While it seems clear that drug-resistant microorganisms often have point mutation(s) in drug-target molecule genes (Walsh, 2000), no reports have yet described how and why such mutations occur in clinically important drug-resistant bacteria. We have reported that oxygen can Cyclopamine mw induce DNA damage, causing mutations in rpoB and Rif resistance, in a strict anaerobe (Takumi et al., 2008). In the environment, there are numerous mutagens capable of damaging DNA and inducing mutation. Clinical drugs, such as some used in cancer therapy, may also be mutagenic (Kunz & Mis, 1989). Cigarette smoke also

contains many mutagenic chemicals (Fujita & Kamataki, 2001; Yim & Hee, 2001). Environmental microorganisms, especially indigenous microorganisms, may frequently be exposed to mutagens. Pseudomonas aeruginosa is an indigenous bacterium and emerging drug resistance in this bacterium is a growing concern (Jalal & Wretlind, check details 1998; Mouneimne et al., 1999; Akasaka et al., 2001; Wydmuch et al., 2005). In this study, we exposed P. aeruginosa to mutagens that are known to induce point mutation. The environmental concentrations of mutagens are similar or even higher than those we have used in the present experiments. The concentration of EMS in the Viracept case was 2.3 mg mL-1. (Gerber & Toelle, 2009), that of 1,6-DNP in soot was 0.41–0.71 μg g−1 (Schauer et al., 2004), and that of BCNU was 4 μg mL−1 in human plasma and

3.3 mg mL−1 in injection fluid (Petros et al., 2002). We have set the exposure time at 24 h because indigenous bacteria may be exposed 3-oxoacyl-(acyl-carrier-protein) reductase to these mutagens continuously in

the environment. We selected Rif and CPFX, because the emergence of microorganisms resistant to Rif and to CPFX is a growing concern (Jalal & Wretlind, 1998; Wydmuch et al., 2005). In addition, both antibacterial agents have obvious target molecules and mutations related to these target molecules are known to confer drug resistance (Campbell et al., 2001; Mariam et al., 2004). Pseudomonas aeruginosa is inherently relatively resistant to Rif (Yee et al., 1996), but has been susceptible to high concentrations of Rif. At the same time, the emergence of Rif-resistant M. tuberculosis is also a growing concern (Yee et al., 1996; Murphy et al., 2006). CPFX has been highly effective in treating P. aeruginosa infections, but recently, CPFX-resistant P. aeruginosa has become a growing problem (Jalal & Wretlind, 1998). Rif- and CPFX-resistant P. aeruginosa emerged after exposure to EMS and MNU. Meanwhile, BCNU induced Rif resistance, and 1,6-DNP induced CPFX resistance. NNN did not increase Rif- or CPFX-resistant P. aeruginosa. While BP induced mutation in S. Typhimurium TA100, Rif- or CPFX-resistant P. aeruginosa did not result. Susceptibility to BP differs considerably among strains (Jemnitz et al., 2004). We supposed that the P. aeruginosa was not susceptible to the mutagenic action of BP metabolites.

We assigned these enzymes to group 2. Further analysis revealed several microorganisms (Agrobacterium vitis S4, Bordetella petrii DSM 12804, Vibrio vulnificus YJ016, Sideroxydans lithotrophicus ES-1) whose IDO homologues are expressed only in combination with a specific efflux pump (RhtA/RhtB exporter family) without AR in the same operon regulated by a LysR-type repressor. These dioxygenases were assigned to the third group [Fig. 1 (5, 6, 7)]. By way of

analogy to B. thuringiensis, we proposed that the operons from the Enzalutamide manufacturer first, second and third groups could be involved in the synthesis and excretion of special derivatives of the hydroxylated free l-amino acids produced by their corresponding IDO homologues. In several microbes that we assigned to the fourth group (e.g. Gluconacetobacter diazotrophicus PAl 5 and GSI-IX mouse Pseudomonas fluorescens Pf0-1), the IDO homologue genes belong to the operons assumed to be involved in the synthetic

process, one stage of which is hydroxylation of an unknown substrate [Fig. 1 (8, 9)]. In some bacteria (e.g. Burkholderia oklahomensis EO147, Burkholderia pseudomallei 668, Photorhabdus luminescens ssp. laumondii TTO1 and Photorhabdus asymbiotica ATCC 43949), the IDO is thought to be co-expressed with polyketide/nonribosomal peptide synthetase-like protein. We proposed that these dioxygenases can be involved in the synthesis of peptide antibiotics containing hydroxylated l-amino acid residues and may also hydroxylate free l-amino acids [Fig. 1 (10)]. We assigned these dioxygenases to the fifth group. Many bacteria encode IDO homologues that are not part of an operon structure and can hydroxylate unpredictable substrates, including free l-amino acids; we included these enzymes in the sixth group. Based on the data obtained thus far, we assumed that the free amino acid dioxygenases were likely to belong to any group except group number four. Eight members of the PF10014 family – IDO (group

1, as a control enzyme); PAA (group 2); AVI, BPE (group 3); PLU (group 5); MFL, GOX and GVI (group 6) – were arbitrarily chosen for cloning and expression in E. coli to examine their substrate specificities with regard to canonical l-amino acids (Table 1). Using standard methods, we expressed selected enzymes as his6-tag proteins and purified them to near homogeneity using conventional aminophylline IMAC. Because our goal was to identify enzymes possessing high hydroxylase activities with potential for biotechnology applications, we first performed a high-throughput analysis for dioxygenase substrate specificity with 20 canonical l-amino acids using TLC analysis of the reaction mixture products (Fig. 2a,b). We found that new amino group-containing substances are formed by hydroxylation reactions with l-isoleucine (IDO, PAA), l-leucine (all enzymes with exception of GVI and PLU), l-methionine (all enzymes, but the activity of PLU was rather low) and l-threonine (BPE, AVI) (Fig. 2c).

It is likely that clinically isolated heme-auxotrophic SCVs are able to obtain heme from the host via heme transport systems, which may contribute to the pathogenesis and persistence of these strains. Characterization of a heme-auxotrophic, heme transport–defective mutant in appropriate in vivo infection models would enable the contribution of heme transport in these SCVs to be assessed. With this in mind, we set out to construct a ΔhemBΔhtsAΔisdE S. aureus strain to investigate the role of heme acquisition via these transport systems in a heme-auxotrophic SCV. Characterization of this strain in vitro demonstrates that S. aureus is still able to acquire heme

added to the growth medium in the form of either hemin or hemoglobin in the absence of both htsA and isdE. This learn more lends support to the hypothesis that the Hts system is responsible only for the transport of staphyloferrin A and contradicts the argument that IsdE AZD4547 may transfer heme to the HtsBC permease (Hammer & Skaar, 2011). Furthermore, these data strongly suggest that additional, as yet uncharacterized, heme transport system components operate in S. aureus. This may take the form of an additional lipoprotein that is able to transport heme in conjunction with

HtsBC or IsdDF, or possibly another transport system altogether. Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli was grown on Luria–Bertani (LB) agar or in LB broth, supplemented with 100 μg mL−1 ampicillin and 10 μg mL−1 chloramphenicol where appropriate, at 37 °C under aerobic conditions. Staphylococcus aureus was cultured on tryptone soy agar (TSA) or in tryptone soy broth (TSB), supplemented with 10 μg mL−1 chloramphenicol where required, at 37 °C under aerobic conditions. Gene deletion mutants were constructed in S. aureus LS-1 according to the method of Bae and Schneewind (Bae & Schneewind,

2006). DNA fragments flanking the gene of interest of S. aureus LS-1 were amplified by PCR using primers listed in Table 2 and cloned into the vector pKOR1 in E. coli DH5α. Staphylococcus aureus RN4220 was used to passage plasmids prior Branched chain aminotransferase to transformation of target S. aureus strains. Double- and triple-deletion mutant strains were constructed by sequential allelic replacement using the plasmid constructs listed in Table 1. Gene deletions were confirmed by PCR amplification and DNA sequencing using the primers listed in Table 2, which flank the manipulated regions. The hemB gene was amplified by PCR from S. aureus LS-1 genomic DNA using primers JAW418 and JAW419 (Table 2) to yield a product of 996 bp, then purified, and digested with BamHI and XbaI. Plasmid pSK236 was digested with SacI and XbaI, and pHCMC05 was digested with BamHI and SacI to excise the Pspac promoter.

It is likely that clinically isolated heme-auxotrophic SCVs are able to obtain heme from the host via heme transport systems, which may contribute to the pathogenesis and persistence of these strains. Characterization of a heme-auxotrophic, heme transport–defective mutant in appropriate in vivo infection models would enable the contribution of heme transport in these SCVs to be assessed. With this in mind, we set out to construct a ΔhemBΔhtsAΔisdE S. aureus strain to investigate the role of heme acquisition via these transport systems in a heme-auxotrophic SCV. Characterization of this strain in vitro demonstrates that S. aureus is still able to acquire heme

added to the growth medium in the form of either hemin or hemoglobin in the absence of both htsA and isdE. This selleckchem lends support to the hypothesis that the Hts system is responsible only for the transport of staphyloferrin A and contradicts the argument that IsdE Antidiabetic Compound Library datasheet may transfer heme to the HtsBC permease (Hammer & Skaar, 2011). Furthermore, these data strongly suggest that additional, as yet uncharacterized, heme transport system components operate in S. aureus. This may take the form of an additional lipoprotein that is able to transport heme in conjunction with

HtsBC or IsdDF, or possibly another transport system altogether. Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli was grown on Luria–Bertani (LB) agar or in LB broth, supplemented with 100 μg mL−1 ampicillin and 10 μg mL−1 chloramphenicol where appropriate, at 37 °C under aerobic conditions. Staphylococcus aureus was cultured on tryptone soy agar (TSA) or in tryptone soy broth (TSB), supplemented with 10 μg mL−1 chloramphenicol where required, at 37 °C under aerobic conditions. Gene deletion mutants were constructed in S. aureus LS-1 according to the method of Bae and Schneewind (Bae & Schneewind,

2006). DNA fragments flanking the gene of interest of S. aureus LS-1 were amplified by PCR using primers listed in Table 2 and cloned into the vector pKOR1 in E. coli DH5α. Staphylococcus aureus RN4220 was used to passage plasmids prior cAMP inhibitor to transformation of target S. aureus strains. Double- and triple-deletion mutant strains were constructed by sequential allelic replacement using the plasmid constructs listed in Table 1. Gene deletions were confirmed by PCR amplification and DNA sequencing using the primers listed in Table 2, which flank the manipulated regions. The hemB gene was amplified by PCR from S. aureus LS-1 genomic DNA using primers JAW418 and JAW419 (Table 2) to yield a product of 996 bp, then purified, and digested with BamHI and XbaI. Plasmid pSK236 was digested with SacI and XbaI, and pHCMC05 was digested with BamHI and SacI to excise the Pspac promoter.