The positions of molecular weight markers in base pairs are shown to the check details right. Purified chromosomal DNA from S. aureus subsp. aureus (from now on called S. aureus) strain NCTC 8325-4 [26] was sonicated into fragments mainly 250 to 1000 bp in length (Figure 1B). The polished, blunt-ended DNA fragments were ligated into pSRP18/0 and transformed into the secretion-competent strain E. coli MKS12 to generate a primary genomic library including more than 80 000 colonies.

By colony PCR, the cloning efficiency, i.e. the% insert-carrying transformants of all transformants, was estimated from 200 randomly picked colonies to be 60% and the average insert size of 200 randomly picked insert-containing clones was estimated to be approximately 400 bp. The PCR primers

used are shown in Figure 1A. Generation of the final FLAG-tag positive (Ftp) library in E. coli The 80 000 colonies of the primary genomic library were screened by colony blotting using anti-FLAG BAY 73-4506 datasheet antibodies for exclusion of transformants carrying an empty vector or insertions out-of-frame in relation to the FLAG tag. Totally 1663 clones were confirmed to carry gene products with C-terminal FLAG tags and these were included into the final Ftp library. Colony-blot analysis showed that MKS12 (pSRP18/0) with the empty vector reacted with monoclonal anti-FLAG antibodies as weakly as MKS12 carrying no plasmid (data not shown), thus confirming that the Ftp colonies did possess an insertion in their plasmids. Sequence analysis of the Ftp library The coverage of the Ftp library was determined by sequencing the inserted DNA fragments in both directions in all FAD the 1663 Ftp

library clones. The sequencing primers are shown in Figure 1A. The sequence of the insert was successfully determined in 1514 clones using the 017F primer and in 1564 clones with the 071R primer. When projected over the genome sequence of S. aureus NCTC 8325 using genomic blast searches [27], the 1514 sequences obtained using the 017F primer corresponded to 708963 nt in total and covered 435809 nt of the genome. For the later 1564 sequences obtained with the 071R oligonucleotide, the corresponding values were 769323 nt and 462172 nt, respectively. The sequenced inserts overlapped totally 345890 nts of the genome, thus the overlap of the Ftp library was 63%. Comparison of the Ftp library sequences with the gene sequences of S. aureus NCTC 8325 using BLASTN revealed a significant match for 1325 and 1401 of the 1514 and 1564 determined insertion sequences. The inserts showed homology to 808 and 845 gene sequences, respectively, and covered in total 950 gene sequences in S. aureus NCTC 8325. The matches were distributed randomly and evenly over the staphylococcal chromosome (Figure 2). Based on genomic and proteomic data, the theoretical number of encoded proteins in S. aureus NCTC 8325 is 2891 [28, 29], which indicates that our final Ftp library covers approximately 32% of the staphylococcal proteome.