culbertsoni or A. castellanii supernatants obtained in PAS may likewise lead to increased bacterial counts (Fig. 3). The results were similar with supernatants obtained from filtered tap water (data not shown). This could be due, particularly for A. culbertsoni, to the death and the lysis of amoeba, especially when they were co-cultivated with A. baumanii, which could provide nutrients for the bacteria to grow. Among microorganisms related to amoebae, some bacteria

that may be human pathogens have evolved in a way that allows them to resist destruction by protozoa either because they are not internalized or else because they are able to survive, grow and exit amoebae following internalization (Greub & Raoult, 2004). We have evaluated the growth and survival of the bacterium in a poor medium such as encystment medium with and without amoebae. The bacterial count showed that the presence of amoebae (A. castellanii or A. culbertsoni) Fulvestrant research buy allows for increased bacterial find more growth, while A. baumanii in the same medium without amoebae is able to survive, but at lower concentrations. After 60 days in this medium, the survival of the bacteria is favored by the presence of amoebae (Fig. 4) In electron microscopy after 11 days of incubation, some cysts already contained intracellular

A. baumanii, located only in the space between the double walls (Fig. 2), which is similar to the location of Pseudomonas in Acanthamoeba astronyxis (Marciano-Cabral & Cabral, 2003), Mycobacterium avium in A. polyphaga (Steinert et al., 1998), Mycobacterium sp. (Sharbati-Tehrani et al., 2005; Ben Salah & Drancourt, 2010), V. cholerae Oxalosuccinic acid (Abd et al., 2005) or Vibrio mimicus (Abd et al.,

2010) in A. castellanii. The significance of both this location within the cyst structures, but outside of the cytoplasm and the fact that the bacteria seem clustered together remain to be determined. The survival of other bacteria such as F. tularensis or Shigella sp. in A. castellanii cysts has also been reported, but the bacteria are intracellular and not associated with the outer surface (Abd et al., 2003; Saeed et al., 2009). According to Ben Salah & Drancourt (2010), the cellulase encoded by some bacteria may play a role in their exocyst location. Moreover, this location could allow the bacterium to more rapidly escape from the cyst. In this study, we have shown that the presence of A. castellanii or A. culbertsoni may allow increase of A. baumanii growth, whatever the co-culture medium, PAS or filtered water. The presence of A. baumanii did not influence the viability of A. castellanii, but did dramatically decrease the viability of A. culbertsoni. When the cells were incubated in a hostile medium such as the encystment medium, the presence of the amoebae (A. castellanii or A. culbertsoni) increased the viable counts of bacteria, even after 60 days of incubation.

culbertsoni or A. castellanii supernatants obtained in PAS may likewise lead to increased bacterial counts (Fig. 3). The results were similar with supernatants obtained from filtered tap water (data not shown). This could be due, particularly for A. culbertsoni, to the death and the lysis of amoeba, especially when they were co-cultivated with A. baumanii, which could provide nutrients for the bacteria to grow. Among microorganisms related to amoebae, some bacteria

that may be human pathogens have evolved in a way that allows them to resist destruction by protozoa either because they are not internalized or else because they are able to survive, grow and exit amoebae following internalization (Greub & Raoult, 2004). We have evaluated the growth and survival of the bacterium in a poor medium such as encystment medium with and without amoebae. The bacterial count showed that the presence of amoebae (A. castellanii or A. culbertsoni) BI 6727 manufacturer allows for increased bacterial PD98059 mouse growth, while A. baumanii in the same medium without amoebae is able to survive, but at lower concentrations. After 60 days in this medium, the survival of the bacteria is favored by the presence of amoebae (Fig. 4) In electron microscopy after 11 days of incubation, some cysts already contained intracellular

A. baumanii, located only in the space between the double walls (Fig. 2), which is similar to the location of Pseudomonas in Acanthamoeba astronyxis (Marciano-Cabral & Cabral, 2003), Mycobacterium avium in A. polyphaga (Steinert et al., 1998), Mycobacterium sp. (Sharbati-Tehrani et al., 2005; Ben Salah & Drancourt, 2010), V. cholerae Docetaxel order (Abd et al., 2005) or Vibrio mimicus (Abd et al.,

2010) in A. castellanii. The significance of both this location within the cyst structures, but outside of the cytoplasm and the fact that the bacteria seem clustered together remain to be determined. The survival of other bacteria such as F. tularensis or Shigella sp. in A. castellanii cysts has also been reported, but the bacteria are intracellular and not associated with the outer surface (Abd et al., 2003; Saeed et al., 2009). According to Ben Salah & Drancourt (2010), the cellulase encoded by some bacteria may play a role in their exocyst location. Moreover, this location could allow the bacterium to more rapidly escape from the cyst. In this study, we have shown that the presence of A. castellanii or A. culbertsoni may allow increase of A. baumanii growth, whatever the co-culture medium, PAS or filtered water. The presence of A. baumanii did not influence the viability of A. castellanii, but did dramatically decrease the viability of A. culbertsoni. When the cells were incubated in a hostile medium such as the encystment medium, the presence of the amoebae (A. castellanii or A. culbertsoni) increased the viable counts of bacteria, even after 60 days of incubation.

, 2008) and rtxA1 was used to determine the location of CTX prophage in the large chromosome (Colombo et al., 1994; O’Shea et al., 2004). The rtxA gene encodes a presumptive cytotoxin that Ku-0059436 supplier is a part of the RTX (repeats in toxin) gene cluster containing GD-rich repeated motifs, which represent a family of toxin well disseminated in Gram-negative bacteria and has been reported to be present in the large chromosome adjacent to ctx genes (Lin et al., 1999; Sheahan et al., 2004). Vibrio cholerae O1 strains devoid of a CTX prophage in the small chromosome but possessed of the same in the large chromosome without

any direct repeat sequence (RS) element connecting the core downstream of ctx genes will yield an amplicon of nearly 2.4 kb. Another combination of primers zotF and rtxA1 was this website used to determine the presence of CTX prophages lacking the ctxAB operon and lying downstream of the RS1 element adjacent to rtx genes, which will produce an expected amplicons of ∼2.35 kb. Purified genomic DNA was treated with suitable restriction endonuclease enzymes and separated by electrophoresis in 0.8% agarose

gels. DNA fragments were denatured by treatment with alkali and subsequently transferred to a nylon membrane (Hybond-N+; Amersham Pharmacia Biotech), according to the procedure of De et al. (2005), and hybridized with a DNA probe. CTX typing was performed by digesting the genomic Thiamet G DNA with HindIII, PstI, AvaI and BglII (Takara). A 540-bp XbaI–ClaI fragment of ctxA

was ligated with the EcoRI linker and subsequently the ligated product was cloned into the EcoRI site of pKTN901 that served as a probe for ctxA (Kaper et al., 1988). The specific probes of cep (core-encoded pilus) encoding a putative colonization factor present in the core (Pearson et al., 1993), rstRET and rstRcalc, which are cloned in the plasmids pSC01, pSC06 and pSC10, respectively, were obtained by digesting the plasmids individually with EcoRI (Chatterjee et al., 2007). DNA probes were labelled with chemiluminescent dye (Amersham Biosciences) and hybridization reactions were developed following the manufacturer’s protocol and recognition patterns recorded on X-ray film. The results of MAMA PCR showed that all V. cholerae O139 strains isolated up to 1995 yielded amplicons with El Tor allelic primer pair of ctxB only. But 54% and 18% of the V. cholerae O139 strains isolated during 1996 produced amplicons with El Tor and classical specific ctxB primer pairs, respectively, while 28% of the tested strains yielded amplicon with both classical and El Tor primer pairs of ctxB (Table 2). The same trend was continued among V. cholerae O139 strains isolated in 1997. Strains isolated during 1998 did not produce amplicons using only the El Tor ctxB primer pair, but 68% produced amplicon with classical specific ctxB primers and 32% yielded amplicons with both classical and El Tor-specific ctxB primer pairs.

g. pSLGP, pSPHCH01, pSWIT01),

which presumably are not involved in the degradation of organic compounds, although the relevant annotations suggest that plasmids pSLPG and pSPHCH01 carry several genes, which are related to the resistance against toxic metals such as Cu or Hg. Unfortunately, there is some confusion in the annotation of the genes encoding the rep genes in this group. Thus, these genes have been annotated as repA in the case of plasmids pCHQ1, pLA1 EPZ-6438 manufacturer and pSLGP, but as repB for plasmids pISP0, pSWIT01 and pSPHCH01. This differentiation is not reflected by the phylogenetic trees obtained in the course of the sequence comparisons and thus should be avoided (see e.g. Fig. 1). The coexistence of plasmids pSWIT01 and pSWIT02

in S. wittichii RW1 suggests that also the plasmids belonging to the ‘Mega-RPA-group’ (pSWIT02) and ‘Mega-Rep3-group’ (pSWIT01) represent different incompatibility groups within the sphingomonads. The sequence comparisons also suggested that the smaller plasmids in general code for Rep proteins which either belong to the HTH-36 superfamily or the RPA superfamily (Table 1). [But it should be kept in mind that Pfam 10134 (=RPA superfamily) and Pfam 01051(=Rep_3 superfamily) define closely related sequences.] The dendrogram also suggested that pISP2 and pUT1, pISP3 and pSY2, and pISP4 and pYAN-2, respectively, carry closely related selleck Rep proteins. As plasmids pISP2, pISP3 and pISP4 are able to coexist in Sphingomonas sp. MM-1, this might indicate that these groups represent three additional ‘incompatibility groups’ within the sphingomonads which might mainly enclose smaller plasmids. The identification of Rep proteins belonging to the RepA_C-, Rep_3- and RPA-superfamilies P-type ATPase clearly demonstrated that the plasmids from sphingomonads are closely related to plasmids from other bacterial groups. Thus, RepA proteins belonging to the RepA_C family

have previously been described for plasmids from the incompatibility group IncW. The members of this incompatibility group (e.g. plasmids R388 or pSa) are known as broad-host-range plasmids and have already been isolated from Alphaproteobacteria (Fernández-Lopez et al., 2006). Similarly, Rep proteins belonging to the Rep_3 family have been identified in broad-host-range plasmids belonging to the IncN family (such as e.g. plasmid R46). Furthermore, a recent ‘metagenomic’ survey of rep genes obtained from activated sludge communities demonstrated that these three types of rep genes are rather prevalent among the rep genes observed in these complex communities (Sentchilo et al., 2013). The analysis of the large ‘megaplasmids’ pNL1 and pCAR3 had demonstrated that on these plasmids, parA and parB genes are located in close proximity to the repA genes (Romine et al., 1999; Shintani et al.

IMC captures heat flow in the microwatt (μW) range and enables detection of the metabolic heat evolved from ca. 10 000 mammalian cells or ca. 100 000 bacteria (Braissant et al., 2010). Thus, IMC has the potential to provide real-time quantitative data on metabolic activity, aggregation, and biomass formation in biofilms in situ. The sensitivity of IMC has been exploited in evaluating C646 mw metabolism and growth of living cells in culture in medical and environmental microbiology (Howell et al., 2012). While IMC

has been applied to study the co-aggregation of different strains of biofilm-forming bacteria (Postollec et al., 2003), studies that focus on the use of this technique for investigating in vitro multispecies biofilms are scarce. The purpose of this study was to characterize a peri-implantitis-related biofilm by well-established commonly used microscopic methods and to complement this information using IMC to determine various measures 3-deazaneplanocin A of the metabolic activity. A three-species biofilm was allowed to form on surfaces of protein-coated titanium disks in a newly developed anaerobic flow chamber system. The selected bacterial species were an early colonizer, Streptococcus sanguinis; a pathogenic bridging organism, Fusobacterium nucleatum; and a common periodontal and peri-implant pathogen, Porphyromonas gingivalis (Quirynen et al.,

Cell press 2006; Fürst et al., 2007; Heuer et al., 2007). Streptococcus sanguinis (DSM 20068), F. nucleatum (ATCC 10953), and P. gingivalis (DSM 20709) were used for the biofilm formation. A 10 μL inoculum of S. sanguinis in skim milk solution (stored at −20 °C) was suspended in 5 mL Schaedler broth (BBL™; Becton Dickinson, Basel, Switzerland) and incubated aerobically at 37 °C for 8 h. The bacterial suspension was used

as an inoculum for a new subculture (1 : 50), which was incubated aerobically at 37 °C for 16 h. The culture was ultrasonicated for 30 s (22.5 W; Vibracell, Sonics & Materials, Newtown, CT), centrifuged at 5700 g for 5 min at room temperature, washed with physiological saline, and harvested by centrifugation. The S. sanguinis cells were resuspended in simulated body fluid (Cho et al., 1995) to a density of 1.1 × 108 ± 6.2 × 107 CFU mL−1. Fusobacterium nucleatum and P. gingivalis were maintained in Microbank® blue vials (Chemie Brunschwig AG, Basel, Switzerland) at −70 °C. One pearl of each frozen culture was inoculated into 10 mL thioglucolate aliquots (Biomerieux SA, Geneva, Switzerland), enriched with 5 μg mL−1 hemin (Fluka, Buchs, Switzerland) and 0.5 μg mL−1 menadione (VWR International, Dietikon, Switzerland), and incubated anaerobically at 37 °C for 96 h. The cultures were harvested; F. nucleatum and P. gingivalis were suspended to a density of 3.2 × 107 ± 1.9 × 106 CFU mL−1 and 2.1 × 109 ± 9.3 × 108 CFUmL−1, respectively.

IMC captures heat flow in the microwatt (μW) range and enables detection of the metabolic heat evolved from ca. 10 000 mammalian cells or ca. 100 000 bacteria (Braissant et al., 2010). Thus, IMC has the potential to provide real-time quantitative data on metabolic activity, aggregation, and biomass formation in biofilms in situ. The sensitivity of IMC has been exploited in evaluating Small molecule library metabolism and growth of living cells in culture in medical and environmental microbiology (Howell et al., 2012). While IMC

has been applied to study the co-aggregation of different strains of biofilm-forming bacteria (Postollec et al., 2003), studies that focus on the use of this technique for investigating in vitro multispecies biofilms are scarce. The purpose of this study was to characterize a peri-implantitis-related biofilm by well-established commonly used microscopic methods and to complement this information using IMC to determine various measures ICG-001 in vitro of the metabolic activity. A three-species biofilm was allowed to form on surfaces of protein-coated titanium disks in a newly developed anaerobic flow chamber system. The selected bacterial species were an early colonizer, Streptococcus sanguinis; a pathogenic bridging organism, Fusobacterium nucleatum; and a common periodontal and peri-implant pathogen, Porphyromonas gingivalis (Quirynen et al.,

OSBPL9 2006; Fürst et al., 2007; Heuer et al., 2007). Streptococcus sanguinis (DSM 20068), F. nucleatum (ATCC 10953), and P. gingivalis (DSM 20709) were used for the biofilm formation. A 10 μL inoculum of S. sanguinis in skim milk solution (stored at −20 °C) was suspended in 5 mL Schaedler broth (BBL™; Becton Dickinson, Basel, Switzerland) and incubated aerobically at 37 °C for 8 h. The bacterial suspension was used

as an inoculum for a new subculture (1 : 50), which was incubated aerobically at 37 °C for 16 h. The culture was ultrasonicated for 30 s (22.5 W; Vibracell, Sonics & Materials, Newtown, CT), centrifuged at 5700 g for 5 min at room temperature, washed with physiological saline, and harvested by centrifugation. The S. sanguinis cells were resuspended in simulated body fluid (Cho et al., 1995) to a density of 1.1 × 108 ± 6.2 × 107 CFU mL−1. Fusobacterium nucleatum and P. gingivalis were maintained in Microbank® blue vials (Chemie Brunschwig AG, Basel, Switzerland) at −70 °C. One pearl of each frozen culture was inoculated into 10 mL thioglucolate aliquots (Biomerieux SA, Geneva, Switzerland), enriched with 5 μg mL−1 hemin (Fluka, Buchs, Switzerland) and 0.5 μg mL−1 menadione (VWR International, Dietikon, Switzerland), and incubated anaerobically at 37 °C for 96 h. The cultures were harvested; F. nucleatum and P. gingivalis were suspended to a density of 3.2 × 107 ± 1.9 × 106 CFU mL−1 and 2.1 × 109 ± 9.3 × 108 CFUmL−1, respectively.

, 2003; Burch-Smith et al., 2004). Recently, a bean pod mottle virus (BPMV)-based vector was developed for foreign gene expression and endogenous gene silencing in Fabaceae plants (Zhang & Ghabrial, 2006; Zhang et al., 2010). The development of the BPMV viral vector facilitated investigation of the molecular interaction in the common bean–P. syringae system. Here, a BPMV-based vector was used to study the virulence function of HopF1 in bean cultivar Tendergreen based on background researches of HopF2 functioning in Arabidopsis. Our studies

Doxorubicin supplier displayed similarities and differences for the virulence mechanisms between the two homologs of the HopF family effector. Common bean (Phaseolus vulgaris L.) plants

of Tendergreen were grown in the greenhouse with day and night temperatures of 25 and 20 °C, respectively. Bacterial strains and plasmids used are listed in Supporting Information, Table S1. Isolates and modified strains of Psp were cultured at 28 °C in King’s medium B with corresponding antibiotics. Plant inoculation and bacterial growth assays were performed according to Tsiamis et al. (2000) and Fu et al. (2009). Fully expanded leaves of bean cultivar Tendergreen were vacuum-infiltrated with a bacterial suspension of 1 × 106 CFU mL−1 for bacterial population counts or syringe-infiltrated with a bacterial suspension of 5 × 108 CFU mL−1 for phenotypic tests. Bean leaves to be detected were first sliced PF-02341066 ic50 into 1-mm strips and then kept in double distilled water (ddH2O) in a 96-well plate for 12 h. The ddH2O was then aspirated and replaced with a fresh solution

containing 1 μM flg22, 10 μg mL−1 horseradish peroxidase (Sigma) and 20 μM luminol in dimmed light. Luminescence was measured and calculated with a Modulus microplate luminometer (Turner Biosystems). Full expanded primary leaves of bean without infection ROS1 or infection with BPMV vectors for gene overexpression or silence were vacuum-infiltrated with 1 μM flg22 or ddH2O. Whole leaves were collected 24 h post infiltration (or as indicated in Fig. 1c), stained with 0.1% (w/v) aniline blue for 15 min (Hauck et al., 2003), mounted in 50% glycerol and examined with a UV epifluorescence microscope (Olympus BX51). The amount of callose deposits was counted with image j software (http://www.uhnresearch.ca/wcif ). Primary fully expanded bean leaves were sprayed with 2 μM flg22 or ddH2O for inoculation at the indicated time points. After treatment, protein was immediately extracted for in-gel kinase assay performed as described previously (Zhang et al., 2007). Ten micrograms of total protein was electrophoresed on sodium dodeclysulfate-polyacrylamide gels embedded with 0.25 mg mL−1 of myelin basic protein (Invitrogen) in the separating gel as a substrate for the kinase.

Infrequent diagnoses (those

with a frequency of <10 cases) were also recorded. Continuous variables were expressed as the mean and standard deviation when normally distributed, as the median and interquartile range (IQR) if distribution was skewed, and discrete variables as percentages. The Student's independent samples t-test was used to compare continuous variables and the Mann–Whitney U-test for continuous variables without a normal distribution. The association between categorical variables was evaluated using a chi-squared test (when samples were of sufficient size) or with a Fisher's exact test. Magnitude of the DZNeP effect was expressed as a 95% confidence interval. A p value of <0.005 was considered statistically

significant. A total of 2,993 travelers were included in the study; 11 of them were excluded because destination did not correspond with the areas included in the study. The total number of travelers analyzed was 2,982. In total, 47.8% were women; median age was 35 years (IQR 28 to 40). Median time elapsed from return to consultation was 30 days (IQR 13 to 90). Geographical areas of travel and number of travelers to each area are shown in Figure 1. The duration of travel in order of frequency was: short term in 1,594 (53.4%), long term in 710 (23.8%), and medium term in 678 (22.7%) cases. The type of travel in order of frequency was: type A in 979 (32.8%), type B in 511 (17.1%), type C in 508 (17%), and type D in 984 (33%) Selleck APO866 cases. The age of the traveler, duration, and type of travel depending on the geographic area visited are shown in Table 1. In total, 2,062 had received a travel-related vaccine (69.1%), and the median number of vaccines received was two (IQR: 1 to

4). In order of frequency, vaccines received were: yellow fever (79.1%), typhoid fever (55.9%), tetanus–diphtheria (44%), hepatitis B (40.6%), and hepatitis A (31.8%). Complete information was available regarding malarial chemoprophylaxis in 2,568 (86.08%) cases. In total, 1,059 (35.5%) had taken malarial chemoprophylaxis, with variations according to geographical area of travel: prophylaxis was used by 54.4% of travelers to sub-Saharan Africa, 33.5% to Central Asia Southeast, 19.4% to South America, 11.5% Tyrosine-protein kinase BLK to the Caribbean–Central America, and 5.1% to other destinations (p < 0.05). Of these 1,059, 623 (58.8%) took chemoprophylaxis correctly. This proportion varied depending on the drug used: 57 of 71 (80.3%) taking atovaquone–proguanil did so correctly, 274 of 409 (67%) taking mefloquine, 23 of 43 (53.5%) taking doxycycline, 193 of 379 (50.9%) taking chloroquine–proguanil, and 85 of 176 (48.3%) taking chloroquine; χ2 = 43.3 (p < 0.001). More than 75% of the cases had one of the following five presenting syndromes: 1,028 (34.5%) febrile syndrome, 872 (29.

The effect of HIV coinfection Selleck RGFP966 on the risk of cirrhosis and hepatocellular carcinoma in U.S. veterans with hepatitis C. Am J Gastroenterol 2005; 100: 56–63. 26  Tedaldi E, Peters L, Neuhaus J et al. Opportunistic disease and mortality in patients coinfected with hepatitis B or C virus in the strategic management of antiretroviral therapy (SMART) study. Clin Infect Dis 2008; 47: 1468–1475. 27  van der Helm J, Geskus R, Sabin C et al. Effect of HCV infection on cause-specific mortality after HIV seroconversion, before and after 1997. Gastroenterology 2013; 144: 751–760. 28  Smit C, van den Berg C, Geskus R, Berkhout B, Coutinho R, Prins M. Risk

of hepatitis-related mortality increased among hepatitis C virus/HIV-coinfected drug users compared with drug users infected only with hepatitis C virus: a 20-year prospective study. J Acquir Immune Defic Syndr 2008; 47: 221–225. selleck chemicals llc 29  Weber R, Ruppik M, Rickenbach M et al. for the Swiss HIV Cohort Study (SHCS). Decreasing mortality and changing patterns of causes of death in the Swiss HIV Cohort Study. HIV Med 2013: 14: 195–207. 30  Thomson EC, Nastouli E, Main J et al. Delayed anti-HCV antibody

response in HIV-positive men acutely infected with HCV. AIDS 2009; 23: 89–93. 31  Nastouli E, Thomson EC, Karayiannis P, Main J, McClure MO, Muir D. Diagnosing acute hepatitis C in HIV-infected patients: Nucleic acid testing compared with antibody and antigen–antibody detecting methods. J Clin Virol 2009; 44: 78–80. 32  Yaphe S, Bozinoff N, Kyle R, Shivkumar S, Pai NP, Klein M. Incidence of acute hepatitis C virus infection among men who have sex with men with and without HIV infection: a

systematic review. Sex Transm Infect 2012; 88: 558–564. 33  Gamage DG, Read TR, Bradshaw CS et al. Incidence of hepatitis-C among HIV infected men who have sex with men (MSM) attending a sexual health L-gulonolactone oxidase service: a cohort study. BMC Infect Dis 2011; 11: 39. 34  Lambers FA, Prins M, Thomas X et al. Alarming incidence of hepatitis C virus re-infection after treatment of sexually acquired acute hepatitis C virus infection in HIV-infected MSM. AIDS 2011; 25: F21–F27. 35  Larsen C, Chaix ML, Le Strat Y et al. Gaining greater insight into HCV emergence in HIV-infected men who have sex with men: the HEPAIG Study. PLoS ONE 2011; 6: e29322. 36  Jones R, Brown D, Nelson M et al. Re-emergent hepatitis C viremia after apparent clearance in HIV-positive men who have sex with men: reinfection or late recurrence? J Acquir Immune Defic Syndr 2010; 53: 547–550 (and correction JAIDS 2010; 54; 112). 37  Peters L, Mocroft A, Soriano V et al. Hepatitis C virus reappearance in HIV-infected patients with spontaneous HCV-RNA clearance. J Hepatol 2009; 50(Suppl 1): S155. 38  Martin T, Martin N, Hickman M et al. HCV reinfection incidence among HIV-positive men who have sex with men. 19th Annual Conference of the British HIV Association. Manchester, UK. April 2013 [Abstract O7].

The Inhibitor Library manufacturer methoxymalonyl-ACP biosynthesis locus, which is composed of fkbGHIJK, was first reported in the FK520 biosynthetic gene cluster (Wu et al., 2000). The roles of fkbGHIJK orthologs in the biosynthesis of methoxymalonyl-ACP

were then substantiated by genetic and biochemical experiments (Fig. 1b) (Kato et al., 2002; Chan et al., 2006; Dorrestein et al., 2006). The methoxymalonyl-ACP biosynthesis locus is generally colocalized with the related modular PKS gene cluster. In the present study, a gene disruption experiment establishes that a methoxymalonyl-ACP biosynthesis locus (galGHIJK) is involved in the biosynthesis of galbonolide A in S. galbus. It is also shown that orf4, which is proximal to galGHIJK and contains a KAS domain, is involved in the biosynthesis of galbonolides A and B. Notably, there is no multimodular PKS gene cluster flanking Adriamycin these loci, however. Streptomyces galbus KCCM 41354 was acquired from the Korean Culture Center of Microorganisms. Escherichia coli DH5α was used as the host for general subcloning. For total DNA isolation, S. galbus was grown in tryptic soy broth with 10 mM MgCl2·6H2O and 0.5% w/v glycine. Glucose–yeast extract–malt extract (GYM) agar was used for S. galbus spore preparation. Escherichia

coli ET12567 (dam−, dcm−, hsdS−)/pUZ8002 was the nonmethylating plasmid donor strain for the intergeneric conjugative transfer to S. galbus (Flett et al., 1997). GYM agar supplemented with 10 mM MgCl2 was used for the conjugation experiment. Genetic procedures including the gene disruption Suplatast tosilate experiment were performed using standard procedures (Kieser et al., 2000). Southern hybridization

was performed with digoxigenin DNA labeling and a detection kit from Roche Diagnostics (Pleasanton, CA) by following the procedure outlined by the manufacturer. An 800-bp DNA fragment of an fkbI homologue was amplified from the S. galbus chromosome using the PCR and the primer set of 5′-CAGGGCATGGCCGCSTG GACSGT-3′ and 5′-GATGATCTCCATSAGCTTSGCRTC-3′ (Li et al., 2006), which was then cloned into the pGEM-T Easy vector (Promega, Madison, WI). This DNA clone was named pHJK1001. A cosmid library of S. galbus KCCM 41354 genomic DNA was constructed using SuperCos I and the Gigapack III Gold packaging extract kit according to the manufacturer’s handbook (Stratagene, La Jolla, CA). A 3.2-kb KpnI DNA fragment was isolated from the S. galbus genome using the 800-bp fragment in pHJK1001 as a probe, and the presence of methoxymalonyl-ACP biosynthetic genes (galGHI and a truncated galJ) was confirmed by nucleotide sequencing. The 3.2-kb KpnI DNA fragment was then used as a probe in screening the cosmid library, which resulted in the isolation of a positive clone pHJK1011. The 800-bp fragment internal to galI was isolated as an EcoRI fragment from pHJK1001 and ligated into pKC1139 to generate pD-galI, a galI-disruption plasmid. The conjugative plasmid pKC1139 contains an E.