Many bioactive natural products are glycosylated compounds in which the sugar components usually participate in interaction and molecular recognition of the cellular target. Therefore, the presence of sugar moieties is important, in some cases essential, for bioactivity. Searching for novel glycosylated bioactive compounds is an important aim in the field of the research for natural products from actinomycetes. A great majority of these sugar moieties belong to the 6-deoxyhexoses and share two common biosynthetic steps catalyzed by a NDP-D-glucose synthase (GS) and a NDP-D-glucose 4,6-dehydratase (DH). Based on this fact, seventy one Streptomyces strains isolated from the integument of ants of the Tribe Attini were screened for the presence of biosynthetic gene clusters (BGCs) for glycosylated compounds. Total DNAs were analyzed by PCR amplification using oligo primers for GSs and DHs and also for a NDP-D-glucose-2,3-dehydratases. Amplicons were used in gene disruption experiments to generate non-producing mutants in the corresponding clusters. Eleven mutants were obtained and comparative dereplication analyses between the wild type strains and the corresponding mutants allowed in some cases the identification of the compound coded by the corresponding cluster (lobophorins, vicenistatin, chromomycins and benzanthrins) and that of two novel macrolactams (sipanmycin A and B). Several strains did not show UPLC differential peaks between the wild type strain and mutant profiles. However, after genome sequencing of these strains, the activation of the expression of two clusters was achieved by using nutritional and genetic approaches leading to the identification of compounds of the cervimycins family and two novel members of the warkmycins family. Our work defines a useful strategy for the identification new glycosylated compounds by a combination of genome mining, gene inactivation experiments and the activation of silent biosynthetic clusters in Streptomyces strains.

Plasmids, when transferred by conjugation in natural environments, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida. Expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. ardC was also required if the hsdRMS system was present in E. coli recipient cells. Thus, ArdC has antirestriction activity against the HsdRMS system and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and presence of Mn 2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity was not needed for the antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggered an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies and settle into new hosts.

Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix–turn–helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies.

Plasmids, when transferred by conjugation, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida, but not from E. coli to E. coli. Surprisingly, expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. Thus, ArdC has antirestriction activity against HsdRMS system, and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and in the presence of Mn 2+ . ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity is not needed for antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggers an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies. ResultsardC is required for R388 conjugation from E. coli to P. putida R388 plasmid is composed by three functional sectors ( Fig EV1). One for general maintenance (modules of replication, stable inheritance and establishment) located in the leading region, a sector for Ab R and integration and a third one for conjugation (modules of DNA transfer replication and mating pore formation) [11]. We expected the stable inheritance and establishment region to be required in interspecies conjugation. pSU2007, a Kn R R388 derivative, is transferred with different efficiencies from E. coli BW27783-Nx R to other bacteria ( Fig EV2). All conjugation experiments were done at the recipient cells optimal growing temperature except for P. putida KT2440 to which we observed that the conjugation was more effective at 37 °C. The transfer of pIC10 (R388∆kfrA-orf14), an R388 derivative without the stability and maintenance region, is similar to pSU2007 in all species but in P. putida KT2440 where the conjugation frequencies dropped around 1000 times. We have performed the conjugation experiments at both the optimal E. coli growing temperature (37 °C) or P. putida growing temperature (30 °C). In the stability and maintenance gene region deleted in pIC10 there are 13 genes that code for proteins homologous to some with predicted function of: fertility inhibition in a different system (kfrA, nuc1, nuc2 and osa) [12], hypothetical proteins of unknown function (orf7, orf8, orf9, orf12 and orf14), transcriptional regulators (ardk and klcB), ssDNA binding

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