Establishment of apiary-level risk of American foulbrood through the detection of Paenibacillus larvae spores in pooled, extracted honey in Saskatchewan.

Paenibacillus larvae, the causative agent of American foulbrood (AFB), produces spores that may be detectable within honey. We analyzed the spore content of pooled, extracted honey from 52 large-scale (L) and 64 small-scale (S) Saskatchewan beekeepers over a two-year period (2019-2020). Our objectives were: (i) establish reliable prognostic reference ranges for spore concentrations in extracted honey to determine future AFB risk at the apiary level; (ii) identify management practices as targets for mitigation of risk. P. larvae spores were detected in 753 of 1476 samples (51%). Beekeepers were stratified into low (< 2 spores/gram), moderate (2- < 100 spores/gram), and high (≥ 100 spores/gram) risk categories. Of forty-nine L beekeepers sampled in 2019, those that reported AFB in 2020 included 0/26 low, 3/18 moderate, and 3/5 high risk. Of twenty-seven L beekeepers sampled in 2020, those that reported AFB in 2021 included 0/11 low, 2/14 moderate, and 1/2 high risk. Predictive modelling included indoor overwintering of hives, purchase of used equipment, movement of honey-producing colonies between apiaries, beekeeper demographic, and antimicrobial use as risk category predictors. Saskatchewan beekeepers with fewer than 2 spores/gram in extracted honey that avoid high risk activities may be considered at low risk of AFB the following year.

Comparison of individual hive and apiary-level sample types for spores of Paenibacillus larvae in Saskatchewan honey bee operations.

Three commercial honey bee operations in Saskatchewan, Canada, with outbreaks of American foulbrood (AFB) and recent or ongoing metaphylactic antibiotic use were intensively sampled to detect spores of Paenibacillus larvae during the summer of 2019. Here, we compared spore concentrations in different sample types within individual hives, assessed the surrogacy potential of honey collected from honey supers in place of brood chamber honey or adult bees within hives, and evaluated the ability of pooled, extracted honey to predict the degree of spore contamination identified through individual hive testing. Samples of honey and bees from hives within apiaries with a recent, confirmed case of AFB in a single hive (index apiaries) and apiaries without clinical evidence of AFB (unaffected apiaries), as well as pooled, apiary-level honey samples from end-of-season extraction, were collected and cultured to detect and enumerate spores. Only a few hives were heavily contaminated by spores in any given apiary. All operations were different from one another with regard to both the overall degree of spore contamination across apiaries and the distribution of spores between index apiaries and unaffected apiaries. Within operations, individual hive spore concentrations in unaffected apiaries were significantly different from index apiaries in the brood chamber (BC) honey, honey super (HS) honey, and BC bees of one of three operations. Across all operations, BC honey was best for discriminating index apiaries from unaffected apiaries (p = 0.001), followed by HS honey (p = 0.06), and BC bees (p = 0.398). HS honey positively correlated with both BC honey (rs = 0.76, p < 0.0001) and bees (rs = 0.50, p < 0.0001) and may be useful as a surrogate for either. Spore concentrations in pooled, extracted honey seem to have predictive potential for overall spore contamination within each operation and may have prognostic value in assessing the risk of future AFB outbreaks at the apiary (or operation) level.

Investigation of Macrolide Resistance Genotypes in Mycoplasma bovis Isolates from Canadian Feedlot Cattle.

Mycoplasma bovis is associated with bovine respiratory disease (BRD) and chronic pneumonia and polyarthritis syndrome (CPPS) in feedlot cattle. No efficacious vaccines for M. bovis exist; hence, macrolides are commonly used to control mycoplasmosis. Whole genome sequences of 126 M. bovis isolates, derived from 96 feedlot cattle over 12 production years, were determined. Antimicrobial susceptibility testing (AST) of five macrolides (gamithromycin, tildipirosin, tilmicosin, tulathromycin, tylosin) was conducted using a microbroth dilution method. The AST phenotypes were compared to the genotypes generated for 23S rRNA and the L4 and L22 ribosomal proteins. Mutations in domains II (nucleotide 748; E. coli numbering) and V (nucleotide 2059 and 2060) of the 23S rRNA (rrl) gene alleles were associated with resistance. All isolates with a single mutation at Δ748 were susceptible to tulathromycin, but resistant to tilmicosin and tildipirosin. Isolates with mutations in both domain II and V (Δ748Δ2059 or Δ748Δ2060) were resistant to all five macrolides. However, >99% of isolates were resistant to tildipirosin and tilmicosin, regardless of the number and positions of the mutations. Isolates with a Δ748 mutation in the 23S rRNA gene and mutations in L4 and L22 were resistant to all macrolides except for tulathromycin.

Metabolites Involved in Immune Evasion by Batrachochytrium dendrobatidis Include the Polyamine Spermidine.

Amphibians have been declining around the world for more than four decades. One recognized driver of these declines is the chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Amphibians have complex and varied immune defenses against B. dendrobatidis, but the fungus also has a number of counterdefenses. Previously, we identified two small molecules produced by the fungus that inhibit frog lymphocyte proliferation, methylthioadenosine (MTA) and kynurenine (KYN). Here, we report on the isolation and identification of the polyamine spermidine (SPD) as another significant immunomodulatory molecule produced by B. dendrobatidis SPD and its precursor, putrescine (PUT), are the major polyamines detected, and SPD is required for growth. The major pathway of biosynthesis is from ornithine through putrescine to spermidine. An alternative pathway from arginine to agmatine to putrescine appears to be absent. SPD is inhibitory at concentrations of ≥10 µM and is found at concentrations between 1 and 10 µM in active fungal supernatants. Although PUT is detected in the fungal supernatants, it is not inhibitory to lymphocytes even at concentrations as high as 100 µM. Two other related polyamines, norspermidine (NSP) and spermine (SPM), also inhibit amphibian lymphocyte proliferation, but a third polyamine, cadaverine (CAD), does not. A suboptimal (noninhibitory) concentration of MTA (10 µM), a by-product of spermidine synthesis, enhances the inhibition of SPD at 1 and 10 µM. We interpret these results to suggest that B. dendrobatidis produces an "armamentarium" of small molecules that, alone or in concert, may help it to evade clearance by the amphibian immune system.

Pyrazines from bacteria and ants: convergent chemistry within an ecological niche.

Ants use pheromones to coordinate their communal activity. Volatile pyrazines, for instance, mediate food resource gathering and alarm behaviors in different ant species. Here we report that leaf-cutter ant-associated bacteria produce a family of pyrazines that includes members previously identified as ant trail and alarm pheromones. We found that L-threonine induces the bacterial production of the trail pheromone pyrazines, which are common for the host leaf-cutter ants. Isotope feeding experiments revealed that L-threonine along with sodium acetate were the biosynthetic precursors of these natural products and a biosynthetic pathway was proposed.

Tryptorubin A: A Polycyclic Peptide from a Fungus-Derived Streptomycete.

Fungus-growing ants engage in complex symbiotic relationships with their fungal crop, specialized fungal pathogens, and bacteria that provide chemical defenses. In an effort to understand the evolutionary origins of this multilateral system, we investigated bacteria isolated from fungi. One bacterial strain (Streptomyces sp. CLI2509) from the bracket fungus Hymenochaete rubiginosa, produced an unusual peptide, tryptorubin A, which contains heteroaromatic links between side chains that give it a rigid polycyclic globular structure. The three-dimensional structure was determined by NMR and MS, including a 13C-13C COSY of isotopically enriched material, degradation, derivatives, and computer modeling. Whole genome sequencing identified a likely pair of biosynthetic genes responsible for tryptorubin A's linear hexapeptide backbone. The genome also revealed the close relationship between CLI2509 and Streptomyces sp. SPB78, which was previously implicated in an insect-bacterium symbiosis.

Linear Peptides Are the Major Products of a Biosynthetic Pathway That Encodes for Cyclic Depsipeptides.

Three new dentigerumycin analogues are produced by Streptomyces sp. M41, a bacterium isolated from a South African termite, Macrotermes natalensis. The structures of the complex nonribosomal peptide synthetase-polyketide synthase (NRPS/PKS) hybrid compounds were determined by 1D- and 2D-NMR spectroscopy, high-resolution mass spectrometry, and circular dichroism (CD) spectroscopy. Both cyclic and linear peptides are reported, and the genetic organization of the NRPS modules within the biosynthetic gene cluster accounts for the observed structural diversity.

Selvamicin, an atypical antifungal polyene from two alternative genomic contexts.

The bacteria harbored by fungus-growing ants produce a variety of small molecules that help maintain a complex multilateral symbiosis. In a survey of antifungal compounds from these bacteria, we discovered selvamicin, an unusual antifungal polyene macrolide, in bacterial isolates from two neighboring ant nests. Selvamicin resembles the clinically important antifungals nystatin A1 and amphotericin B, but it has several distinctive structural features: a noncationic 6-deoxymannose sugar at the canonical glycosylation site and a second sugar, an unusual 4-O-methyldigitoxose, at the opposite end of selvamicin's shortened polyene macrolide. It also lacks some of the pharmacokinetic liabilities of the clinical agents and appears to have a different target. Whole genome sequencing revealed the putative type I polyketide gene cluster responsible for selvamicin's biosynthesis including a subcluster of genes consistent with selvamicin's 4-O-methyldigitoxose sugar. Although the selvamicin biosynthetic cluster is virtually identical in both bacterial producers, in one it is on the chromosome, in the other it is on a plasmid. These alternative genomic contexts illustrate the biosynthetic gene cluster mobility that underlies the diversity and distribution of chemical defenses by the specialized bacteria in this multilateral symbiosis.

Gene Flow and Molecular Innovation in Bacteria.

The small molecules produced by environmental bacteria have been mainstays of both chemical and biological research for decades, and some have led to important therapeutic interventions. These small molecules have been shaped by natural selection as they evolved to fulfill changing functional roles in their native environments. This minireview describes some recent systematic studies providing illustrative examples that involve the acquisition and alteration of genetic information for molecular innovation by bacteria in well-defined environments. Two different bacterial genera are featured, Pseudonocardia and Salinispora, and, although the small-molecule repertoires of both have benefited from horizontal gene transfer, Pseudonocardia spp. have relied on plasmid-based tactics while Salinispora spp. have relied on chromosomally integrated genomic islands.

Genome Sequence of Streptomyces sp. Strain RTd22, an Endophyte of the Mexican Sunflower.

We report here the complete genome sequence of Streptomyces sp. strain RTd22, an endophytic actinobacterium that was isolated from the roots of the Mexican sunflower Tithonia diversifolia The bacterium's 11.1-Mb linear chromosome is predicted to encode a large number of unknown natural products.

Whole-Genome Sequence of Bacillus sp. SDLI1, Isolated from the Social Bee Scaptotrigona depilis.

We announce the complete genome sequence ofBacillussp. strain SDLI1, isolated from larval gut of the stingless beeScaptotrigona depilis The 4.13-Mb circular chromosome harbors biosynthetic gene clusters for the production of antimicrobial compounds.

A Rebeccamycin Analog Provides Plasmid-Encoded Niche Defense.

Bacterial symbionts of fungus-growing ants occupy a highly specialized ecological niche and face the constant existential threat of displacement by another strain of ant-adapted bacteria. As part of a systematic study of the small molecules underlying this fraternal competition, we discovered an analog of the antitumor agent rebeccamycin, a member of the increasingly important indolocarbazole family. While several gene clusters consistent with this molecule's newly reported modification had previously been identified in metagenomic studies, the metabolite itself has been cryptic. The biosynthetic gene cluster for 9-methoxyrebeccamycin is encoded on a plasmid in a manner reminiscent of plasmid-derived peptide antimicrobials that commonly mediate antagonism among closely related Gram-negative bacteria.

Variable genetic architectures produce virtually identical molecules in bacterial symbionts of fungus-growing ants.

Small molecules produced by Actinobacteria have played a prominent role in both drug discovery and organic chemistry. As part of a larger study of the actinobacterial symbionts of fungus-growing ants, we discovered a small family of three previously unreported piperazic acid-containing cyclic depsipeptides, gerumycins A-C. The gerumycins are slightly smaller versions of dentigerumycin, a cyclic depsipeptide that selectively inhibits a common fungal pathogen, Escovopsis. We had previously identified this molecule from a Pseudonocardia associated with Apterostigma dentigerum, and now we report the molecule from an associate of the more highly derived ant Trachymyrmex cornetzi. The three previously unidentified compounds, gerumycins A-C, have essentially identical structures and were produced by two different symbiotic Pseudonocardia spp. from ants in the genus Apterostigma found in both Panama and Costa Rica. To understand the similarities and differences in the biosynthetic pathways that produced these closely related molecules, the genomes of the three producing Pseudonocardia were sequenced and the biosynthetic gene clusters identified. This analysis revealed that dramatically different biosynthetic architectures, including genomic islands, a plasmid, and the use of spatially separated genetic loci, can lead to molecules with virtually identical core structures. A plausible evolutionary model that unifies these disparate architectures is presented.

A P450 fusion library of heme domains from Rhodococcus jostii RHA1 and its evaluation for the biotransformation of drug molecules.

The actinomycete Rhodococcus jostii RHA1 contains a multitude of oxygenase enzymes, consonant with its remarkable activities in the catabolism of hydrophobic xenobiotic compounds. In the interests of identifying activities for the transformation of drug molecules, we have cloned genes encoding 23 cytochrome P450 heme domains from R. jostii and expressed them as fusions with the P450 reductase domain (RhfRED) of cytochrome P450Rhf from Rhodococcus sp. NCIMB 9784. Fifteen of the fusions were expressed in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Strains expressing the fusions of RhfRED with genes ro02604, ro04667, ro11069, ro11320, ro11277, ro08984 and ro04671 were challenged with 48 commercially available drugs revealing many different activities commensurate with P450-catalyzed hydroxylation and demethylation reactions. One recombinant strain, expressing the fusion of P450 gene ro11069 (CYP257A1) with RhfRED, and named Ro07-RhfRED, catalyzed the N-demethylation of diltiazem and imipramine. This observation was in accord with previous reports of this enzyme's activity as a demethylase of alkaloid substrates. Ro07-RhfRED was purified and characterised, and applied in cell-free biotransformations of imipramine (7 µM) giving a 63% conversion to the N-desmethyl product.

A substrate-assisted mechanism of nucleophile activation in a Ser-His-Asp containing C-C bond hydrolase.

The meta-cleavage product (MCP) hydrolases utilize a Ser-His-Asp triad to hydrolyze a carbon-carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ES(red), which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ES(red) decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2 (ßnuc ∼ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His-Asp pair does not play an essential role. The data further suggest that ES(red) represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.

The lid domain of the MCP hydrolase DxnB2 contributes to the reactivity toward recalcitrant PCB metabolites.

DxnB2 and BphD are meta-cleavage product (MCP) hydrolases that catalyze C-C bond hydrolysis of the biphenyl metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). BphD is a bottleneck in the bacterial degradation of polychlorinated biphenyls (PCBs) by the Bph catabolic pathway due in part to inhibition by 3-Cl HOPDAs. By contrast, DxnB2 from Sphingomonas wittichii RW1 catalyzes the hydrolysis of 3-Cl HOPDAs more efficiently. X-ray crystallographic studies of the catalytically inactive S105A variant of DxnB2 complexed with 3-Cl HOPDA revealed a binding mode in which C1 through C6 of the dienoate are coplanar. The chlorine substituent is accommodated by a hydrophobic pocket that is larger than the homologous site in BphDLB400 from Burkholderia xenovorans LB400. The planar binding mode observed in the crystalline complex was consistent with the hyper- and hypsochromically shifted absorption spectra of 3-Cl and 3,9,11-triCl HOPDA, respectively, bound to S105A in solution. Moreover, ES(red), an intermediate possessing a bathochromically shifted spectrum observed in the turnover of HOPDA, was not detected, suggesting that substrate destabilization was rate-limiting in the turnover of these PCB metabolites. Interestingly, electron density for the first α-helix of the lid domain was poorly defined in the dimeric DxnB2 structures, unlike in the tetrameric BphDLB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the α/ß-hydrolase core domain, as a determinant in the oligomeric state and suggests its involvement in catalysis. Finally, an increased mobility of the DxnB2 lid may contribute to the enzyme's ability to hydrolyze PCB metabolites, highlighting how lid architecture contributes to substrate specificity in α/ß-hydrolases.

The catalytic serine of meta-cleavage product hydrolases is activated differently for C-O bond cleavage than for C-C bond cleavage.

meta-Cleavage product (MCP) hydrolases catalyze C-C bond fission in the aerobic catabolism of aromatic compounds by bacteria. These enzymes utilize a Ser-His-Asp triad to catalyze hydrolysis via an acyl-enzyme intermediate. BphD, which catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) in biphenyl degradation, catalyzed the hydrolysis of an ester analogue, p-nitrophenyl benzoate (pNPB), with a k(cat) value (6.3 ± 0.5 s(-1)) similar to that of HOPDA (6.5 ± 0.5 s(-1)). Consistent with the breakdown of a shared intermediate, product analyses revealed that BphD catalyzed the methanolysis of both HOPDA and pNPB, partitioning the products to benzoic acid and methyl benzoate in similar ratios. Turnover of HOPDA was accelerated up to 4-fold in the presence of short, primary alcohols (methanol > ethanol > n-propanol), suggesting that deacylation is rate-limiting during catalysis. In the steady-state hydrolysis of HOPDA, k(cat)/K(m) values were independent of methanol concentration, while both k(cat) and K(m) values increased with methanol concentration. This result was consistent with a simple model of nucleophilic catalysis. Although the enzyme could not be saturated with pNPB at methanol concentrations of >250 mM, k(obs) values from the steady-state turnover of pNPB at low methanol concentrations were also consistent with a nucleophilic mechanism of catalysis. Finally, transient-state kinetic analysis of pNPB hydrolysis by BphD variants established that substitution of the catalytic His reduced the rate of acylation by more than 3 orders of magnitude. This suggests that for pNPB hydrolysis, the serine nucleophile is activated by the His-Asp dyad. In contrast, rapid acylation of the H265Q variant during C-C bond cleavage suggests that the serinate forms via a substrate-assisted mechanism. Overall, the data indicate that ester hydrolysis proceeds via the same acyl-enzyme intermediate as that of the physiological substrate but that the serine nucleophile is activated via a different mechanism.

Identification of an acyl-enzyme intermediate in a meta-cleavage product hydrolase reveals the versatility of the catalytic triad.

Meta-cleavage product (MCP) hydrolases are members of the α/ß-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C-C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6 Å resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme's catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of 'oxyanion hole' residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of (18)O into the benzoate produced during hydrolysis in H(2)(18)O. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over 5 orders of magnitude. The orange-colored catalytic intermediate, ES(red), previously detected in the wild-type enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q. In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophilic attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad.