) by 31 P nuclear magnetic resonance (NMR) and mass spectrometry following strong acid hydrolysis of the purified molecules. Partial acid hydrolysis of the purified EPS from Glycomyces yielded 2-HEP in ester linkage to the O -5 or O -6 position of a hexose and a 2-HEP mono(2,3-dihydroxypropyl)ester. Partial acid hydrolysis of Stackebrandtia EPS also revealed the presence of 2-HEP mono(2,3-dihydroxypropyl)ester. Examination of the genome sequences of the two strains revealed similar pepM -containing gene clusters that are likely to be required for phosphonoglycan synthesis.

Maize ChitA chitinase is composed of a small, hevein-like domain attached to a carboxy-terminal chitinase domain. During fungal ear rot, the hevein-like domain is cleaved by secreted fungal proteases to produce truncated forms of ChitA. Here, we report a structural and biochemical characterization of truncated ChitA (ChitA ΔN), which lacks the hevein-like domain. ChitA ΔN and a mutant form (ChitA ΔN-EQ) were expressed and purified; enzyme assays showed that ChitA ΔN activity was comparable to the full-length enzyme. Mutation of Glu62 to Gln (ChitA ΔN-EQ) abolished chitinase activity without disrupting substrate binding, demonstrating that Glu62 is directly involved in catalysis. A crystal structure of ChitA ΔN-EQ provided strong support for key roles for Glu62, Arg177, and Glu165 in hydrolysis, and for Ser103 and Tyr106 in substrate binding. These findings demonstrate that the hevein-like domain is not needed for enzyme activity. Moreover, comparison of the crystal structure of this plant class IV chitinase with structures from larger class I and II enzymes suggest that class IV chitinases have evolved to accommodate shorter substrates.

Background: Chitin synthases are stimulated by N-acetylglucosamine (GlcNAc). Results: GlcNAc and 2-acylamido analogues of GlcNAc stimulate formation of chitin oligosaccharides by yeast chitin synthase, and GlcNAc is transferred to the 2-acylamido analogues. Conclusion: Chitin synthases use GlcNAc analogues as primers and transfer one GlcNAc at a time. Significance: Results are new insights into polysaccharide synthase mechanism and suggest ways of synthesizing novel modified polysaccharides., Chitin, a homopolymer of β1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N′-diacetylchitobiose (GlcNAc2) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc2 and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc2 as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues.

Lactic acid bacteria (LAB) frequently contaminate commercial fuel ethanol fermentations, reducing yields and decreasing profitability of biofuel production. Microorganisms from environmental sources in different geographic regions of Thailand were tested for antibacterial activity against LAB. Four bacterial strains, designated as ALT3A, ALT3B, ALT17, and MR1, produced inhibitory effects on growth of LAB. Sequencing of rRNA identified these strains as species of Bacillus subtilis (ALT3A and ALT3B) and B. cereus (ALT17 and MR1). Cell mass from colonies and agar samples from inhibition zones were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The spectra of ALT3A and ALT3B showed a strong signal at m/z 1,060, similar in mass to the surfactin family of antimicrobial lipopeptides. ALT3A and ALT3B were analyzed by zymogram analysis using SDS-PAGE gels placed on agar plates inoculated with LAB. Cell lysates possessed an inhibitory protein of less than 10 kDa, consistent with the production of an antibacterial lipopeptide. Mass spectra of ALT17 and MR1 had notable signals at m/z 908 and 930 in the whole cell extracts and at m/z 687 in agar, but these masses do not correlate with those of previously reported antibacterial lipopeptides, and no antibacterial activity was detected by zymogram. The antibacterial activities produced by these strains may have application in the fuel ethanol industry as an alternative to antibiotics for prevention and control of bacterial contamination.

Liamocins are unique heavier-than-water "oils" produced by certain strains of the fungus Aureobasidium pullulans. Liamocins have antibacterial activity with specificity for Streptococcus sp. Previous studies reported that liamocin yields were highest from strains of A. pullulans belonging to phylogenetic clades 8, 9, and 11, cultured on medium containing sucrose. In this study, 27 strains from these clades were examined for the first time for production of liamocins from agricultural biomass substrates. Liamocin yields were highest from strains in phylogenetic clade 11, and yields were higher from cultures grown on sucrose than from those grown on pretreated wheat straw. However, when supplementary enzymes (cellulase, β-glucosidase, and xylanase) were added, liamocin production on pretreated wheat straw was equivalent to that on sucrose. Liamocins produced from wheat straw were free of the melanin contamination common in sucrose-grown cultures. Furthermore, MALDI-TOF MS analysis showed that liamocins produced from wheat straw were under-acetylated, resulting in higher proportions of the mannitol A1 and B1 species of liamocin, the latter of which has the highest biological activity against Streptococcus sp.

Fermentable oligosaccharides, particularly those found in soybean meal (SBM), may modulate fermentation in the ceca, thus affecting intestinal immune responses to intestinal pathogens. We hypothesized that fermentable oligosaccharides found in SBM would positively affect cecal fermentation and intestinal immune status in chicks challenged with an acute coccidiosis (Eimeria acervulina) infection and fed either a SBM-based diet or a semi-purified soy protein isolate- (SPI) based diet. Using a completely randomized design, 1-d-old broiler chicks (n = 200; 5 replications/treatment; 5 chicks/replication) were assigned to 1 of 4 SBM- or SPI-based diets containing either dietary cellulose (4%) or a fermentable carbohydrate, galactoglucomannan oligosaccharide-arabinoxylan (GGMO-AX) complex (4%). On d 9 posthatch, an equal number of chicks on each diet were inoculated with either distilled water (sham control) or E. acervulina (1 × 10(6) oocysts) and then euthanized on d 7 postinoculation. Overall, body weight gain and feed intake were greater (P < 0.01) for SBM-fed chicks, regardless of infection status. Gain:feed ratio was greater (P ≤ 0.05) for SPI-fed chicks except during d 3-7 postinoculation. Infection status, but not fiber source, affected propionate, isobutyrate, isovalerate, and total branched-chain fatty acid concentrations (P ≤ 0.02). Soybean meal-based diets resulted in greater (P ≤ 0.04) short-chain fatty acid and branched-chain fatty acid concentrations than SPI-based diets. Messenger RNA fold changes relative to uninfected SBM-cellulose-fed chicks of all duodenal cytokines were greater (P ≤ 0.01) for infected chicks, and SBM-fed chicks had greater (P < 0.01) interferon-γ and interleukin-12β expression compared with SPI-fed chicks. Cecal tonsil cytokine expression was also affected (P ≤ 0.02) by infection; however, protein source only affected (P < 0.01) interleukin-1β expression in this tissue. Overall, a SBM-based diet, compared with a semi-purified SPI-based diet with a different ingredient composition, resulted in greater weight gain, feed intake, and short-chain fatty acid production regardless of infection status, and also greater duodenal cytokine expression in E. acervulina- infected chicks, which is hypothesized to be related to the nutrients and oligosaccharides found in SBM.

Fermentable carbohydrates may enhance the ability of the gastrointestinal tract to defend against a pathogenic infection. We hypothesized that a galactoglucomannan oligosaccharide-arabinoxylan (GGMO-AX) complex would positively affect immune status and prevent colonization and shedding in Salmonella typhimurium-infected chicks. Using a completely randomized design, 1-d-old commercial broiler chicks (n = 240 chicks; 4 replications/treatment; 5 chicks/replication) were assigned to 1 of 6 dietary treatments differing in concentration of GGMO-AX (0, 1, 2, or 4%) or containing 2% Safmannan or 2% short-chain fructooligosaccharides. Cellulose was used to make diets iso-total dietary fiber. On d 10 posthatch, an equal number of chicks on each diet were inoculated with either phosphate-buffered saline (sham control) or Salmonella typhimurium (1 × 10(8) cfu). All birds were euthanized on d 10 postinoculation (PI) for collection of intestinal contents and select tissues. Body weight gain and feed intake of chicks were greater (P < 0.05) in infected chicks PI for all time periods, except for weight gain on d 0 to 3 PI. Gain:feed was affected (P < 0.05) by diet, with Safmannan-fed chicks having the highest G:F and 1% GGMO-AX-fed chicks having the lowest. The GGMO-AX substrate demonstrated effects similar to a prebiotic substrate as indicated by increased cecal short-chain fatty acid concentrations, decreased cecal pH, and increased populations of Lactobacillus spp. and Bifidobacteria spp. as dietary GGMO-AX concentration increased. Excreta Salmonella typhimurium populations on d 5 and 10 PI, and ileal and cecal Salmonella typhimurium populations, tended to be affected (P < 0.10) by the main effect of diet. Messenger RNA expression of IFN-γ in the cecal tonsils was the only cytokine independently affected by infection and diet (P < 0.01). Chicks fed 2 and 4% GGMO-AX had similar expressions of IFN-γ and IL-1β, regardless of infection, suggesting that Salmonella typhimurium virulence was suppressed. Dietary supplementation with GGMO-AX resulted in prebiotic-like effects but did not limit Salmonella typhimurium intestinal colonization or shedding, but possibly decreased the virulence of Salmonella typhimurium within the digestive tract.

SUMMARY Plant class IV chitinases have a small amino-terminal chitin-binding domain and a larger chitinase domain, and are involved in plant defence against fungal infection. Our previous work on the chitinases ChitA and ChitB from the model monocotyledon Zea mays showed that the chitin-binding domain is removed by secreted fungal proteases called fungalysins. In this article, we extend this work to dicotyledons. The effects of fungalysin-like proteases on four class IV chitinases from the model dicotyledon Arabidopsis thaliana were analysed. Four Arabidopsis chitinases were heterologously expressed in Pichia pastoris, purified and shown to have chitinase activity against a chitohexaose (dp6) substrate. The incubation of these four chitinases with Fv-cmp, a fungalysin protease secreted by Fusarium verticillioides, resulted in the truncation of AtchitIV3 and AtchitIV5. Moreover, incubation with secreted proteins from Alternaria brassicae, a pathogen of A. thaliana and brassica crops, also led to a similar truncation of AtchitIV3 and AtchitIV4. Our finding that class IV chitinases from both dicotyledons (A. thaliana) and monocotyledons (Z. mays) are truncated by proteases secreted by specialized pathogens of each plant suggests that this may be a general mechanism of plant–fungal pathogenicity.

Chitinase-modifying proteins (cmps) are proteases secreted by fungal pathogens that truncate the plant class IV chitinases ChitA and ChitB during maize ear rot. cmp activity has been characterized for Bipolaris zeicola and Stenocarpella maydis, but the identities of the proteases are not known. Here, we report that cmps are secreted by multiple species from the genus Fusarium, that cmp from Fusarium verticillioides (Fv-cmp) is a fungalysin metalloprotease, and that it cleaves within a sequence that is conserved in class IV chitinases. Protein extracts from Fusarium cultures were found to truncate ChitA and ChitB in vitro. Based on this activity, Fv-cmp was purified from F. verticillioides. N-terminal sequencing of truncated ChitA and MALDI-TOF-MS analysis of reaction products showed that Fv-cmp is an endoprotease that cleaves a peptide bond on the C-terminal side of the lectin domain. The N-terminal sequence of purified Fv-cmp was determined and compared with a set of predicted proteins, resulting in its identification as a zinc metalloprotease of the fungalysin family. Recombinant Fv-cmp also truncated ChitA, confirming its identity, but had reduced activity, suggesting that the recombinant protease did not mature efficiently from its propeptide-containing precursor. This is the first report of a fungalysin that targets a nonstructural host protein and the first to implicate this class of virulence-related proteases in plant disease.

Summary Biosurfactants are diverse molecules with numerous biological functions and industrial applications. A variety of environments were examined for biosurfactant-producing bacteria including soil, water and leaf surfaces. Biosurfactant production was assessed with an atomized oil assay for a large number of bacterial isolates and compared with a commonly used drop collapse assay from broth and plate cultures. The atomized oil assay detected every strain that produced a biosurfactant detectable by the drop collapse test, and also identified additional strains that were not detected with the drop collapse assay because they produced low levels of surfactant or hydrophobic (low water solubility) surfactants such as pumilacidins. Not all strains that produced a biosurfactant detectable by the drop collapse when cultured on agar surfaces produced surfactants detectable by drop collapse when cultured in broth, and vice versa. Many bacterial strains exhibited preferential production of surfactants when grown on an agar surface compared with broth cultures, and such surface enhancement of production could also be stimulated by increasing the viscosity of liquid culture media. Surface induction of surfactant pro- duction in the epiphyte Pseudomonas syringae was regulated at the transcriptional level.

A high-throughput matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI–TOF MS) assay is described for determination of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates and is readily achievable on a microliter scale (2 μl of total volume containing 2 μg of substrate and 1 ng of protein). The speed and sensitivity of the assay make it potentially well suited for the high-throughput screening of chitinase inhibitors. The mass spectrum is acquired in approximately 2 min, as opposed to typically 30–40 min for a single run with a high-performance liquid chromatography (HPLC)-based assay. By using the multiple-place MALDI MS targets, we estimate that 100 assays could be run in approximately 2–3 h without needing to remove the target from the instrument. In addition, because the substrate and product chitomers are visualized simultaneously in the TOF spectrum, this gives immediate information about the cleavage site and mechanism of the enzyme under study. The assay was used to monitor the purification and transgenic expression of plant class IV chitinases. By performing the assay with chitomer substrates and C -glycoside chitomer analogs, the enzyme mechanism of the class IV chitinases is described for the first time.

From a survey of more than 50 diverse strains of Aureobasidium pullulans, 21 produced extracellular heavy oils. Most oil producers fell into phylogenetic clades 8, 9, and 11. Oil colors ranged from bright yellow to malachite. More than half of the strains produced oil that was fluorescent. In medium containing 5% (w/v) sucrose, oil yields ranged from 0.5 to 6 g oil/l. Strain CU 43 reached stationary growth phase at day 4 while oil yields were maximal at day 6. CU 43 produced bright yellow, highly fluorescent oil that also was visible as intracellular droplets under fluorescent microscopy. Oil was surface active, suggesting that it functions as a biosurfactant. Oil from two strains (CU 43 and NRRL Y-12974) differentially inhibited mammalian cancer cell lines. MALDI-TOF MS spectra suggested that A. pullulans strains produce a family of related oil structures.

It is of interest to discover new fermentable carbohydrate sources that function as prebiotics. This study evaluated the hydrolytic digestibility, fermentative capacity, and microbiota modulating properties of Temulose molasses, four hydrolyzed fractions of Temulose molasses, short-chain fructooligosaccharides (scFOS), and a yeast cell wall preparation (Safmannan). These substrates resisted in vitro hydrolytic digestion. Each substrate was fermented in vitro using dog fecal inoculum, and fermentation characteristics were quantified at 0 and 12 h. All Temulose molasses substrates decreased pH by at least 0.64 unit and resulted in greater (P < 0.05) butyrate and total short-chain fatty acid (SCFA) production compared to scFOS and Safmannan. Temulose molasses substrates resulted in higher (P < 0.01) or equal Bifidobacterium spp. concentrations compared to scFOS. Temulose molasses substrate and its fractions demonstrated prebiotic characteristics as indicated by low hydrolytic digestibility, high fermentability, and enhanced growth of microbiota considered to be beneficial to health.

A galactoglucomannan oligosaccharide (GGMO) obtained from fiberboard production was evaluated as a dietary supplement for dogs. The GGMO substrate contained increased concentrations of oligosaccharides containing mannose, xylose, and glucose, with the mannose component accounting for 35% of DM. Adult dogs assigned to a 6 × 6 Latin square design were fed 6 diets, each containing a different concentration of supplemental GGMO (0, 0.5, 1, 2, 4, and 8%) that replaced dietary cellulose. Total tract DM and OM apparent digestibilities increased (P < 0.001) linearly, whereas total tract CP apparent digestibility decreased (P < 0.001) linearly as dietary GGMO substrate concentration increased. Fecal concentrations of acetate, propionate, and total short-chain fatty acids increased (P ≤ 0.001) linearly, whereas butyrate concentration decreased (P ≤ 0.001) linearly with increasing dietary concentrations of GGMO. Fecal pH decreased (P ≤ 0.001) linearly as dietary GGMO substrate concentration increased, whereas fecal score increased quadratically (P ≤ 0.001). Fecal phenol (P ≤ 0.05) and indole (P ≤ 0.01) concentrations decreased linearly with GGMO supplementation. Fecal biogenic amine concentrations were not different among treatments except for phenylethylamine, which decreased (P < 0.001) linearly as dietary GGMO substrate concentration increased. Fecal microbial concentrations of Escherichia coli, Lactobacillus spp., and Clostridium perfringens were not different among treatments. A quadratic increase (P ≤ 0.01) was noted for Bifidobacterium spp. as dietary GGMO substrate concentration increased. The data suggest positive nutritional properties of supplemental GGMO when incorporated in a good-quality dog food.

Sophorolipids are carbohydrate-based, amphiphilic biosurfactants that are of increasing interest for use in environmentally benign cleaning agents. Sophorolipid production was tested for 26 strains representing 19 species of the Starmerella yeast clade, including Starmerella bombicola and Candida apicola, which were previously reported to produce sophorolipids. Five of the 19 species tested showed significant production of sophorolipids: S. bombicola, C. apicola, Candida riodocensis, Candida stellata and a new species, Candida sp. NRRL Y-27208. A high-throughput matrix-assisted laser desorption/ionization-time of flight MS assay was developed that showed S. bombicola and C. apicola to produce a lactone form of sophorolipid, whereas C. riodocensis, C. stellata and Candida sp. NRRL Y-27208 produced predominantly free acid sophorolipids. Phylogenetic analysis of sequences for the D1/D2 domains of the nuclear large subunit rRNA gene placed all sophorolipid-producing species in the S. bombicola subclade of the Starmerella clade.

Two actinomycete strains, NRRL B-24165(T) and NRRL B-24166(T), isolated from lesions on equine placentas in Kentucky, USA, were analysed using a polyphasic taxonomic approach. On the basis of phylogenetic analysis of 16S rRNA gene sequences, morphological observations and the presence of ll-diaminopimelic acid as the diagnostic diamino acid in whole-cell hydrolysates, the new isolates clearly belonged to the genus Streptomyces. Analyses of the phylogenetic positions of strains NRRL B-24165(T) and NRRL B-24166(T) based on 16S rRNA gene sequences of all recognized species of the genus Streptomyces, as well as evaluation of morphological and physiological characteristics, demonstrated that the new isolates could be differentiated from all recognized species and therefore represented novel species. It is proposed that the new strains represent two novel species for which the names Streptomyces atriruber sp. nov. (type strain NRRL B-24165(T)=DSM 41860(T)=LDDC 6330-99(T)) and Streptomyces silaceus sp. nov. (NRRL B-24166(T)=DSM 41861(T)=LDDC 6638-99(T)) are proposed. The species names are based on the distinctive colours of the substrate mycelium of these strains, dark red and deep orange-yellow, respectively.

The Bacillus subtilis species complex is a tight assemblage of closely related species. For many years, it has been recognized that these species cannot be differentiated on the basis of phenotypic characteristics. Recently, it has been shown that phylogenetic analysis of the 16S rRNA gene also fails to differentiate species within the complex due to the highly conserved nature of the gene, yet DNA-DNA hybridization values fall well below 70 % for the same species comparisons. As a complementary approach, we propose that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Indeed, our phylogenetic analyses revealed the existence of a previously unknown group of strains closely related to, but distinct from, Bacillus subtilis subsp. spizizenii. Results of matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the group produces a novel surfactin-like lipopeptide with mass m/z 1120.8 that is not produced by the other currently recognized subspecies. In addition, the group displayed differences in the total cellular content of the fatty acids C(16 : 0) and iso-C(17 : 1)omega10c that distinguish it from the closely related B. subtilis subsp. spizizenii. Consequently, the correlation of these novel phenotypic traits with the phylogenetic distinctiveness of this previously unknown subspecies group showed that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Therefore, we propose that this new group should be recognized as representing a novel taxon, Bacillus subtilis subsp. inaquosorum subsp. nov., with the type strain NRRL B-23052(T) (=KCTC 13429(T)=BGSC 3A28(T)).

Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp cluster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteomic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.

Skatole (3-methylindole) is a malodorous chemical in stored swine manure and is implicated as a component of foul-tasting pork. Definitive evidence for the skatole pathway is lacking. Deuterium-labeled substrates were employed to resolve this pathway in the acetogenic bacterium Clostridium drakei and Clostridium scatologenes and to determine if a similar pathway is used by microorganisms present in stored swine manure. Indoleacetic acid (IAA) was synthesized from tryptophan by both bacteria, and skatole was synthesized from both IAA and tryptophan. Microorganisms in swine manure produced skatole and other oxidation products from tryptophan, but IAA yielded only skatole. A catabolic mechanism for the synthesis of skatole is proposed.

Tunicamycins (TUN) are potent inhibitors of polyprenyl phosphate N-acetylhexosamine 1-phosphate transferases (PPHP), including essential eukaryotic GPT enzymes and bacterial HexNAc 1-P translocases. Hence, TUN blocks the formation of eukaryotic N-glycoproteins and the assembly of bacterial call wall polysaccharides. The genetic requirement for TUN production is well-established. Using two genes unique to the TUN pathway (tunB and tunD) as probes we identified four new prospective TUN-producing strains. Chemical analysis showed that one strain, Streptomyces niger NRRL B-3857, produces TUN plus new compounds, named quinovosamycins (QVMs). QVMs are structurally akin to TUN, but uniquely in the 1″,11'-HexNAc sugar head group, which is invariably d-GlcNAc for the known TUN, but is d-QuiNAc for the QVM. Surprisingly, this modification has only a minor effect on either the inhibitory or antimicrobial properties of QVM and TUN. These findings have unexpected consequences for TUN/QVM biosynthesis, and for the specificity of the PPHP enzyme family.

To compare production of antibacterial liamocins (polyol lipids) by diverse strains of Aureobasidium pullulans grown on different culture media. Liamocins produced by strains of A. pullulans have potential agricultural and pharmaceutical applications as antibacterials with specificity against Streptococcus spp. Six strains of A. pullulans were characterized for liamocin production on four different culture media. The choice of strain and culture medium affected growth, liamocin yields, and production of contaminating pigments. Best growth and highest liamocin yields were obtained using A. pullulans strain NRRL 50384 grown on a sucrose basal medium. Unexpectedly, the choice of strain and culture medium also affected the structure of liamocins produced, providing novel types of liamocins. Liamocins varied not only in the ratios of trimer and tetramer polyester tail groups, but also in the nature of the polyol headgroup, which could include mannitol, arabitol, or glycerol. The ability to conveniently produce novel types of liamocins in good yields will provide novel antibacterials for applied uses, and facilitate structure-function studies on the mechanism of antibacterial activity.

Schizophyllum commune strain ATCC 38548 grew well on a medium containing alkaline H2O2 -pretreated corn fiber as a sole carbon source, and clarified the culture medium within 7 days. The strain preferentially utilized the starch component of corn fiber for growth and production of schizophyllan. Culture supernatants contained approx. 50 mg schizophyllan and 200 mg arabinoxylan per g corn fiber. These polysaccharides were recovered separately by differential precipitation with ethanol.

Tunicamycins are potent inhibitors of UDP-N-acetyl-D-hexosamine:polyprenol-phosphate N-acetylhexosamine-1-phosphate translocases (D-HexNAc-1-P translocases), a family of enzymes involved in bacterial cell wall synthesis and eukaryotic protein N-glycosylation. Structurally, tunicamycins consist of an 11-carbon dialdose core sugar called tunicamine that is N-linked at C-1' to uracil and O-linked at C-11' to N-acetylglucosamine (GlcNAc). The C-11' O-glycosidic linkage is highly unusual because it forms an alpha/beta anomeric-to-anomeric linkage to the 1-position of the GlcNAc residue. We have assigned the (1)H and (13)C NMR spectra of tunicamycin and have undertaken a conformational analysis from rotating angle nuclear Overhauser effect (ROESY) data. In addition, chirally deuterated tunicamycins produced by fermentation of Streptomyces chartreusis on chemically synthesized, monodeuterated (S-6)-[(2)H(1)]glucose have been used to assign the geminal H-6'a, H-6'b methylene bridge of the 11-carbon dialdose sugar, tunicamine. The tunicamine residue is shown to assume pseudo-D-ribofuranose and (4)C(1) pseudo-D-galactopyranosaminyl ring conformers. Conformation about the C-6' methylene bridge determines the relative orientation of these rings. The model predicts that tunicamycin forms a right-handed cupped structure, with the potential for divalent metal ion coordination at 5'-OH, 8'-OH, and the pseudogalactopyranosyl 7'-O ring oxygen. The formation of tunicamycin complexes with various divalent metal ions was confirmed experimentally by MALDI-TOF mass spectrometry. Our data support the hypothesis that tunicamycin is a structural analogue of the UDP-D-HexNAc substrate and is reversibly coordinated to the divalent metal cofactor in the D-HexNAc-1-P translocase active site.