the blank (179 cpm) were at the origin (Fig. 3a). This result confirmed that it was the MurG activity of the membrane preparation that was deficient. MurG was assayed under similar conditions. Lipid II was synthesized when 10 ng of pure E. coli MurG was added to these membranes along with Triton X-100 (Table 2). The identity of the product was confirmed by paper chromatography analysis (Fig. 3b) where radioactivity was detected

at the solvent front (Rf ~ 0.9) ABT-737 where lipid II migrates. Thus, the MurG activity in the MurG-deficient membranes could be reconstituted, and this assay for convenience is further referred to as the ‘reconstituted MurG assay’. In the reconstituted MurG assay, the product formed was dependent on the quantity of MurG added and the time of the reaction (Fig. 4). Using 10 ng of MurG, the reaction was linear up to ~ 30 min. Synthesis of lipid II was linear to ~ 20 ng and saturated above 100 ng. In membrane-based assays of MurG, both the quantity of the substrate, lipid I, and the quantity of enzyme are undefined (Mengin-Lecreulx et al., 1991; Ravishankar et al., 2005). However,

in the reconstituted MurG assay, the quantity of enzyme is defined, allowing the specific activity of MurG with the natural substrate to be defined for the first time. In the SPA, the efficiency of counting and capture is difficult to estimate, and hence, results are reported in cpm and not nmols. However, using the paper chromatography analysis, presuming the efficiency of counting of lipid II on the paper is similar to Fluorouracil solubility dmso that of UDP-[3H]GlcNAc (~ 10%), and the specific activity of E. coli MurG was 1.4 nmol min−1 mg−1; some batches had activity five times higher than this. Interestingly, the specific activity appears similar to that reported (Ha et al., 2000), Anidulafungin (LY303366) despite

the fact that the published assay used a synthetic lipid analogue and MurG was in solution. MurG activity in the reconstituted MurG assay was 60- to 100-fold higher in the presence of Triton X-100 than in its absence. In contrast, peptidoglycan synthesis activity of the MurG-reconstituted membranes was inhibited by Triton X-100. This is not unexpected, because peptidoglycan synthesis in wild-type membranes was inhibited 50% by 0.05% TritonX-100, most likely due to the inhibition of the transglycosylase (Branstrom et al., 2000; Chandrakala et al., 2001). Triton X-100 did not stimulate MurG in wild-type membranes, so it is likely that the detergent improved accessibility of the purified soluble MurG to the lipid substrate and other components present in the membranes. Nisin and vancomycin inhibited the reconstituted MurG assay with IC50s of 3.5 μg mL−1 and 32 μM, respectively; these were similar to the IC50s for MurG in wild-type membranes (nisin:10 μg mL−1 and vancomycin: 30 μM). Thus, the reconstituted MurG assay closely resembles the assay of MurG in wild-type membranes.

5 h. HRP-conjugated donkey anti-goat was used as the secondary antibody and the reaction was developed with a TMB substrate (Tiangen). After 15 min of color development, the stop solution (8.5 M acetic acid, 2.5 M H2SO4) was added and the A450 nm

was recorded. The binding of HDL to the GAS (Type M6 and M41) was tested using GAS cells that were either immobilized onto microplate wells or were in suspension. GAS cell suspensions were added to microplate wells and incubated at room temperature for 1.5 h. Wells were washed and blocked overnight with 200 μL of 1% bovine serum albumin (BSA) in TBS or TBST at 4 °C. HDL binding was performed as described above in the rScl1 binding ELISA. Trichostatin A For the HDL binding to GAS cells in suspension, cells were incubated for 1.5 h with 1% BSA in TBS or TBST. After washing with TBS or TBST, 100 μL of human plasma was added to 1 mL of the cells. Following a 1-h incubation at room temperature, bacterial cells were pelleted and washed three times with TBS or TBST. After the final centrifugation, cell-bound proteins were dissociated 20s Proteasome activity from the cells by the incubation with 200 μL of 0.1 M glycine-HCl solution, pH 2, for 15 min. Bacterial cells were then removed by centrifugation and the proteins in the supernatant were precipitated with 10% TCA and analyzed by SDS-PAGE and immunoblotting. Electroblotting was carried out at a constant voltage

of 30 V for 1 h to transfer ApoAI and at a constant current of 300 mA for 3 h to transfer ApoB100. The immunodetection of ApoAI was performed with a goat anti-ApoAI antibody, followed by HRP-conjugated donkey CYTH4 anti-goat secondary antibody as described above. The presence of ApoB100 was tested with a goat anti-LDL antibody (Chemicon, CA), followed by HRP-conjugated donkey anti-goat antibody (R&D Systems), and the detection was performed with chemiluminescence reagent (Tiangen). Results were expressed as mean±SD. Statistical significance was calculated

using two-tailed Student’s t-test for comparisons of two groups and Student–Neuman–Keuls for comparison of multiple groups, respectively. It was reported previously that several Scl1 proteins interact with LDL/ApoB100 via globular noncollagenous V regions (Han et al., 2006a). Here, we are testing the hypothesis that the Scl1.41 variant possess binding ability to HDL. Recombinant Scl1 (rScl1) proteins C176, C176V, and C176T were constructed, which are derived from Scl1.41 protein of GAS M41-type strain ATCC12373. PCR-amplified DNA fragments corresponding to a full-length or a partial scl1.41-gene sequence were cloned, expressed, and purified in E. coli BL21 (Table 1; Fig. 1a). rScl1 proteins were immobilized onto Strep-Tactin columns through their C-terminal tags (Strep-tag II) and these affinity columns were used to detect Scl1 ligands in human plasma. Human plasma (0.5 mL) was applied to these columns, including the control column without the rScl1 protein.

Erm proteins catalyze either monomethylation (type I) or dimethylation (type II) reactions at the exocyclic N6 position of a specific adenine residue (A2058, Escherichia coli rRNA nucleotide numbering) in 23S rRNA to reduce the affinity of MLSB antibiotics to the peptidyl transferase center, the most problematic MLSB-resistance mechanism adopted by many clinically selleck kinase inhibitor isolated, resistant

pathogens (Weisblum, 1995). KsgA, another posttranscriptional rRNA methylation enzyme, catalyzes two consecutive dimethylation reactions, resulting in two adjacent, dimethylated adenines at the 3′ end of 16S rRNA in bacteria (Helser et al., 1972; Poldermans et al., 1979; O’Farrell et al., 2004). In contrast to Erm, the inactivation of the ksgA gene confers resistance to the aminoglycoside antibiotic kasugamycin. KsgA enzymes and the resulting methylated adenine bases GKT137831 mouse appear to be conserved

in all three domains of life (O’Farrell et al., 2004; Xu et al., 2008; Park et al., 2009), while Erm is found in limited species of microorganisms that are considered to be either the target or the producers of MLSB antibiotics (Weisblum, 1995). This finding suggests that KsgA might be an essential enzyme for survival, but Erm is necessary only in the presence of antibiotic pressure. However, KsgA is not absolutely essential in bacteria. Mutant E. coli (i.e., KsgA−) exhibits a longer doubling Selleck Osimertinib time, but survival does not appear to be affected by mutation (O’Farrell et al., 2004). Recent studies have demonstrated that KsgA binds to translationally inactive 30S ribosomal subunits and acts as a checkpoint in ribosome biogenesis by ensuring that only mature small subunits proceed to translation (Desai and Rife, 2006; Connolly

et al., 2008; Mangat and Brown, 2008; Xu et al., 2008). On the other hand, the eukaryotic ortholog of KsgA, Dim1, is found to be essential in yeast, where its most important role is the cleavage of 33S pre-rRNA rather than rRNA methylation (Lafontaine et al., 1994, 1995; Pulicherla et al., 2009). The sequence homology between Erm and KsgA was first recognized in the mid-1980s (van Buul and van Knippenberg, 1985). These two protein families also have a very similar basic architecture; both consist of two domains, a conserved Rossman-fold N-terminal domain and a less-conserved C-terminal domain, and carry out very similar catalytic reactions (Yu et al., 1997; Schluckebier et al., 1999; O’Farrell et al., 2004). Recent crystal structures of Aquifex aeolicus KsgA in complex with RNA and cofactor revealed that Erm and KsgA showed a very similar mode in cofactor binding, but a different mode in the details of RNA binding (Tu et al., 2009).

Although C. pneumoniae-specific antibody responses have been characterized by immunoblotting, only few major surface proteins (MOMP, Omp2, and CrpA; Iijima et al., 1994; Klein et al., GDC-0199 order 2003; Mygind et al., 1998) and some Inc proteins (Cpj0146, Cpj0147, and Cpj0308) have been detected (Hongliang et al., 2010). However, these antigens have yielded variable results with respect to the consistency and accuracy of C. pneumoniae identification. Taken together, very little information is available regarding specific detection of C. pneumoniae. We determined the sequence of the whole genome of C. pneumoniae J138 isolated

in Japan (Shirai et al., 2000) and found that this strain features putative protein coding from its 1069 open reading frames (ORFs). A comprehensive bioinformatics approach was applied for annotation taxonomy, and about half of the predicted genes were found to encode proteins without any known functions. To identify novel specific antigens from C. pneumoniae, we screened 455 genes without any known functions. A fusion protein expression library of C. pneumoniae was constructed in Saccharomyces cerevisiae. Protein extracts of the recombinant yeast cells expressing the green fluorescent protein (GFP)-tagged C. pneumoniae proteins were subjected to Western blot analysis using serum samples from C. pneumoniae-infected patients as the primary

antibodies. This study sought to identify specific and highly immunodominant antigens, which are required for the development of new serodiagnostic assays, and hopefully, vaccines, in the future. Thirteen serum samples were collected from eight patients before (age: range, 4–11 years; Selleck PD0325901 Table 1), who had been clinically diagnosed with primary acute C. pneumoniae infection. The levels of C. pneumoniae-specific immunoglobulin (Ig) IgA, IgG, and IgM in these patients were evaluated using two different

ELISA kits: (1) HITAZYME C. pneumoniae kits for IgA, IgG, and IgM that utilize the soluble elementary body (EB)-outer membrane complex, without the lipopolysaccharide, as the antigen (Hitachi Chemical, Japan) and (2) C. pneumoniae-ELISA plus Medac kits for IgA and IgG and C. pneumoniae-sELISA Medac kit for IgM, which utilize the purified cell wall membrane proteins as the antigen (Medac Diagnostika, Germany). Eight serum samples from 0-year-old healthy children were used as negative controls. Chlamydophila pneumoniae genomic DNA was obtained from the EBs of the C. pneumoniae J138-infected HEp-2 cells (Miura et al., 2001). We used a gene expression system controlled by a Tet-off promoter in S. cerevisiae. The ORFs of 455 genes from C. pneumoniae J138, including genes of unknown function (Supporting Information, Table S1), were cloned into a pMT830 vector, which was constructed as previously described (Tabuchi et al., 2009). This vector system allows a protein of interest to be expressed with GFP fused to the C-terminus.

, 2009). In our study, tet(40) was located in tandem with tet(O). Sequence homology search showed that the ARGs we identified in this study

were of diverse bacterial origin, including nonpathogenic species such as Bifidobacterium longum, as well as opportunistic pathogens such as Streptococcus suis and Staphylococcus pseudintermedius. Because the potential for gene transfer in the human gut is very high due to the dense microbial population (Kazimierczak & Scott, 2007), it is worth addressing in the future to what extent these bacteria serve as donors, disseminating the ARGs to other bacteria, especially the incoming pathogenic bacteria. The GSI-IX fosmid-based method has some potential disadvantages in ARG screening. Genes on smaller plasmids (< 30 kb) might not be represented in the metagenomic library. Moreover, only ARGs that are properly expressed in E. coli with their own promoters will be identified. However, the fosmid-based

method also has advantages. The larger insert size increases the likelihood PF-02341066 mouse of cloning complete ARGs. In fact, nearly one-third of resistant fosmid clones could not be subcloned, even after several trials. This could be because different vectors were used for cloning (pCC2FOS) and subcloning (pUC118 or pHSG298) or because some resistant determinants are out of the range of length chosen for subcloning (1–5 kb). Our further work will focus on whole-length sequencing to elucidate the resistance mechanisms conferred by the clones that failed to be subcloned.

It is worth noting that although the human subjects we used in this study were not exposed to antibiotic treatment for at least SDHB 6 months prior to sampling, we cannot exclude their antibiotic consumption history. As antibiotic-resistant strains can persist in the human host environment in the absence of selective pressure for a long time (Jernberg et al., 2010), the ARGs we identified cannot be considered intrinsic; they are probably the results of selective pressure conferred by antibiotics that the gut microbes previously encountered and somehow managed to maintain in the gut. In summary, we constructed a metagenomic library from four human gut microbiota and screened for ARGs, uncovering diverse new genes, including a new kanamycin resistance gene fusion. This work helps us to further understand the ARG reservoir of the human gut microbiota, and we believe that other new ARGs will be mined from human gut in the near future. However, to what degree these ARGs in our gut are linked to the potential emergence and dissemination of antimicrobial resistance genes in human pathogens is unclear. This work was supported in part by the National Basic Research Program of China (973 Program grants 2007CB513002 and 2009CB522605). Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors.

2b. Therefore, they may be responsible for Selisistat order the hydrolysis of RNA by a mechanism similar to RNase A. However, due to localization of aspartic acid (D535) on the surface of catalytic domain as shown in Fig. 2b, its role in RNA hydrolysis by mechanism similar to barnase and colicin E3 cannot be ruled out. Therefore, to determine individual role of conserved amino acid residues in the putative active site of catalytic domain of

xenocin, site-directed mutagenesis was performed. All the conserved amino acid residues were mutated to alanine, and endogenous toxicity assay was performed with each mutant strain. Growth profile of JSR4 strain–containing vector alone was considered as 100% and compared with growth profile of D535A, H538A, E542A, H551A, K564A and R570A strains. From the predicted structure of catalytic domain of xenocin as shown in Fig. 2b, it was observed that H538 was the most surface-exposed histidine residue among the four other present in the catalytic domain. Endogenous assay showed that mutation at H538 position results in the reduction of toxicity by more than 90% after 8 h postinduction as shown in Fig. 2c, which confirmed the role D535 as an important residue of the putative active site. As second conserved histidine residues H551 was nearer to H538 and exposed on the surface, it

may behave as the second histidine residue required for the hydrolysis of RNA by a mechanism similar to RNase A ribonuclease. Therefore, INCB018424 mouse H551 was mutated to alanine, and endogenous assay was performed. Results showed that there was only 50% reduction in endogenous toxicity in H551A strain after 8 h of induction as shown in Fig. 2c. One reason for such minimum reduction in endogenous toxicity in H551A strain is that it could be due to partial exposure of H551 to the surface as compared to H538 as revealed by the surface view model of catalytic domain as Progesterone shown in

Fig. 2b. This result indicates that RNA hydrolysis mechanism of catalytic domain of xenocin is different from RNase A ribonuclease. D535 and E542 are two acidic amino acid residues that are conserved, exposed to surface as well as close to the H538 as shown in Fig. 2a and b. These two residues may be responsible for the hydrolysis of RNA by mechanism similar to barnase and colicin E3. Therefore, these two residues were mutated to alanine and analysed by endogenous assay. Endogenous toxicity assay result showed that toxicity was reduced by 70% after 8 h postinduction in E542A strain as shown in Fig. 2c. Structural studies showed that E542 was also a part of cleft formed by D535 and H538, which is exposed to the surface as shown in Fig. 2b. However, studies with D535 strain showed significant reduction (88%) in the endogenous toxicity after 8 h postinduction as shown in Fig. 2c; moreover, D535 was the closest amino acid residue with respect to H538 as shown in Fig. 2a.

Polyketides can selleck compound also be extracted from different algae, dinoflagellates and plants (Hopwood & Sherman, 1990; Austin & Noel, 2003), for which those compounds apparently serve as defensive substances against natural enemies (Manojlovic et al., 2000; Choi et al., 2004).

The probably most diverse group of polyketide producers are marine organisms like sponges, tunicates, and bryozoans. Such animals are a source of natural compounds with strong cytotoxic properties that are extremely interesting from a medical point of view (Piel, 2004, 2006; Moore, 2005, 2006; Piel et al., 2005). These substances belong to the pederin family, which currently comprises 36 members from eight different invertebrate animal genera (Narquizian & Kocienski, 2000; Simpson et al., 2000; Vuong et al., 2001; Paul et al., 2002). MLN0128 solubility dmso Polyketides are produced by hitherto uncultured, highly adapted bacterial endosymbionts. Cultivation of the pederin-producing bacterial endosymbionts of female Paederus rove beetles is not yet possible, and although chemical synthesis of pederin has been successfully reported by some groups

(Matsuda et al., 1988; Kocienski et al., 2000; Takemura et al., 2002; Jewett & Rawal, 2007), its low availability represents a serious impediment to drug development (Munro et al., 1999; Piel, 2002, 2004, 2006). Thus, tools are required for custom tailoring growth media for the enrichment and isolation of Paederus endosymbionts. Kellner (1999, 2001a, b, 2002a) demonstrated that a Pseudomonas-like endosymbiont is associated with the transfer of pederin production capabilities to the female progeny of Paederus beetles via endosymbiont-harbouring eggs. Analysis of metagenomic DNA from Paederus fuscipes beetles revealed the existence of a mixed modular polyketide synthase (pks)-gene cluster that is responsible for pederin biosynthesis (Piel, 2002). Specific PCR primers were designed from conserved regions of single cluster modules and utilized for the amplification of pks-gene fragments from endosymbionts in beetle or egg specimens (Piel, 2002).

However, direct evidence for the localization of Pseudomonas-like endosymbionts on eggs is lacking, and it is still unresolved, where such endosymbionts are located within Paederus beetles. FISH is an appropriate tool Farnesyltransferase for the in situ localization of specific phylogenetically defined groups of bacteria (Amann et al., 2001; Amann & Fuchs, 2008). Thus, the objectives were to (1) design and evaluate a specific 16S rRNA gene-targeted oligonucleotide probe for Pseudomonas-like Paederus riparius endosymbiont detection; (2) localize endosymbionts within serial egg thin-sections by FISH; and (3) determine where within the host symbionts are transferred to eggs by surface comparison of different egg stadiums using electron microscopy and pks-targeted PCR.

More than for any other infection, patients receiving ART require their doctor to have a clear understanding of the basic principles of pharmacology to ensure effective and appropriate prescribing. This is especially the case in four therapeutic areas. We recommend that potential adverse pharmacokinetic interactions between ARV drugs and other concomitant medications are checked before administration (with tools such as http://www.hiv-druginteractions.org) find more (GPP). Record in patient’s

notes of potential adverse pharmacokinetic interactions between ARV drugs and other concomitant medications. The importance of considering the potential for drug interactions in patients receiving ART cannot be overemphasized. DDIs may involve positive or negative interactions between ARV agents or between these and drugs used to treat other coexistent conditions. A detailed list is beyond the remit of these guidelines but clinically important interactions to consider when co-administering with ARV drugs

include interactions with the following drugs: methadone, oral contraceptives, anti-epileptics, antidepressants, lipid-lowering agents, acid-reducing agents, certain antimicrobials (e.g. clarithromycin, minocycline and fluconazole), some anti-arrhythmics, TB therapy, anticancer drugs, immunosuppressants, phosphodiesterase inhibitors and anti-HCV therapies. Most of these interactions can be managed safely (i.e. with/without dosage Regorafenib in vivo modification, together with enhanced clinical vigilance) but in some cases (e.g. rifampicin and PIs, proton pump inhibitors and ATV, and didanosine and HCV therapy)

the nature of the interaction is such that co-administration must be avoided. Importantly, patient education on the risks of drug interactions, including over-the-counter or recreational drugs, should be undertaken and patients should be encouraged to check with pharmacies or their healthcare professionals filipin before commencing any new drugs, including those prescribed in primary care. Large surveys report that about one-in-three-to-four patients receiving ART is at risk of a clinically significant drug interaction [1-6]. This suggests that safe management of HIV drug interactions is only possible if medication recording is complete, and if physicians are aware of the possibility that an interaction might exist. Incomplete or inaccurate medication recording has resulted from patient self-medication, between hospital and community health services [7] and within hospital settings particularly when multiple teams are involved, or when medical records are fragmented (e.g. with separate HIV case sheets) [8]. More worryingly, one survey in the UK reported that even when medication recording is complete, physicians were only able to identify correctly one-third of clinically significant interactions involving HIV drugs [4].

Inoculations were carried out from precultures grown for 24 h in trace iron GPP at inoculation rates of 0.1% v/v to minimize carryover of iron. The total initial cell counts of cultures

thus inoculated typically were 5 × 104 mL−1 and 3 × 103 mL−1 for C. albicans and C. vini, respectively. Incubation of flask cultures was carried out aerobically in a temperature-regulated shaker at 30 °C and 200 r.p.m. Media and stock solutions were kept in sterile plastic ware (polypropylene, Nalgene) for this work. Glassware used Galunisertib solubility dmso for incubations was first washed with a conventional detergent (Alconox, Fisher), followed by 24-h soaking in a 3% v/v solution of a commercial trace metal removal detergent (Citronox, Fisher) and nine rinses in deionized water. The growth of microorganisms was measured by following the OD600 nm of cultures in 1-cm light path cuvettes. For dry weight determinations, cells were harvested by centrifugation at 1200 g for 10 min and washed twice with deionized water. Then, the cell mass was determined after drying at 100 °C for 24 h, with cooling in a vacuum dessicator containing a granular desiccant (Drierite, Xenia, OH) on preweighed aluminium dishes

to a constant weight. The total cell counts were carried out using a 0.1-mm depth haemocytometer see more with improved Neubauer ruling (Brightline, Hausser Scientific, Horsham, PA). Trace iron and other trace metal concentrations in the media before and after extraction were determined in quadruplicate by high-resolution magnetic-sector aminophylline ICP-MS at the Environmental Chemistry & Technology and Wisconsin State Laboratory of Hygiene, University of Wisconsin-Madison. Table 1 shows the concentrations of iron and several other metals in the chemically defined medium prepared without any Fe addition before and after Fe extraction. Using an insoluble resin in a batch-contacting process, it was possible to reduce iron concentrations by >80% to 1.2 μg L−1 (0.021 μM) in the chemically defined medium used. The residual Fe content in the Fe-extracted medium was found to result in Fe-restricted growth for both C. albicans and C.

vini with increased lag phases and lower specific growth rates as compared with cultivations with added iron (Fig. 1a and b, respectively). Candida vini appeared to be more affected by low Fe concentrations than C. albicans. Accordingly, the maximum growth yields (Ymax) determined after 44-h growth exhibited a stronger dose dependence for added iron in the case of C. vini (Fig. 2). At the lowest iron concentration tested (0.02 μM), the maximum growth yield attained by C. vini was less than half the Ymax value obtained for C. albicans. The comparison of the effects of several iron chelators including the clinically relevant desferrioxamine and deferiprone at relatively low concentrations (0.25 g L−1) showed that the growth of C. albicans was not inhibited by desferrioxamine in comparison with the control treatment with no added iron chelator (Fig. 3).

(2003) with slight modifications. Cosmids of S. coelicolor containing the genes for replacement were introduced by transformation into E. coli BW25113 (pIJ790). Electrocompetent cells were prepared and electroporated with a PCR product (containing an aac(3)IV gene) using a GenePulser II (Bio-Rad Inc.). The PCR-targeted constructs were introduced by electroporation into E. coli ET12567 (pUZ8002) and then transferred by conjugation into S. coelicolor. Thiostrepton-resistant colonies were selected for single

crossing PD0332991 chemical structure over between the cosmid and the host chromosome. After sporulating on MS medium without antibiotic selection, thiostrepton-sensitive but apramycin-resistant colonies were screened to obtain double-crossover clones. To remove the aac(3)IV marker for the next round of gene disruption and replacement, cosmid with the aac(3)IV gene inserted in a FRT-aac(3)IV-FRT cassette was introduced by electroporation into E. coli BT340 containing a flp gene encoding Flp recombinase to remove the cassette. Clones containing a double-crossover allelic exchange in S. coelicolor were confirmed by PCR analysis and some clones (i.e. ZM4) by microarray hybridization analysis performed in the Shanghai Biochip Inc. To delete a large segment (e.g. > 40 kb)

on the S. coelicolor chromosome, two fragments (e.g. > 5 kb) from different cosmids of the ordered library plus a kanamycin resistance gene (kan) were cleaved and cloned in the polylinker of pHAQ31 or pHY642. The

resulting plasmid was introduced by electroporation into E. coli ET12567 (pUZ8002) and ADP ribosylation factor then Ixazomib cost transferred by conjugation into S. coelicolor. Thiostrepton-resistant colonies were selected for single crossing over, and thiostrepton-sensitive but kanamycin-resistant colonies for double crossing over. Clones containing a double-crossover allelic exchange in S. coelicolor were further confirmed by PCR analysis. A 2.6-kb fragment (digested with XbaI and NheI) containing a phiC31 integrase gene was cloned in a pHAQ31-derived cosmid containing the entire actinorhodin biosynthetic gene cluster. The resulting plasmid, pCWH74, was introduced by conjugation into Streptomyces strains. To quantitate the production of actinorhodin, strains were inoculated into R2YE (lacking CaCl2, KH2PO4, and L-proline) liquid medium, 1 mL culture was harvested, and spun at 15 294 g for 1 min to collect the supernatant, which was further treated with KOH and scanned at 640 nm. Measurements of actinorhodin production were carried out by the method of Kieser et al. (2000). PCR-targeting of cosmids is a precise and efficient method for gene disruption and replacement in Streptomyces. Because two long segments (e.g. > 5 kb) on a suicide plasmid are employed for homologous recombination with chromosomal sequences, high frequencies of single- and double-crossover events can usually be obtained by screening a few clones (Gust et al., 2003).