The presence of tetracycline resistance (Tc(r)) genes and class I integrons (in-1), and their ability to cotransfer were investigated in Tc(r) gram-negative (185 strains) and gram-positive (72 strains) bacteria from Danish farmland and pigsties. The isolates belonged to the groups or species Escherichia coli, Enterobacter spp., Arthrobacter spp., Alcaligenes spp., Pseudomonas spp., and Corynebacterium glutamicum. The 257 isolates were screened for in-1. Eighty-one of the gram-negative isolates were also screened for the Tc(r) genes tet(A), tet(B), and tet(C), and all (n = 72) gram-positive isolates were screened for tet(33). Fourteen (7%) of the soil isolates and eleven (25%) of the pigsty isolates contained in-1. All isolates that contained tet genes also contained in-1, except one gram-negative isolate from a pigsty that contained tet(B). All gram-positive isolates with in-1 also contained tet(33). No isolates contained more than one tet gene. The in-1-positive isolates were tested for resistance to selected antimicrobial agents and showed resistance to three to nine drugs. Filter-mating experiments showed cotransfer of Tc(r) and class I integrons from soil isolates to Escherichia coli and/or Pseudomonas putida. We conclude that soil bacteria in close contact to manure or pigsty environment may thus have an important role in horizontal spread of resistance. Use of tetracyclines in food animal production may increase not only Tc(r) but also multidrug resistance (caused by the presence tet genes and in-1) in bacteria.

Glycoside hydrolase family GH85 is a family of endo-beta-N-acetylglucosaminidases that is responsible for the hydrolysis of beta-1,4 linkage in the N,N-diacetylchitobiose core of N-linked glycans. The endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) is of particular interest, given its increasing use for the chemoenzymatic synthesis of bespoke N-glycans using N-glycan oxazolines as glycosyl donors. The E173Q variant of Endo-A is especially attractive for synthesis, as it is hydrolytically impaired but still able to catalyze N-glycan synthesis by transglycosylation using activated oxazoline donors. Here we present the three-dimensional structure of the A. protophormiae Endo-A E173Q variant, solved by multiple-wavelength anomalous scattering methods and refined at 1.8 A resolution. The structure reveals that GH85 enzymes display a trimodular architecture in which a (beta/alpha)(8) catalytic domain occurs with two ancillary beta-sheet modules. The active centre is fully consistent with the known neighboring-group catalytic mechanism in which E173 acts as the catalytic acid/base for reaction via an oxazoline intermediate. Of note is the presence of an asparagine in the active centre, in a position likely to interact with the acetyl NH group that, in all other known families of glycosidase using this mechanism, is an aspartate or glutamate residue. The substrate-binding surface reveals an open topography, consistent with the ability to accept a large range of glycoprotein substrates and the ability to transglycosylate other acceptors. The three-dimensional structure of this important biocatalyst reveals that residues implicated in the enhancement of transglycosylation and synthetic capacity are proximal to the active centre, where they may act to favor binding of acceptor substrates.

Endo-beta-N-acetylglucosaminidases (ENGases) are dual specificity enzymes with an ability to catalyze hydrolysis and transglycosylation reactions. Recently, these enzymes have become the focus of intense research because of their potential for synthesis of glycopeptides. We have determined the 3D structures of an ENGase from Arthrobacter protophormiae (Endo-A) in 3 forms, one in native form, one in complex with Man(3)GlcNAc-thiazoline and another in complex with GlcNAc-Asn. The carbohydrate moiety sits above the TIM-barrel in a cleft region surrounded by aromatic residues. The conserved essential catalytic residues - E173, N171 and Y205 are within hydrogen bonding distance of the substrate. W216 and W244 regulate access to the active site during transglycosylation by serving as "gate-keepers". Interestingly, Y299F mutation resulted in a 3 fold increase in the transglycosylation activity. The structure provides insights into the catalytic mechanism of GH85 family of glycoside hydrolases at molecular level and could assist rational engineering of ENGases.