3a). The observed localization was quite similar to that of the proteins involved in endocytosis, such as AoEnd4 (Higuchi et al., 2009b). Moreover, we confirmed the colocalization of AipA with AoAbp1 in A. oryzae, suggesting that AipA also plays a role in endocytosis (Fig. 3a). Furthermore, to test whether the localization of AipA was dependent on actin similar to AoAbp1, analysis using Lat B, an inhibitor of actin polymerization, was performed. After Lat B treatment, both EGFP-AipA and AoAbp1-mDsRed were dispersed into the cytoplasm,

suggesting that PI3K Inhibitor Library nmr the localization of both proteins was dependent on actin (Fig. 3b). To analyze the function of AipA, we generated aipA disruptants in A. oryzae and then compared the growth of the control and ΔaipA strains (Fig. S3). However, we did not observe any remarkable phenotype in the ΔaipA strains compared with the control strain under several culture conditions. Moreover, the staining of hyphae with FM4-64, a fluorescent dye that

labels the endocytic pathway, showed no significant defects of endocytosis in ΔaipA strains compared with the control strain (data not shown). To further analyze the function of AipA, we generated an aipA-overexpressing strain, which expresses aipA under the control of PamyB at the niaD locus. The aipA-overexpressing strain showed retarded growth and a wider hyphal morphology (Fig. 4a and b). In S. cerevisiae, K197A and E233Q mutants of Vps4p, a AAA ATPase functioning in the formation of MVB, an endocytic organelle, have defective ATPase activity and, thus, do not function see more Glutathione peroxidase correctly (Babst et al., 1997, 1998). We determined that the ATPase domains of AipA, Sap1p, Yta6p,

and Vps4p are highly conserved (Fig. 4d). For the phenotypic analysis of mutations to the ATPase domain of AipA, strains expressing either aipAK542A or aipAE596Q, the counterpart of vps4K197A or vps4pE233Q, respectively, under control of PamyB were generated. Moreover, we also created egfp-fused WT aipA- and mutant aipA-overexpressing strains and confirmed that their growth was nearly identical to the strains overexpressing WT aipA and mutant aipA without egfp. Microscopic observation verified that there was EGFP fluorescence in most hyphae of these strains (data not shown). Furthermore, by Western blot analysis, it was confirmed that both mutant AipAs fused with EGFP were expressed as EGFP-fused WT AipA was, indicating that mutant AipAs were not degraded (Fig. 4c). In contrast to the aipA-overexpressing strain, mutated aipA-overexpressing strains did not show defective growth or aberrant hyphal morphology, suggesting that ATPase activity is essential for the function of AipA (Fig. 4a and b). To monitor the endocytic process in the aipA-overexpressing strain, we performed a time-course experiment with FM4-64.

The plasmid construct was confirmed by DNA sequencing. A CR plate assay was performed as described previously (Barnhart & Chapman, 2006). To determine curli assembly, each E. coli test strain was grown at 28 °C for 48 h on YESCA plates

containing 50 μg mL−1 CR and 10 μg mL−1 Coomassie blue. As an initial attempt to identify the regulatory role of MlrA on the csgD promoter, we examined the possible influence of its overexpression on the csgD promoter–lacZ reporter fusion. In wild-type background, overexpression of MlrA led to an approximately 2.5-fold increase in csgD–lacZ expression (Fig. 1a). In the csgD knockout mutant, this increase was more than 4.5-fold BIBF 1120 in vivo (Fig. 1b), indicating that MlrA is a positive factor of

the csgD promoter. As overexpression of MlrA did not affect the promoter of the divergently transcribed csgBC operon, the effect of MlrA on curli production is solely attributable to activation of the csgD promoter, ultimately leading to activation of the csgB promoter. The expression level of MlrA in both wild-type E. coli and the csgD mutant was essentially the same as detected by immunoblot analysis of whole-cell lysates (data not shown). To gain insight into how the csgD promoter is activated by MlrA, we next examined the DNA-binding activity of purified MlrA and determined the site of recognition and binding on the csgD promoter. Gel-shift assays using various types of csgD probes selleck chemicals covering various portions of the csgD promoter and its upstream region (for DNA segments see Ogasawara et al., 2010), only the CD6 (−67 to −335 from csgDp1) probe was found to bind stable complexes with MlrA (Fig. 2a), but MlrA did not bind to CD7 (−335 to −615) (Fig. 2b). For identification of the MlrA-binding

sequence, DNase I-footprinting mapping was performed using the purified His-tagged MlrA protein and the csgD promoter DNA fragment. A single unique protection sequence was identified (Fig. 2b and c) covering a 33-bp sequence (−113 to−146) including an inverted repeat sequence of AAAGTTGTACA(12N)TGCACAATTTT (Fig. 2c). This MlrA-binding sequence consisting of a 11-bp-long inverted repeat with a 12-bp interval is unique with respect to the length (total 32 bp long) of the consensus sequence of other characterized transcription Etofibrate factors from E. coli (Ishihama, 2010). The MlrA-binding site is located between the promoter-distal IHF-binding site in the transcription factor-binding hot-spot I and the OmpR-binding site in the hot-spot II along the csgD promoter (Ogasawara et al., 2010). After searching the predicated MlrA-box sequence along the whole E. coli genome, we identified four additional MlrA-binding target sequences (Fig. 3a), which all carry a well-conserved MlrA-box sequence (Fig. 3b). A gel shift assay indicated MlrR binding to the predicted target sequences (data not shown).

The plasmid construct was confirmed by DNA sequencing. A CR plate assay was performed as described previously (Barnhart & Chapman, 2006). To determine curli assembly, each E. coli test strain was grown at 28 °C for 48 h on YESCA plates

containing 50 μg mL−1 CR and 10 μg mL−1 Coomassie blue. As an initial attempt to identify the regulatory role of MlrA on the csgD promoter, we examined the possible influence of its overexpression on the csgD promoter–lacZ reporter fusion. In wild-type background, overexpression of MlrA led to an approximately 2.5-fold increase in csgD–lacZ expression (Fig. 1a). In the csgD knockout mutant, this increase was more than 4.5-fold this website (Fig. 1b), indicating that MlrA is a positive factor of

the csgD promoter. As overexpression of MlrA did not affect the promoter of the divergently transcribed csgBC operon, the effect of MlrA on curli production is solely attributable to activation of the csgD promoter, ultimately leading to activation of the csgB promoter. The expression level of MlrA in both wild-type E. coli and the csgD mutant was essentially the same as detected by immunoblot analysis of whole-cell lysates (data not shown). To gain insight into how the csgD promoter is activated by MlrA, we next examined the DNA-binding activity of purified MlrA and determined the site of recognition and binding on the csgD promoter. Gel-shift assays using various types of csgD probes GSI-IX solubility dmso covering various portions of the csgD promoter and its upstream region (for DNA segments see Ogasawara et al., 2010), only the CD6 (−67 to −335 from csgDp1) probe was found to bind stable complexes with MlrA (Fig. 2a), but MlrA did not bind to CD7 (−335 to −615) (Fig. 2b). For identification of the MlrA-binding

sequence, DNase I-footprinting mapping was performed using the purified His-tagged MlrA protein and the csgD promoter DNA fragment. A single unique protection sequence was identified (Fig. 2b and c) covering a 33-bp sequence (−113 to−146) including an inverted repeat sequence of AAAGTTGTACA(12N)TGCACAATTTT (Fig. 2c). This MlrA-binding sequence consisting of a 11-bp-long inverted repeat with a 12-bp interval is unique with respect to the length (total 32 bp long) of the consensus sequence of other characterized transcription Janus kinase (JAK) factors from E. coli (Ishihama, 2010). The MlrA-binding site is located between the promoter-distal IHF-binding site in the transcription factor-binding hot-spot I and the OmpR-binding site in the hot-spot II along the csgD promoter (Ogasawara et al., 2010). After searching the predicated MlrA-box sequence along the whole E. coli genome, we identified four additional MlrA-binding target sequences (Fig. 3a), which all carry a well-conserved MlrA-box sequence (Fig. 3b). A gel shift assay indicated MlrR binding to the predicted target sequences (data not shown).

In recent years, the dental profession has increasingly become concerned by the seemingly very widespread nature of DE. Dental LDK378 solubility dmso erosion is a multifactorial condition, and the possible aetiological factors of erosion are chemical, biological and behavioural in origin[4]. Sources of erosive acids can be either intrinsic or extrinsic. Intrinsic acid sources include acids of gastric origin. These acids come in contact with teeth in cases of

gastro-oesophageal reflux, excessive vomiting or rumination, drug side effects, nervous system disorders and bowl diseases[5]. Extrinsic acid sources can be classified into dietary acids, medications and environmental acids[2, 6-10]. Addressing the aetiology of DE is a challenging aspect. Researches have continually demonstrated the high susceptibility of dental hard tissue to acidic challenge, which can be modified by the interplay

between chemical, biological and behavioural factors. It is likely that many potential supposed factors can occur simultaneously or sequentially, which makes identification of a definite Trichostatin A in vivo aetiological factor almost impossible. The multifactorial and complex aetiology may actually be used to explain the variation in the presentation, distribution and severity of defects seen clinically in individuals with DE. It is therefore important to identify those at risk of developing clinical problems, so they can be targeted through preventive programmes. Most studies of the aetiology of DE have been carried out mostly in Western European countries[8, 11-13]. Recent reports have included the United States of America [14, 15] and Asia[16], but representative studies on DE prevalence

in Arab countries are scarce[17, 18]. No studies were found to address the risk indicators of DE in Jordan. The aim of this study was to identify potential risk indicators of DE among Jordanian school children. The Institutional Review Board (IRB) at Jordan University of Science and Technology approved the study protocol. In this cross-sectional study, a cluster random sample was selected from Amman, Irbid, and Al-Karak Lepirudin governorates which represent the Northern, Middle, and Southern parts of Jordan, respectively. A multistage cluster random sampling was adopted to select the students. Firstly, the Ministry of Education in Jordan supplied a list of schools teaching 6th, 7th, and 8th grade children. The total number of schools in the three governorates was 1514: 851 schools were in Amman, 450 were in Irbid, and 213 were in Al-Karak. A random selection of 5% of each type of the schools (governmental, private, and United Nations Relief and Works Agency (UNRWA)), (males, females, and mixed)) was performed using the random tables. A total number of 81 schools were selected: 45 from Amman, 25 from Irbid, and 11 from Al-Karak.

Twenty transtibial amputees (16 male) aged 60.1 years (range

45–80 years), and 20 age- and gender-matched healthy adult controls were recruited. Single-pulse transcranial magnetic stimulation assessed corticomotor excitability. Two indices of corticomotor excitability were calculated. An index of corticospinal excitability (ICE) determined relative excitability of ipsilateral and contralateral corticomotor projections to alpha-motoneurons innervating the quadriceps muscle (QM) of selleckchem the amputated limb. A laterality index (LI) assessed relative excitability of contralateral projections from each hemisphere. Spatial-temporal gait analysis was performed to calculate step-time variability. Amputees had lower ICE values, indicating relatively greater excitability of ipsilateral corticomotor Lapatinib mouse projections than controls (P = 0.04). A lower ICE value was associated with increased step-time variability for amputated (P = 0.04) and non-amputated limbs (P = 0.02). This association suggests corticomotor projections

from ipsilateral M1 to alpha-motoneurons innervating the amputated limb QM may interfere with gait. Cortical excitability in amputees was not increased bilaterally, contrary to our hypothesis. There was no difference in excitability of contralateral M1 between amputees and controls (P = 0.10), and no difference in LI (P = 0.71). It appears both hemispheres control one QM, with predominance of contralateral corticomotor excitability in healthy adults. Following lower-limb amputation, putative ipsilateral corticomotor excitability is relatively increased in some amputees and may negatively impact on function. “
“The lack of axonal regeneration in the adult central nervous system is in part attributable to the presence of inhibitory molecules present in the environment of injured axons such as the myelin-associated proteins Nogo-A and MAG and the repulsive guidance molecules Ephrins, Netrins and Semaphorins. In the present study, we hypothesized that EphA4 and one

of its potential binding partners EphrinA3 may participate in the inhibition of adult axon regeneration in the model of adult mouse optic nerve injury. Axonal regeneration DOK2 was analysed in three dimensions after tissue clearing of EphA4 knockout (KO), EphrinA3 KO and wild-type (WT) optic nerves. By immunohistochemistry, EphA4 was highly expressed in Müller glia endfeet in the retina and in astrocytes in the retina and the optic nerve, while EphrinA3 was present in retinal ganglion cells and oligodendrocytes. Optic nerve crush did not cause expression changes. Significantly more axons grew in the crushed optic nerve of EphA4 KO mice than in WT or EphrinA3 KO animals. Single axon analysis revealed that EphA4 KO axons were less prone to form aberrant branching than axons in the other mouse groups.

Par (=partition) proteins are encoded by various plasmids and are essential for the proper partition of (especially larger) plasmids to the bacterial daughter

cells. In these systems, ParB binds in a sequence-specific way to the plasmid DNA, and ParA is acting as an ATPase involved in plasmid partition (Funnell & Slavcev, 2004). Sequence comparisons demonstrated that parA and parB genes are present in close proximity to the respective repA genes not only in pNL1 and pCAR3 but also on the two other groups of large plasmids identified above (Table 1). To further confirm the suggested classification of the ‘megaplasmids’ from sphingomonads, phylogenetic trees were constructed derived from the RepA, ParA and ParB sequences. These comparisons demonstrated Metformin in vitro for the three independently constructed dendrograms, a rather similar organization

(Fig. 2). Thus, in all three dendrograms, pCAR3, pNL1, pSWIT02 and Mpl (=‘Mega-RepAC’) clustered together. Furthermore, also pCHQ1, pSLCP, pSPHCH01, pISP0 and pLA1 (=‘Mega-Rep3’) formed a clearly defined cluster. There was only some variability regarding the ‘Mega-RPA’-group, as the ParA and ParB sequences from plasmid pISP1 did not cluster together with the sequences from plasmids pNL2 and Lpl in the dendrograms. Nevertheless, the relevant sequences from these three plasmids were always clearly separated from the two other groups (Fig. 2). For the smaller plasmids pUT1, pISP2, pISP3, pISP4 and pDL2, only

parA genes had been annotated in close proximity to the respective repA genes. The parA genes from these plasmids are significantly smaller compared with those found in the three groups of ‘megaplasmids’ and encode selleckchem only for proteins of about Erastin concentration 210 aa (Table 1). The sequence comparisons showed for plasmids pUT1, pISP2 and pPDL2 that in each case between the genes annotated as repA or parA, an additional small open reading frame (ORF) was present. These ORFs coded for proteins of 94–95 amino acid residues. An alignment of these sequences from pUT1, pISP2 and pPDL2 (YP_003543404, YP_006965787, YP_006965787) demonstrated that the encoded proteins are almost completely identical (92 identical amino acid residues). The conservation of the sequence and the position of these ORFs suggest that the encoded small proteins function as ParB. Similar combinations of ParA proteins with sizes of about 210–220 aa and ParB proteins with sizes of 70–95 aa have previously been described for plasmids related to plasmid pTAR from Agrobacterium tumefaciens (Kalnin et al., 2000; Funnell & Slavcev, 2004). It has been suggested recently that the structural coupling of a repA (or repB) gene together with a parAB operon, an origin of replication (oriV) and a palindromic centromere seems to be typical for replicons from Alphaproteobacteria. In this context, it also has been suggested that the replicons from this group of bacteria could be classified into only four different systems.

The total cell envelopes obtained from 50 mL cultures were suspended in 200 μL of water, and treated with an equal volume of 90% phenol at 65 °C for 15 min, followed by centrifugation at 16 000 g. The aqueous phase was extracted once with ethyl ether and mixed in a 1 : 1 ratio with a tracking dye solution (125 mM Tris-HCl, pH 6.8, 2% SDS, 20% glycerol, 0.002% bromophenol blue and 10% mercaptoethanol), and then boiled for 5 min. The lipopolysaccharides check details were loaded onto a 15% polyacrylamide gel containing 4 M urea and the gel was stained by silver staining solution. The kanR from pUC4K was inserted into the BglII site of pYJ-2 to disrupt MSMEG_4947, yielding pYJ-3

(Table 1). pYJ-3 was digested by SpeI and NotI and the DNA fragment of MSMEG_4947∷kanR was ligated to the SpeI and NotI sites of pPR27-xylE to yield a conditional replication plasmid

pYJ-4 (Table 1). pYJ-1 was digested by NdeI and BamHI and Rv1302 was ligated to the NdeI and BamHI sites of pET23b-Phsp60 to generate pYJ-5 (Table 1). pYJ-5 was digested by XbaI and BamHI and the Phsp60-Rv1302 was ligated to the XbaI and BamHI sites of pCG76 to yield a rescue plasmid pYJ-6 (Table 1). Mycobacterium smegmatis mc2155 electrocompetent cells were prepared as described Pexidartinib chemical structure (Pelicic et al., 1996). The pYJ-4 was electroporated to the competent cells with Electroporator 2510 (Eppendorf). Transformants were grown on LB agar plates containing kanamycin and gentamicin at 30 °C. One colony was propagated in LB broth containing 0.05% Tween 80, kanamycin and gentamicin at 30 °C and the cells were spread on LB agar plates containing kanamycin and gentamicin at 42 °C. The mc2155 mutant strains with the first single crossover event were selected using a Southern Low-density-lipoprotein receptor kinase blot, as described (Li et al., 2006). The rescue plasmid pYJ-6

was electroporated into the mc2155 mutant. Transformants were grown on LB agar plates containing kanamycin and streptomycin at 30 °C. One colony was inoculated into LB broth containing kanamycin and streptomycin, and incubated at 30 °C. The cells were spread on an LB agar plate containing 10% sucrose, kanamycin and streptomycin. The MSMEG_4947 knockout mutant strains (Table 1) with the second single crossover event were selected via a Southern blot. Five MSMEG_4947 knockout mutants (nos 1–5) were inoculated into LB broth containing 0.05% Tween 80 and appropriate antibiotics, and incubated at both 30 and 42 °C. The wild-type mc2155 carrying pCG76 was used as a control. A600 nm was detected at intervals of 24 h and the growth curves at both 30 and 42 °C were obtained. The MSMEG_4947 knockout mutant (no. 2) was grown in LB broth containing 0.05% Tween 80 and kanamycin at 30 °C for 24 h (A600 nm was 0.064), and then transferred to a 42 °C incubator for continuous growth. The cells grown at 42 °C for 72 and 144 h were harvested and fixed in ice-cold 2.5% glutaraldehyde.

To assess the sensitivity of immunofluorescence staining in sections briefly fixed by immersion after ACSF fixation, we investigated the distribution of the GABAAR α1 and α3 subunits in relation to neuroligin 2, gephyrin and VGAT in the cerebellum of adult mice. In addition, to determine which neuron

type expresses the α3 subunit, we analysed sections from GlyT2-GFP mice (Zeilhofer et al., 2005), in which a large subpopulation of Golgi cells are distinctly eGFP-positive. The results, illustrated in Fig. 5, revealed that upon brief immersion-fixation BKM120 (60–90 min), postsynaptic GABAergic markers exhibit a bright, punctate staining, with high signal-to-noise ratio, thereby precisely revealing the distribution of presumptive GABAergic synapses on the cell body of Purkinje cells and in the molecular layer. While the GABAAR α1 subunit was colocalised extensively with neuroligin 2 (Fig. 5A and A′), the α3 subunit was present in only a small subset of GABAergic synapses located on dendrites in the molecular layer (Fig. 5B). Co-staining with gephyrin confirmed the postsynaptic localisation of the α3 subunit (Fig. 5C and C′), whereas parvalbumin double-labeling a marker of both, Purkinje cells and molecular layer interneurons

(Celio, 1990) confirmed that the α3 subunit-immunoreactivity was not located in parvalbumin-positive neurons (Fig. 5D). Examination of sections from GlyT2-GFP mice revealed that the α3 subunit Selleckchem Belnacasan Fludarabine in vivo clusters were present on the soma and dendrites of glycinergic Golgi cells (Fig. 5E and E′; Simat et al., 2007), and their postsynaptic localisation was confirmed by staining with VGAT (Fig. 5F and F′). As noted above for Fig. 2B and C, a longer immersion-fixation time (up to 3 h) was deleterious for the detection of synaptic proteins, albeit the effects were variable among

the antibodies tested. Presynaptic markers, such as VGAT and VGluT1, showed little influence of fixation time, whereas postsynaptic proteins, such as neuroligin 2 and gephyrin, were highly sensitive to the duration of fixation. Therefore, in our hands, immersion-fixation for 60–90 min represented an optimal duration for good quality staining and preservation of sections after the staining procedure. Taken together, these results underline the remarkable sensitivity of immunofluorescence staining and morphological preservation obtained in sections from ACSF-perfused mice, immersion-fixed for a short duration. Until now, the subcellular distribution of postsynaptic α3 subunit clusters could not be resolved satisfactorily in the cerebellum (Fritschy & Panzanelli, 2006), whereas here it is unambiguously demonstrated in a subpopulation of Golgi cells.

Correlating with inhibitory effects on central amygdala GR gene expression, fluoxetine also decreased amygdala corticotropin-releasing hormone gene expression, an effect not previously observed with MAOIs or TCAs. These actions may be relevant to the efficacy of SSRIs in treating a range of depression and anxiety disorders. “
“Beta amyloid (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease. Aβ is the major constituent of senile plaques, but

there is a significant presence of Aβ in the brain in soluble forms. IWR 1 The results of functional studies indicate that soluble Aβ interacts with the α7 nicotinic acetylcholine receptor (nAChR) complex with apparent high affinity. However, conflicting data exist as to the nature of the Aβ–α7 nAChR interaction, and whether it is the result of specific binding. Moreover, both agonist-like and antagonist-like effects have been reported.

In particular, agonist-like effects have been observed for presynaptic nAChRs. Here, we demonstrate Aβ1-42-evoked stimulatory changes in presynaptic Ca2+ level via exogenous α7 nAChRs expressed in the axonal varicosities of differentiated hybrid neuroblastoma NG108-15 cells as a model, presynaptic system. The Aβ1-42-evoked RG 7204 responses were concentration-dependent and were sensitive to the highly selective α7 nAChR antagonist α-bungarotoxin. Voltage-gated Ca2+ channels and internal Ca2+ stores were both involved in Aβ1-42-evoked increases in presynaptic Ca2+ following activation of α7 nAChRs. In addition, disruption of lipid rafts by cholesterol depletion led to substantially attenuated responses to Aβ1-42, whereas responses to nicotine were largely intact. These results directly implicate the nicotinic receptor complex as a target for the agonist-like action of pico- to nanomolar concentrations of soluble Aβ1-42 on the presynaptic nerve terminal, including the possible involvement

of receptor-associated lipid rafts. This interaction probably plays an important neuromodulatory role in synaptic dynamics. “
“β-Amyloid learn more (Aβ) peptides are thought to play a major role in the pathogenesis of Alzheimer’s disease. Compounds that disrupt the kinetic pathways of Aβ aggregation may be useful in elucidating the role of oligomeric, protofibrillar and fibrillar Aβ in the etiology of the disease. We have previously reported that scyllo-inositol inhibits Aβ42 fibril formation but the mechanism(s) by which this occurs has not been investigated in detail. Using a series of scyllo-inositol derivatives in which one or two hydroxyl groups were replaced with hydrogen, chlorine or methoxy substituents, we examined the role of hydrogen bonding and hydrophobicity in the structure–function relationship of scyllo-inositol–Aβ binding.

3.1 Cycle Sequencing kit (Applied Biosystems) with separation of reactions on an ABI3730 sequencer (Allan Wilson Centre Genome Service Facility, Massey University, NZ). The Tn916 insertion site was mapped to the completed version of the B316T genome sequence, GenBank accession numbers CP001810 (BPc1), CP001811 (BPc2), CP001812 (pCY360) and CP001813 (pCY186). An in-house perl script was used to capture 20 nucleotides upstream and 20 nucleotides downstream of each Tn916 insertion site. Nucleotide sequence clusters from each genetic element were merged in clustalx 2.0 (Thompson et al., 1997) and a complete this website sequence alignment was calculated. The final alignment was then imported into logobar (Pérez-Bercoff

et al., 2006). Plasmid constructs AZD9291 and the conditions for the routine transformation and genetic analysis of the general Butyrivibrio assemblage remain to be determined. However, a previous study demonstrated the conjugal transfer of Tn916 and Tn916ΔErm from an E. faecalis donor to various Butyrivibrio fibrisolvens strains (Hespell & Whitehead, 1991), but there was no analysis of the genomic distribution and consensus sequence associated with transposon insertion sites, and none of those Butyrivibrio strains

had their genome sequenced and fully annotated. With the genome sequence of B316T completed and fully annotated, this study was undertaken to demonstrate Tn916 mutagenesis and to investigate the transposition events in a genome composed of four separate replicons. After exploring a variety of conditions including the selective culture of B. proteoclasticus and the inhibition of the E. faecalis donor strain after conjugation, a total of nine separate conjugation experiments as described in the Materials and methods were performed that gave rise to B316T transconjugants. Attempts were made to standardize conditions to ensure uniformity

of each conjugation experiment with regard to the age of bacterial cultures, the total numbers of donor and recipient bacteria and the incubation time for conjugation. Despite these standardization attempts, Tn916 transfer frequencies still varied over several Sclareol orders of magnitude (approximately 1.0 × 10−5–9.2 × 10−8 transconjugants per recipient). Of the 381 transconjugants that were isolated, 303 were successfully subcultured, frozen at −85 °C and resuscitated for further analysis. Of the 303 transconjugants, 70 (23.1%) had two or more Tn916 inserts, while no inverse PCR amplicon could be obtained from 110 transconjugants. Using inverse PCR and sequence analysis of the resultant products, single transposon insertion sites were established in 123 (32.3%) of the tetracycline-resistant mutants (Fig. 1, Table 2). Initial sequence analysis of the inverse PCR products indicated that 53 insertion sites accounted for the 123 single insertion events. Twenty-nine of the 53 (54.