Excisable Cassettes: New Tools for Functional Analysis of Streptomyces Genomes

Abstract

The functional analysis of microbial genomes often requires gene inactivation. We constructed a set of cassettes consisting of single antibiotic resistance genes flanked by the attL and attR sites resulting from site-specific integration of the Streptomyces pSAM2 element. These cassettes can easily be used to inactivate genes by in-frame deletion in Streptomyces by a three-step strategy. In the first step, in Escherichia coli, the cassette is inserted into a cloned copy of the gene to be inactivated. In the second step, the gene is replaced by homologous recombination in Streptomyces, allowing substitution of the wild-type target gene with its inactivated counterpart. In the third step, the cassette can be removed by expression of the pSAM2 genes xis and int. The resulting strains are marker-free and contain an "attB-like" sequence of 33, 34, or 35 bp with no stop codon if the cassette is correctly chosen. Thus, a gene can be disrupted by creating an in-frame deletion, avoiding polar effects if downstream genes are cotranscribed with the target gene. A set of cassettes was constructed to contain a hygromycin or gentamicin resistance gene flanked by the attL and attR sites. The initial constructions carrying convenient cloning sites allow the insertion of any other marker gene. We tested insertion and excision by inserting a cassette into orf3, the third gene of an operon involved in spiramycin biosynthesis. We verified that the cassette exerted a polar effect on the transcription of downstream genes but that, after excision, complementation with orf3 alone restored spiramycin production.

Glycosylation Steps during Spiramycin Biosynthesis in Streptomyces ambofaciens : Involvement of Three Glycosyltransferases and Their Interplay with Two Auxiliary Proteins

“…These cassettes are similar to the excisable cassettes att1⍀aac, att2⍀aac, and att3⍀aac previously described (33) except that the terminators originating from the ⍀ interposon (31) and flanking the apramycin resistance gene in ⍀aac (1) are absent in these excisable cassettes. Inactivation of srm5, srm6, srm28, srm29, srm30, and srm38 by in-frame deletion in S. ambofaciens.…”

The Absence of Pupylation (Prokaryotic Ubiquitin-Like Protein Modification) Affects Morphological and Physiological Differentiation in Streptomyces coelicolor

“…The replacement of the pup gene with the apramycin resistance cassette was checked by PCR amplification (primers HBP5/L-acc2R and R-acc2F/HBP6). The apramycin resistance cassette was then excised by site-specific recombination, as previously described (39). The resulting pup locus was then amplified by PCR and sequenced (with the HBP5/HBP6 primers) to confirm that 206 bp had been deleted from the pup coding sequence and replaced with a 41-bp sequence.…”

“…We checked for an absence of mutations in the up-and downstream fragments by sequencing. The upstream and downstream fragments were inserted, together with the apramycin resistance cassette att3-aac (39), into the suicide vector pOSV400 (carrying a hygromycin resistance gene), yielding pOSV400-pup. In this plasmid, the pup upstream and downstream fragments flank the apramycin resistance gene.…”

Multiple biosynthetic and uptake systems mediate siderophore-dependent iron acquisition in Streptomyces coelicolor A3(2) and Streptomyces ambofaciens ATCC 23877

“…Details of the mutants constructed in this study are included in Table 3 and Figs 3 and 4. The S. coelicolor and S. ambofaciens mutants were constructed using the M145 (Bentley et al, 2002) and OSC2 strains (Raynal et al, 2006), respectively. The Escherichia coli strain DH5a was used for cloning experiments.…”