, 2008) However, no international clone III isolates were identi

, 2008). However, no international clone III isolates were identified in this study. Since bacterial motility is a known virulence factor in numerous bacterial species (Han et al., 2008; Alarcon et al., 2009; Proft & Baker, 2009), the motility potential of our 52 clinical isolates was examined. The motility phenotypes in this study were determined using the general classifications for both swarming and twitching (Semmler et al.,

1999; Kaiser, 2007). Our data revealed that all international clone I isolates showed significant twitching. A number of other twitching isolates, not part of this clonal lineage, had the ability to form well developed biofilms compared to the international clone I isolates (see below), Selleck PLX3397 with the exception of A. baumannii strain D1279779. This relatively poor biofilm former (OD595 nm<1) also showed a small twitching zone (approximately 12 mm). Swarming motility was Fluorouracil observed in three noninternational clone isolates, including A. baumannii ATCC 17978, a fully sequenced reference strain. Studies using MH and LB media showed that twitching and swarming phenotypes are largely medium dependent. Furthermore, twitching and swarming

were demonstrated to be distinct characteristics, as many twitchers did not swarm, and A. baumannii strain ATCC 17978 swarmed, but did not twitch. PilA showed a high degree of amino acid sequence conservation within twitching isolates, indicating that type IV pili may play a role in motility in this species. Examination of biofilm formation showed that there was a significant difference between international clone

I and II isolates, correlating with previously published data (de Breij et al., 2010). We also found a significant difference (P < 0.05) between international clone I and noninternational clone isolates, indicating that in general international clone I isolates are limited in their ability to form biofilms. We determined the adherence of selected A. baumannii isolates to eukaryotic cells of nasopharyngeal (Detroit 562) and alveolar (A549) origin. Not only were significant differences observed between strains, two Glutamate dehydrogenase isolates, D1279779 and ATCC 17978, showed significantly lower adherence to nasopharyngeal cells compared to lung epithelial cells. Comparison of the ability to form biofilms and eukaryotic cell adherence revealed no relationship between these two phenotypes in the strains tested. This suggests that the mechanism of adherence to either abiotic or biotic surfaces appears to be different and draws a parallel with the results from other studies (Lee et al., 2008; de Breij et al., 2010). Moreover, previous studies have shown that adherence to abiotic surfaces is in part mediated by the csu type I pili cluster in strain ATCC 19606 (Tomaras et al., 2003), however, in a subsequent study using the same csu knockout strain, no difference was observed in the ability to bind bronchial cells (de Breij et al., 2009).

36; 95% confidence interval (CI) 208, 542] Greater than 95% ad

36; 95% confidence interval (CI) 2.08, 5.42]. Greater than 95% adherence to ART (AOR 1.80; 95% CI 1.14–2.84) and having a baseline CD4 count >200 cells/μL (AOR 2.18; 95% CI 1.29–3.68) were also associated Lenvatinib supplier with having the maximum number of possible combinations. This study found that a high proportion of resistance mutations among individuals who initiated ART with NNRTI-based regimens had the potential to markedly reduce the number of future options for second-line drug regimens. This was demonstrated by the median GSS after use of NNRTI-based first-line regimens,

which was 9.8 as compared with 11.0 after boosted PI-based first-line regimens. The odds of having all available active combinations was more than three times higher in

participants who initiated treatment on boosted PIs. The study also showed that the proportion of individuals with more ART combinations for those who initiated boosted PI-based ART was almost twice that for those who initiated ART with NNRTIs. As HIV-positive individuals are now living longer, the availability of alternative drug options in the face of drug resistance becomes an important issue to consider. The clinical significance of this reduced GSS among ART-naïve patients starting with NNRTI-based regimens is that these patients may run out of drug DAPT price options among the readily available drugs in RLSs more rapidly. This problem is made worse by the higher cost of newer antiretroviral drugs. This also may contribute to the many factors leading to unbalanced benefits from ART between developed and the resource-limited settings. Although the absolute difference in GSS was small in terms of the median number of active drugs available in each group (9.8 vs. 11), the distribution

find more of these limitations for the NNRTI group was significant, such that over 40% of these patients had fewer than five drug combinations available to them after only 3 years of treatment. A recent cost-effectiveness analysis found that the use of boosted PI (lopinavir/ritonavir) as first-line therapy was very cost effective, especially in individuals with prior exposure to NNRTIs and those with unknown drug resistance profiles (cost-effectiveness ratio $1520/year of life saved versus first-line nevirapine) [23]. Given that in 2008 45% of HIV-infected women in RLSs had received some form of antiretroviral drugs (mainly nevirapine and/or zidovudine) for the prevention of mother-to-child transmission of HIV [24], and widespread resistance testing is not available in the region, consideration should be given to recommending boosted PIs as first-line therapy. This study confirmed that participants on NNRTI-based first-line regimens are more prone to develop antiretroviral drug resistance mutations as compared with those on boosted PI first-line regimens.

loti chromosome (Fig 1, bottom) The numbering of the genes is f

loti chromosome (Fig. 1, bottom). The numbering of the genes is fixed in the RhizoBase (genome database for Rhizobia, http://bacteria.kazusa.or.jp/rhizobase/). The first enzyme, pyridoxine 4-oxidase, is encoded by the mll6785 gene (Yuan et al., 2004); the second, pyridoxal 4-dehydrogenase, by mlr6807 (Yokochi et al., 2006); the third, 4-pyridoxolactonase, by mlr6805 (Funami et al., 2005); the fourth, 4-pyridoxic acid dehydrogenase, by mlr6792 (Ge et al., 2008); the fifth, 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic

acid (FHMPC) dehydrogenase, by mlr6793 (Yokochi et al., 2009); the sixth, 3-hydroxy-2-methylpyridine-4,5-dicarboxylic acid (HMPC) decarboxylase, by mlr6791 (Mukherjee et al., 2007); the seventh, 3-hydroxy-2-methylpyridine-5-carboxylic acid (HMPC) oxygenase, by mlr6788 (Yuan learn more et al., 2006; McCulloch buy PI3K Inhibitor Library et al., 2009); and the eighth, AAMS amidohydrolase, by mlr6787 (Mukherjee et al., 2008; Yuan et al., 2008). Pyridoxamine is converted into pyridoxal by pyridoxamine-pyruvate aminotransferase

encoded by mlr6806 (Yoshikane et al., 2006). Thus, the genes form a cluster, from mll6785 to mlr6807, including several genes of unknown function. The expression of genes involved in bacterial catabolic pathways is often regulated by one or several transcriptional regulators (Tropel & Van der Meer, 2004). The GntR family proteins are well known transcription factors and comprise more than 8500 members in the Pfam database (Hoskisson & Rigali, 2009). They are distributed throughout the bacterial world. The GntR regulators are subdivided into the AraR, DevA, FadR, HutC, MocR, PlmA, and YtrA subfamilies based on the sequence FER similarity of their C-terminal effector-binding oligomerization domains. The FadR subfamily is the most representative GntR subfamily and can be divided into FadR and VanR subgroups based on the number of α-helices (10 and 9, respectively) in their secondary structures (Rigali et al., 2002). In the cluster of genes involved in the degradation of pyridoxine (Fig. 1b) there is one gene (mll6786) that encodes a probable transcriptional regulator protein. The primary structure and deduced secondary structure suggested that mll6786

encodes a regulator protein that belongs to the VanR subgroup. As far as we know, no study has been done on the regulation mechanism for the degradation pathway for pyridoxine. Here, we identified the protein PyrR encoded by mll6786 as a transcriptional repressor protein. The recombinant repressor protein was over-expressed and characterized as the first step of elucidation of the regulatory mechanism for the pyridoxine-degradation pathway in M. loti cells. Escherichia coli strains BL21(DE3) and JM109 were purchased from Novagen (San Diego, CA) and Takara (Tokyo, Japan), respectively. Escherichia coli S17-1 was obtained from the National Bioresource Project (Mishima, Japan). Mesorhizobium loti MAFF303099 was obtained from the MAFF GenBank (Tsukuba, Japan).

PamI recognition site We also found that activity of this enzyme

PamI recognition site. We also found that activity of this enzyme was lower in E. coli than in P. aminophilus JCM 7686 (CCATGG sites of plasmids pAMI702 selleck chemicals llc and pACYC184/MRW were only partially resistant to NcoI digestion (Fig. 4a) compared with those of the pAMI7 plasmid (see above),

which most probably resulted from the different growth conditions of the strains (37 vs. 30 °C). To examine the putative role of the PamI system in stable maintenance of pAMI7, we tested the stability of two mini-derivatives of this plasmid: (1) pAMI702 (contains PamI) and (2) pAMI703 (deprived of PamI). The retention of both plasmids was determined in plasmid-less P. pantotrophus KL100, a strain routinely used in our laboratory as a host for paracoccal plasmids. The analysis revealed that pAMI702 was stably maintained: 74% of cells still harbored the plasmid http://www.selleckchem.com/products/ipilimumab.html after 30 generations of growth in nonselective medium. In contrast, pAMI703, which lacks PamI, was highly unstable and rapidly lost under analogous growth conditions (3% stability). We also tested whether or not the PamI system is able to stabilize a heterologous replicon. For this purpose the R-M module was cloned into the low-copy-number shuttle vector pABW3 (unstable in Paracoccus spp.) and the stability of the resulting plasmid pABW3-RM

was tested in the strain KL100. Stabilization of pABW3-RM was observed (53% stability; in comparison with 4% stability of ‘empty’ vector pABW3), which confirmed that the PamI system can act as a plasmid stabilization cassette. In this study we show that ORF14 and ORF15 of plasmid pAMI7 of the methylotrophic bacterium P. aminophilus JCM 7686 constitute a functional type II R-M system, designated PamI. Comparative sequence analysis revealed that related R-M systems are present in the genomes of distinct tuclazepam taxonomic groups of Bacteria (e.g. Actinobacteria, Betaproteobacteria, Chlamydiae, Cyanobacteria, Firmicutes) as well as in a member of the Archaea (Fig. 1). This

finding illustrates that horizontal gene transfer contributes significantly to the dissemination of R-M modules – even between domains. In the case of PamI, the transfer may be enhanced by the location of the system within the mobile plasmid pAMI7, whose replication system is functional in many members of the Alphaproteobacteria (Dziewit et al., 2011). One of the R.PamI relatives is restriction endonuclease NcoI that is frequently used in gene cloning experiments. Although the level of sequence similarity between these two enzymes is not strong (33%), our analysis revealed that they are isoschizomers (they share the same sequence specificity). Interestingly, in silico analysis revealed that the R.PamI and NcoI REases are accompanied by MTases, which generate different methylated products (m5C and m4C, respectively); therefore they are members of different MTase subfamilies. This could indicate recombinational shuffling of genes encoding individual components of R-M systems. Furthermore, we showed that M.

Consistently, in vivo, Nogo-A/EphA4 double KO mice show increased

Consistently, in vivo, Nogo-A/EphA4 double KO mice show increased axonal sprouting and regeneration after spinal cord injury as compared with EphA4 KO mice. Our results reveal the upregulation of developmental axon guidance cues following constitutive Nogo-A deletion, e.g. the EphrinA3/EphA4 ligand/receptor pair, and support their role

in restricting neurite outgrowth in the absence of Nogo-A. “
“The relation between informal musical activities at home and electrophysiological indices of neural auditory change detection was investigated in 2–3-year-old children. Auditory event-related potentials were recorded in a multi-feature paradigm that included frequency, duration, intensity, direction, gap deviants FK506 cost and attention-catching novel sounds. Correlations were calculated between these responses find more and the amount of musical activity at home (i.e.

musical play by the child and parental singing) reported by the parents. A higher overall amount of informal musical activity was associated with larger P3as elicited by the gap and duration deviants, and smaller late discriminative negativity responses elicited by all deviant types. Furthermore, more musical activities were linked to smaller P3as elicited by the novel sounds, whereas more paternal singing was associated with smaller reorienting negativity responses to these sounds. These results imply heightened sensitivity to temporal acoustic changes, more mature auditory change detection, and less

distractibility in children with more informal musical activities in their home environment. Our results highlight the significance of informal musical experiences in enhancing the development of highly important auditory abilities in early childhood. In recent years, important advances have been made in demonstrating fast neuroplastic effects of formal musical training in childhood (Hyde et al., 2009; Meyer et al., 2011). For the majority of children, however, musical experience does not predominantly involve formal training Chloroambucil on a musical instrument but mainly consists of informal musical activities such as singing and musical play at home. Little is known about how differences in such musical experiences are related to children’s neural auditory discrimination skills. It is evident that young children are well equipped to benefit from a musically enriched home environment. Behavioural and neuroscientific evidence plainly show that even young children possess the necessary auditory capabilities for perceiving music and display great interest in it (Trehub, 2003; Trainor, 2012). Furthermore, multiple lines of evidence indicate that the brain has a considerable capacity for neuroplastic changes in childhood (Trainor, 2005) and therefore might very well be shaped even by informal exposure to sounds.

Finally, protein–protein interactions were identified by SDS-PAGE

Finally, protein–protein interactions were identified by SDS-PAGE. As shown in Fig. 2a, 16.7 kDa His6–WhcA and 62.2 kDa GST–SpiA coeluted together,

indicating specific binding. Nonspecific binding of the GST–SpiA protein to the beads was not observed (data not shown). Purified maltose-binding protein, which was used to assess nonspecific interactions, did not bind to the bait His6–WhcA (data not shown). However, the band intensity of the GST–SpiA protein was lighter than expected (Fig. 2a), suggesting a weak protein Thiazovivin manufacturer interaction. If protein–protein interactions occurred at a 1 : 1 molar ratio, the band intensity of the GST–SpiA protein, which was three times larger than the His6–WhcA in size, should be approximately three times stronger than that of the His6–WhcA band. This discrepancy could be due

simply to inefficient refolding, leaving only a fraction of Sunitinib manufacturer the bead-bound His6–WhcA in the correct conformation. Alternatively, fractions of the refolded His6–WhcA could have lost their Fe–S cluster during the denaturation–refolding process, thus remaining in an alternative conformation that does not interact with GST–SpiA (see Discussion). Nevertheless, the pull-down assay indicated that WhcA can specifically bind the SpiA protein. So far, we were able to show that WhcA interacts with SpiA via in vivo and in vitro assays. As the WhcA protein was found to play a negative role in the oxidative stress response pathway, we postulated that the protein–protein interaction could be affected by external factors, such as external redox environments. When oxidant diamide was applied to growing HL1387 cells, the interaction between WhcA and SpiA was significantly reduced to 34% relative to those of positive and negative control strains (Fig. 3a). The effect of oxidant menadione

was observable but rather marginal (Fig. 3b), whereas reductant dithiothreitol was not effective at all in disrupting the protein–protein interaction (data not shown). Whereas the thiol-specific oxidant diamide specifically oxidizes sulfhydryl groups (Kosower & Kosower, 1995), the redox-cycling compound menadione exerts its toxic effects via stimulating intracellular production of superoxide radicals and hydrogen peroxide (Hassan & Fridovich, 1979). Phospholipase D1 However, the redox-cycling compound is also known to drain electrons from the reductive pathways, including the thioredoxin system (Holmgren, 1979), thus inducing disulfide bond formation in cells. The differential response of the protein to diamide and menadione may suggest that the cysteine residues of the WhcA protein are involved in disulfide bond formation. To study the effect of diamide on in vitro protein–protein interactions, the pull-down assay was performed in the presence of oxidant diamide, as described in Materials and methods.

Accordingly, the method was then validated recording fluorescence

Accordingly, the method was then validated recording fluorescence at each 0.3 °C step. Tm was directly measured by the internal software (StepOne Software v2.1; Applied Biosystems). Data were exported and processed according to mathematical algorithms for high-resolution DNA melting analysis (Palais & Wittwer, 2009). Briefly, the background was evaluated MEK inhibitor and removed to the negative derivative of the fluorescence data. The results obtained were then normalized and smoothed with the ‘running average’ method. Graphs were generated with Sigma

Plot 5.0 (SSI, CA). To develop the method, we used three different Map strains, carrying three, four and five repeats; unfortunately, we did not have any strains containing the allele with six repeats in our collection, so we used a synthetic single strand DNA amplicon holding six triplets. For this, 1 μg of

the reverse single strand DNA (Eurofins MWG, Ebersberg, Germany) was copied in the presence of forward primer. The synthetic double strand DNA was then diluted to 10 ng prior to PCR. The number of triplet repeats for all strains was confirmed by sequencing with ABI Prism 3100 Avant check details Sequencer (Applied Biosystems), according to Amonsin et al. (2004). The sequences were analysed using the SeqMan Module within the Lasergene Package (DNA Star, Madison, WI). Representative results of HRM analysis are shown in Fig. 1, as derivative melting curves after normalization and exponential background removal. Two melting domains for each sample were observed: one relative to the amplicon homoduplex product (DNA double strand) and another one relative to the heteroduplex single strand DNA/probe. According to the LATE-PCR strategy, the homoduplex

products were Phenylethanolamine N-methyltransferase generated during the first cycles of amplification, whereas the single strand DNA was generated during the late cycles (data not shown). This single strand DNA can match with the probe and generate the heteroduplex single strand DNA/probe. As shown in Fig. 1, analysis of homoduplex amplicons did not allow any differentiation between the various alleles. However, it did reveal approximately three degrees among the adjacent alleles, allowing an unbiased identification. In silico analysis using the software mentioned above revealed that Tm decreased theoretically by 1 °C for every triplet depletion, a difference between two adjacent alleles (ΔTm) smaller than that found in our analysis. An explanation for this discrepancy could be the formation of loops when the probe (six repeats) matches the DNA single strand of the alleles carrying three, four and five repeats. These secondary structures could contribute to further destabilize the heteroduplex, causing a larger ΔTm among two adjacent alleles with respect to that theoretically evaluated by the software (see Supporting Information, Fig. S1).

The relative lower anti-Candida activity of the shorter lipopepti

The relative lower anti-Candida activity of the shorter lipopeptides could be related to their reduced ability to permeate fungal membranes, because of their low hydrophobic character to drive oligomerization (Malina

& Shai, 2005). The effect of various concentrations of the purified anti-Candida compounds on human erythrocytes is reported in Table 4. The compound a1 showed a weak hemolytic activity (50% hemolysis at 68.26 μM) compared with a2 and a3 (50% hemolysis at 37.41 and 22.14 μM, respectively). This could be due to their low hydrophobicity, and therefore, limited ability to oligomerize, which is an important requirement for both the hemolytic and antifungal activity of an antimicrobial peptide. Prior studies showed Hormones antagonist a direct correlation between the fatty acid chain length of surfactin lipopeptides and hemolytic activity (Kracht et al., 1999). It is noticeable that the hemolytic activity of the lipopeptide bacircines is also dependent on the length of the aliphatic side chain and that hemolysis is provoked by the insertion of the fatty acid chain into the phospholipid bilayer (Prokof’eva et al., 1999). Similarly, iturins A are able to lyse human erythrocytes in a dose-dependent manner (100% Alectinib in vitro hemolysis at 25 μM) (Quentin et al., 1982; Aranda et al., 2005). This

limits their potential usage in clinical therapy (Besson & Michel, 1984; Aranda et al., 2005; Oleinikova et al., 2005; Ramarathnam et al., 2007; Chen et al., 2009). Nevertheless, we found that compound a3 with a long fatty acid chain exhibited a strong inhibitory effect (MFC value between 7.38 and 14.76 μM) against Tacrolimus (FK506) most tested strains

of C. albicans causing mucous and cutaneous infections. Note that at these concentrations a3 compound showed a reduced hemolytic activity (17% and 35%). However, when tested against some pathogenic C. albicans strains causing finger nail candidiasis (C. albicans sp. 265 FN and C. albicans sp. 311 FN), compound a3 exhibited both higher MFC values (between 29.53 and 59.07 μM) and hemolytic activity (between 65.91% and 99.64%). Overall, for the treatment of such pathogenic strains causing cutaneous candidiasis, a local application of the a3 compound rather than a systemic or an oral administration is possible. In conclusion, our data have indicated that B. subtilis produce anti-Candida lipopeptides that might be used to treat cutaneous infections. This work was supported by grants from the ‘Ministère de l’Enseignement Supérieur et de la Recherche Scientifique’ of Tunisia. We thank Prof. E. Aouani for valuable discussion and critical reading of the manuscript. “
“The overall purpose of these guidelines is to provide guidance on best clinical practice in the treatment and management of human immunodeficiency virus (HIV)-positive pregnant women in the UK.

Genome analysis of the obligate marine actinomycetes Salinispora

Genome analysis of the obligate marine actinomycetes Salinispora tropica (Udwary et al., 2007) and Salinispora arenicola (Penn et al., 2009) suggested that they possess multiple siderophore-like PD-332991 biosynthetic loci. Four pathways are predicted in S. tropica CNB-440, whereas only two are retained in S. arenicola CNS-205. Both species maintain a des locus that likely codes for desferrioxamine

(DFO) and a sid2 locus related to the gene cluster for yersiniabactin biosynthesis, ybt (Gehring et al., 1998). Intriguingly, ybt is usually encoded on a high pathogenicity island that mobilizes between pathogenic Gram-negative bacteria to confer virulence (Buchrieser et al., 1998; Schubert et al., 1998; Flannery et al., 2009). Salinispora tropica CNB-440 also encodes two additional nonribosomal peptide synthetase (NRPS) pathways, sid3 and sid4, which

are hypothesized to provide unique salicylate-containing iron chelators similar to dihydroaeruginoic acid (Carmi & Carmeli, 1994) and the predicted ‘coelibactin’ (Bentley et al., 2002). DFOs are hydroxamate-type siderophores with a high affinity for iron (Kd ~ 10−31 M) (Keberle, 1964) that are produced by streptomycetes (Müller & Raymond, 1984; Barona-Gómez et al., 2004) and some Gram-negative bacteria (Martinez et al., 2001; Essén et al., 2007). Several analogs have been reported including Selleck Screening Library linear DFOs B, D and G and cyclic DFO E (Fig. 3a), as well as acyl-DFO analogs with terminal branched alkyl chains or aromatic rings (D’Onofrio et al., 2010; Yang et al., 2011). DFOs are biosynthesized via an NRPS-independent mechanism (Challis, Mephenoxalone 2005), encoded by desA-D (Barona-Gómez et al., 2004; Kadi et al., 2007). Transcription from des is repressed by the divalent metal-dependent regulatory protein DmdR1 and derepressed by iron limitation (Flores & Martín, 2004; Tunca et al., 2007). Predicted homologs to desA-D and the ferric-siderophore uptake and utilization genes (desE-F)

are found in both Salinispora genomes (Fig. 1a). Despite bioinformatic predictions on the siderophores produced by Salinispora, no iron chelators have been isolated from this genus. Therefore, we explored the siderophore chemistry of these marine actinomycetes to determine which of the putative siderophore biosynthetic loci play a role in iron acquisition in Salinispora. Salinispora tropica strain CNB-440, S. arenicola strains CNS-205, CNT-088 and CNH-643 and ‘Salinispora pacifica’ strain CNT-133 were cultured at 30 °C with continuous shaking at 200 r.p.m. in iron-limited media (1 g L−1 NH4Cl, 2 g L−1 casamino acids, 28 g L−1 Instant Ocean (Aquarium Systems Inc.), 0.6% v/v glycerol), supplemented with 36 μM FeSO4 when required. PCR targeting (Gust et al., 2003; Eustáquio et al.

felis or A genospecies 2 After 3 days (corresponding to an incr

felis or A. genospecies 2. After 3 days (corresponding to an increase

of OD600 nm from 0.02 to 0.6), the culture was diluted to an OD600 nm of 0.02. This step was repeated at least seven times. Bacteria were grown on agar without antibiotics and five single colonies were selected as templates for colony PCR. The primer pair MCS-2 FP01 and MCS-2 RP01 (Table 1) led to the production of a single polymerization product HSP inhibitor at approximately 800 bp in the presence of the plasmid. Cover slips were coated with poly-l-lysine and left with bacteria in PBS for 30 min at an ambient temperature. Nonattached bacteria were removed by rinsing three times with PBS and samples were fixed with 3% formaldehyde in PBS for 40 min. GFP-bacteria were visualized at 488 nm excitation and 522 nm emission. Monoclonal antibody CSD11 or

rabbit serum were used compound screening assay as primary antibodies in immunofluorescence, together with Alexa488-coupled goat-anti-rabbit or goat-anti-mouse antibodies as secondary antibodies. Slides were mounted with mowiol, examined and photographed using an Axiophot Epifluorescence Microscope (Zeiss, Oberkochen, Germany). Following a published protocol (Riess et al., 2003) for transposome-directed mutagenesis in the closely related B. henselae using commercially available transposome technology, between 450 and 1900 mutants were obtained per microgram of transposome DNA, making this approach extremely costly. Published efficiencies obtained with this transposition system varied from 1200 clones μg−1 DNA with Xylella fastidiosa (Koide et al., 2004) to 107 clones μg−1 DNA with enteric bacteria (Hoffman et al., 2000). As the electrotransformation efficiency of

A. felis using plasmid DNA was within the expected range, one explanation for the low efficiency was the digestion Doxacurium chloride of the introduced DNA fragments by an Afipia DNA restriction system. Recently, a purified phage protein called ‘ocr’ (Walkinshaw et al., 2002) became available. This phage protein is a strong inhibitor for type I endonucleases (Murray, 2000). Adding purified inhibitor to the transformation mixture increased the efficiency from ∼2000 kanamycin-resistant clones per microgram of transposome DNA to >3 × 104 (Fig. 1). Although it is not known whether Afipia spp. have type I restriction enzyme systems, the strong increase in transposon mutant yields using the inhibitor suggests that the transposon sequence contained a restriction site that is recognized by a type I restriction endonuclease of Afipia. Electroporation in the absence of a transposome yielded no colonies, as expected. To test whether all kanamycin-resistant Afipia clones contained a transposon, we performed PCR reactions with the primer pair Tnp FP01/Tnp RP01 internal of the transposon yielding 1109-bp DNA fragments in positive cases. Eighty-five of 86 tested clones contained a transposon.