Horizontal gene transfer and evolution of antibiotic gene clusters

Genome sequencing projects from diverse bacteria and the release of genetically engineered microorganisms make the topic of horizontal gene transfer (HGT) fashionable. Sequence analysis indicates the high frequency and the importance of horizontal gene transfer to the microbial evolution. Much knowledge about horizontal transfer has been derived from studies using soil microcosms, fluorescent-marked donors and recipients and from study of the increase of the antibiotic resistance as a result of use and often abuse of antibiotics. However, little is known about the transfer of chromosomally-located antibiotic biosynthesis genes in natural populations. The streptomycin pathway-specific regulator, strR, has been found in a set of diverse streptomycetes both phenotypically and genetically that previously have already been described to carry the resistance (strA) and one biosynthetic (strBl) gene from the same cluster. Phylogenetic analysis of both 16S rRNA and trpB gene fragments showed that two isolates were closely related to S. coelicolor which are known not to produce streptomycin or to contain any biosynthesis or resistance genes. The remaining pair did appear to be closely related to each other, particularly from the trpB analysis. trpB gene proved to be useful for resolving phylogenetic relationships between strains with highly conserved gamma region of the 16S rRNA gene. However, one of these strains is the only one from the S. griseus distant isolates that produced streptomycin and possessed many other genes from the cluster. The phylogenetic incongruency between `species' tree and `gene' tree can be attributed to horizontal gene transfer. The sequence identity of the detected genes is extremely high (99%) indicating a recent transfer event. Besides the physical proximity of strRABl genes, there is also a well-characterised functional correlation of these genes in the streptomycin producer, S. griseus. However, strRA genes are silent in coelicolor-like isolates ASSF15 and ASB37. Therefore, this transfer may have other ecological role than a simple resistance phenotype such as the evolution of antibiotic cluster. It was not possible to prove the integration site of strRABl genes within ASB37 genome but a possible site was identified. Pathway-specific regulator StrR activates the transcription of strBl and other genes from the streptomycin cluster by binding to the upstream promoter regions. This protein binds as a tetramer. N-terminal deleted StrR derivatives were still able to bind as pseudodimers (two monomers). Two putative domains in the N'-terminus of the protein responsible for the protein dimerisation during the binding process have been identified

The frontline antibiotic vancomycin induces a zinc starvation response in bacteria by binding to Zn(II).

Vancomycin is a front-line antibiotic used for the treatment of nosocomial infections, particularly those caused by methicillin-resistant Staphylococcus aureus. Despite its clinical importance the global effects of vancomycin exposure on bacterial physiology are poorly understood. In a previous transcriptomic analysis we identified a number of Zur regulon genes which were highly but transiently up-regulated by vancomycin in Streptomyces coelicolor. Here, we show that vancomycin also induces similar zinc homeostasis systems in a range of other bacteria and demonstrate that vancomycin binds to Zn(II) in vitro. This implies that vancomycin treatment sequesters zinc from bacterial cells thereby triggering a Zur-dependent zinc starvation response. The Kd value of the binding between vancomycin and Zn(II) was calculated using a novel fluorometric assay, and NMR was used to identify the binding site. These findings highlight a new biologically relevant aspect of the chemical property of vancomycin as a zinc chelator.This work was supported by funding from the Royal Society, UK (516002.K5877/ROG), the Medical Research Council, UK (G0700141). A.Z. was supported from the Said foundation and Cambridge Trust.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep1960

Extracting regulator activity profiles by integration of de novo motifs and expression data: characterizing key regulators of nutrient depletion responses in Streptomyces coelicolor

Determining transcriptional regulator activities is a major focus of systems biology, providing key insight into regulatory mechanisms and co-regulators. For organisms such as Escherichia coli, transcriptional regulator binding site data can be integrated with expression data to infer transcriptional regulator activities. However, for most organisms there is only sparse data on their transcriptional regulators, while their associated binding motifs are largely unknown. Here, we address the challenge of inferring activities of unknown regulators by generating de novo (binding) motifs and integrating with expression data. We identify a number of key regulators active in the metabolic switch, including PhoP with its associated directed repeat PHO box, candidate motifs for two SARPs, a CRP family regulator, an iron response regulator and that for LexA. Experimental validation for some of our predictions was obtained using gel-shift assays. Our analysis is applicable to any organism for which there is a reasonable amount of complementary expression data and for which motifs (either over represented or evolutionary conserved) can be identified in the genome

Determinants of redox sensitivity in RsrA, a zinc-containing anti-sigma factor for regulating thiol oxidative stress response

Various environmental oxidative stresses are sensed by redox-sensitive regulators through cysteine thiol oxidation or modification. A few zinc-containing anti-sigma (ZAS) factors in actinomycetes have been reported to respond sensitively to thiol oxidation, among which RsrA from Streptomyces coelicolor is best characterized. It forms disulfide bonds upon oxidation and releases bound SigR to activate thiol oxidative stress response genes. Even though numerous ZAS proteins exist in bacteria, features that confer redox sensitivity to a subset of these have been uncharacterized. In this study, we identified seven additional redox-sensitive ZAS factors from actinomycetes. Comparison with redox-insensitive ZAS revealed characteristic sequence patterns. Domain swapping demonstrated the significance of the region K33FEHH37FEEC41SPC44LEK47 that encompass the conserved HX3CX2C (HCC) motif. Mutational effect of each residue on diamide responsive induction of SigR target genes in vivo demonstrated that several residues, especially those that flank two cysteines (E39, E40, L45, E46), contribute to redox sensitivity. These residues are well conserved among redox-sensitive ZAS factors, and hence are proposed as redox-determinants in sensitive ZAS. H37A, C41A, C44A and F38A mutations, in contrast, compromised SigR-binding activity significantly, apparently affecting structural integrity of RsrA. The residue pattern around HCC motif could therefore serve as an indicator to predict redox-sensitive ZAS factors from sequence information

Structural analysis of cytochrome P450 105N1 involved in the biosynthesis of the zincophore, coelibactin

Coelibactin is a putative non-ribosomally synthesized peptide with predicted zincophore activity and which has been implicated in antibiotic regulation in Streptomyces coelicolor A3(2). The coelibactin biosynthetic pathway contains a stereo- and regio-specific monooxygenation step catalyzed by a cytochrome P450 enzyme (CYP105N1). We have determined the X-ray crystal structure of CYP105N1 at 2.9 Å and analyzed it in the context of the bacterial CYP105 family as a whole. The crystal structure reveals a channel between the α-helical domain and the β-sheet domain exposing the heme pocket and the long helix I to the solvent. This wide-open conformation of CYP105N1 may be related to the bulky substrate coelibactin. The ligand-free CYP105N1 structure has enough room in the substrate access channel to allow the coelibactin to enter into the active site. Analysis of typical siderophore ligands suggests that CYP105N1 may produce derivatives of coelibactin, which would then be able to chelate the zinc divalent cation

CYP105-diverse structures, functions and roles in an intriguing family of enzymes in 'Streptomyces'

The cytochromes P450 (CYP or P450) are a large superfamily of haem-containing enzymes found in all domains of life. They catalyse a variety of complex reactions, predominantly mixed-function oxidations, often displaying highly regio- and/or stereospecific chemistry. In streptomycetes, they are predominantly associated with secondary metabolite biosynthetic pathways or with xenobiotic catabolism. Homologues of one family, CYP105, have been found in all Streptomyces species thus far sequenced. This review looks at the diverse biological functions of CYP105s and the biosynthetic/catabolic pathways they are associated with. Examples are presented showing a range of biotransformative abilities and different contexts. As biocatalysts capable of some remarkable chemistry, CYP105s have great biotechnological potential and merit detailed study. Recent developments in biotechnological applications which utilize CYP105s are described, alongside a brief overview of the benefits and drawbacks of using P450s in commercial applications. The role of CYP105s in vivo is in many cases undefined and provides a rich source for further investigation into the functions these enzymes fulfil and the metabolic pathways they participate in, in the natural environment

Horizontal gene transfer and evolution of antibiotic gene clusters

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