However, no protein accumulation occurred in the PMS controls.

After 10 days of incubation the DNA Damage inhibitor culture entered the stationary phase. During this period the concentration of chrysene in the medium decreased from 400 to 140 mg L−1, i.e. 60% of the chrysene was degraded during the 12 days of incubation. TLC of the ethyl acetate extract of the supernatants from the washed-cell incubations with chrysene showed the presence of polar metabolites. Metabolic intermediates were tentatively identified by comparing their Rf values with those of the respective standard reference compounds. Chrysene moved along with the solvent front. 1-Hydrox-2-naphthoic acid (Rf 0.43) and salicylic acid (Rf 0.15) were identified as the probable intermediates. A spot with Rf value of 0.86 did not match with any standards tested. The extracts were then analysed by HPLC and the individual spots on TLC were further characterized by LC-ESI-MS. Retention times from HPLC analysis (Fig. 2) and LC-ESI-MS

characteristics of the metabolites are given in Table 1. HPLC retention times of identified metabolites were identical to those of respective standard reference compounds. LC-ESI-MS of metabolite C1 gave a molecular ion (M+) at m/z 138 and GSK2126458 subsequently at 121 (M+– 17, probably due to loss of OH), 110, 93 (M+– 45, loss of COOH), 80, 77 and 63 (Table 1, C1). The fragmentation pattern is identical to that of standard salicylic acid. The mass spectrum of metabolite C2 showed a base peak at 187 (M+– 1), and subsequent ion fragments at m/z 170 (M+– 17, loss of OH), 154, 143 (M+– 45, loss of COOH), 126 (M+– 17 – 45, losses of OH and COOH), 115 and 79 (Table 1, C2). The fragmentation pattern of this metabolite matched well with that of standard 1-hydroxy-2-naphthoic acid. The LC-MS spectrum of metabolite C3 showed an ion fragment at m/z 239 (M+– 1), a base peak m/z 222 (M++1−OH), and subsequent fragments at 204, 193 (M+– COOH) and 176 (phenanthrene ion). This fragmentation pattern is characteristic of hydroxyphenanthroic

acid (Baboshin et al., 2008). The mass spectra of standards and metabolites are cAMP provided as Supporting Information, Figs S1–S3. The enzyme extract prepared from cells grown on different carbon sources showed high activity of 1,2-dihydroxynaphthalene dioxygenase, moderate activity of 1-hydroxy-2-naphthoate hydroxylase and catechol-1,2-dioxygenase, and low activity of salicylaldehyde dehydrogenase; catechol-2,3-dioxygenase and gentisate-1,2-dioxygenase activity was not detected (Table 2). As expected, the crude extract prepared from glucose-grown cells did not show any activity of the above enzymes, thus suggesting the inducible nature of the enzymes involved in the degradation of chrysene. To elucidate the chrysene degradation pathway operating in PNK-04, the expected intermediates of the pathway were supplied as sole source of carbon.

During the same period, it is important to emphasize that the daily increase in mycelial mass remained constant. A positive correlation between cp gene expression and chlamydospores formation was found (Fig. 4). The transcript level increased from the conidium status to the second day of growth, where hyphae were present and chlamydospores just began to be formed. The highest increase occurred at day 3 (Log2 fold change = 6.15), which preceded the maximum increase in chlamydospores concentration observed

at the fourth day post-inoculation. Conidia used to inoculate the plates were known BMN 673 to have a low level of cp transcript (Bernardi et al., 2011) and were used as calibrator (time zero). The analysis of a 1368 bp region upstream of the ATG codon for the presence of putative regulatory motifs revealed a putative TATA box at position −167 and putative CCAAT boxes at positions −634 and −817 (Fig. 5). Moreover, putative motifs involved in the regulation of gene expression in response to stress and developmental cues were identified. Two CATTCY sites bound by transcription factor of the TEA/ATTS family, such as yeast Tec1p (Köhler et al., 2002) and Aspergillus nidulans AbaA, were located at positions −297 and −1258. In A. nidulans, the abaA gene controls the expression of the genes

involved in morphogenesis and developmental regulation and is required in the final stages of conidiophore development and in spore maturation (Andrianopoulos & Timberlake, 1994). Three stress response elements (STRE) were found at positions

−293, −415 and −782 (Marchler NVP-AUY922 cell line et al., 1993) together with a putative binding site Ixazomib ic50 for the Nrg1/Nrg2 Zn finger repressors at position −400. In yeast, these two regulatory sequences are associated with the promoters of many genes that respond to a variety of stress conditions (Vyas et al., 2005). Finally, two recognition sites for the yeast Skn7 regulators involved in the response to stress such as oxidative stress and high osmolarity stress were found at positions −713 and −963 (Morgan et al., 1997; Izumitsu et al., 2007). The present work showed for the first time a significant correlation between regulation of the cp gene and growth of C. platani: when fungal growth was reduced, the cp gene expression was down-regulated; when the growth level was increased, it was instead up regulated. In addition, the expression of the cp gene appeared to be positively correlated with the differentiation process of chlamydospores. The modulation of transcription had already been analysed in some studies concerning cp and other cerato-platanins, but without taking into account the growth level of the fungus and not so extensively as in the present work (Wilson et al., 2002; Chagué et al., 2006; Djonović et al., 2006; Seidl et al.

, 2005; Raman et al., 2009). This turnover

and release of cellulosomes during fermentation may be necessary to allow for the creation of new cellulosomes with modified composition. It has also been suggested that the controlled release of cellulosomes during growth may function as a mechanism to release C. thermocellum from its substrate, leaving deployed cellulosomes to continue hydrolyzing cellulose (Bayer & Lamed, 1986). Although extensive work has been performed analyzing the composition of purified cellulosomes, the composition of the cellulosome in its native microbial context is not well understood. There is an increasing interest in building artificial cellulosomes, which is currently limited by a lack of understanding of structural elements in native cellulosomes click here (Krauss et al., 2012). In order to increase understanding of the cellulosome in its native microbial context, we undertook work to develop a fluorescent probe for labeling type II cohesins based on the commercially available SNAP-tag labeling system (Keppler et al., 2003). The SNAP-tag system was developed by Keppler

et al. as a method of covalently labeling fusion proteins in vivo. SNAP-tag is a mutant of the O6-alkylguanine-DNA alkyl transferase human DNA repair protein which has increased activity against its substrate O6-benzylguanine. The mutated protein binds covalently with benzylguanine-derived AZD3965 fluorophores. To create the probe, we fused a type II dockerin with the commercially available SNAP-tag. We then used this probe to visualize localization of type II cohesin modules in the cellulosome for both wild type and mutants of the cipA scaffolding protein (Supporting Information, Fig. S1). Clostridium thermocellum DSM 1313 (WT) was grown in modified DSM 122 broth (Olson et al.,

2010) with the addition of 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS) sodium salt and 3 g L−1 trisodium citrate (Na3-C6H5O7*2H2O). All manipulations of C. thermocellum were carried out inside an anaerobic chamber (Coy Laboratory Products Inc.) with an atmosphere of 85% nitrogen, acetylcholine 10% carbon dioxide, 5% hydrogen, and < 5 parts per million oxygen. Clostridium thermocellum was grown at 55 °C using 5 g L−1 cellobiose as the primary carbon source. The genotype of strains used in this work is listed in Table 1. Strain construction was performed as described previously (Argyros et al., 2011; Guss et al., 2012; Olson & Lynd, 2012) using plasmids listed in Table 2. Briefly, the regions annotated as ‘5′ flank’ and ‘3′ flank’ are present on both the plasmid and the chromosome. By a series of recombination events, the region flanked by the ‘5′ flank’ and ‘3′ flank’ on the chromosome is replaced by the corresponding region from the plasmid. Plasmid sequences are available from Genbank (accession number in Table 2).

Southern blots probed with DIG-labeled oligonucl-eotides were used to measure the purity of the ssDNA preparations.

Briefly, oligonucleotides gyrBtop2 (5′-GCCATCGACGAAGCACTC) and gyrBbot12 (5′-GGCTTTTTCCAAGGCAAGG) were end labeled with DIG (Roche) following the manufacturer’s instructions. Hybridization, washes, and detection of the Southern blots were performed as per the manufacturer’s instructions (Roche) to determine the relative amounts of ssDNA and RF DNA in the aforementioned preparations. Gonococcal strains were grown for 18 h on GCB plates and resuspended in liquid transformation media [1.5% protease Selleck XL184 peptone no. 3 (Difco), 0.1% NaCl, 200 mM HEPES (Sigma), 5 mM MgSO4 and Kellogg supplements I and II, pH 7.2] to an optical density at 600 nm of approximately 1.5. Thirty microliters of the cell suspension was added RXDX-106 cell line to tubes containing 0.045 pmol of gyrB1 DNA and 200 μL transformation media. DUS12 and DUS0 containing plasmids of gyrB1 (Duffin & Seifert, 2010) were used as transforming dsDNA, and purified recombinant phage DNA was used as transforming ssDNA. Following incubation at 37 °C for 20 min, transformation mixtures were added to pre-warmed 2 mL transformation media and incubated at 37 °C in the presence of 5% CO2 for 4 h. The mixtures were serially diluted 10-fold in transformation media lacking MgSO4 and

Kellogg supplements, and 20-μL serial 10-fold dilutions were spotted on GCB plates in the presence and absence of Nal. Transformation efficiencies are reported as antibiotic resistant CFU divided by total CFU and are the mean of at least three replicates. Efficient transformation in N. gonorrhoeae Fenbendazole requires the presence of the DUS in the transforming DNA and homology to DNA sequences present within the genome (Ambur et al., 2007; Duffin & Seifert, 2010). Many N. gonorrhoeae strains harbor a type IV secretions system and thus secrete ssDNA, which can serve as substrate for transformation (Dillard & Seifert, 2001; Salgado-Pabon et al., 2007). No reports have investigated the potential role

of the DUS in ssDNA transformation, which may clarify its mechanism of action during transformation. Recombinant M13 phage were used to isolate gyrB1 transforming DNA cloned in both orientations, so that the single-stranded DNA would carry either the Watson DUS12 (5′-ATGCCGTCTGAA-3′), the Crick DUS12 (5′-TTCAGACGGCAT-3′), or no DUS (DUS0). As dsDNA RF DNA is produced during the course of M13 infection (Sambrook et al., 2001) and any contaminating dsDNA would transform N. gonorrhoeae, we utilized column purification of the ssDNA following phage isolation (see Methods). We then determined the relative amount of dsDNA in the ssDNA preparations using Southern blots with oligonucleotide probes that bind either the Watson or the Crick strand (Fig. 1). Southern analysis revealed two distinct species of ssDNA: a major band and a minor smaller band (Fig. 1).

With this fusion protein, we established a directed transposon mutagenesis system that is expected Selleck MK2206 to directly integrate close to the fliC operator. The system is composed of three main elements: (1) the target fliC operator flanked by fliC and fliD genes; (2) the IS30–FljA fusion transposase;

(3) and the integration donor sequence containing the (IS30)2 intermediate together with the CmR marker gene. Two essential components of the mutagenesis system required to be constructed: the fusion transposase producer plasmid pFOL1111 and the integration donor pFOL1069 (Fig. 2). The insertion donor plasmid pFOL1069 containing the (IS30)2 intermediate (Olasz et al., 1993; Kiss & Olasz, 1999) represented a highly reactive DNA segment in the presence of the IS30 transposase. The pFOL1069 additionally contained

the CmR marker gene, the mob region necessary for Inhibitor Library cell assay bacterial conjugation and the defective replication origin R6K. Because of the R6K replication origin, the donor plasmid is unable to replicate in Salmonella lacking the pir gene. As a consequence, Salmonella bacteria possessing CmR after the conjugation of pFOL1069 were the ones in which the donor plasmid was integrated into the chromosome. The integration ability of pFOL1069 was verified earlier in E. coli (data not shown). The FljA–IS30 fusion transposase producer plasmid pFOL1111 was constructed by the fusion of the fljA flagellin repressor gene to the C-terminal end of the IS30 transposase gene. The resulting isopropyl-β-d-thiogalactopyranoside-inducible FljA-transposase producer plasmid

also contains the ApR bacterial marker. Because this plasmid codes only for the fusion transposase, but lacking the IS30 inverted repeat ends necessary for transposition, it is not capable of integrating into any target DNA. The inducible expression of the FljA–IS30 fusion protein was verified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (Fig. 3a). No alteration was detected in the amount of the transposase compared with that of the wild type produced by the plasmid pJKI132 (Fig. 3a). The functionality of Ureohydrolase the FljA part of the fusion was tested by introducing pFOL1111 into the wt S. Enteritidis strain 11 and the motility of the transformants was investigated. The pFOL1111 plasmid-harbouring strains (Fig 3b, column 2) showed reduced motility as compared with the plasmid-free bacteria (Fig 3b, column 1). However, a complete elimination of motility never occurred due to the presence of exogenous FljA, and it was always reversible, as the partly motile strains regained their full motility after the plasmid pFOL1111 was eliminated (results not shown). The transposition activity of pFOL1111 was verified similarly as described by Szabo et al. (2008) (data not shown). In summary, it can be stated that all components of the targeting system have proven their expected activity for subsequent immobilization.

The results showed that certain Ca2+ concentrations enhanced the heat resistance of the LAB strains to different

extents, that is produced higher survival and shorter regrowth lag times of the bacterial cells. In some cases, the improvements were dramatic. More scientifically insightful and more intensive instrumental study of the Ca2+ behavior around and in the cells should be carried out in the near future. In the meantime, this work may lead to the development of more cost-effective wall materials with Ca2+ added as a prime AZD0530 factor. “
“Mip (macrophage infectivity potentiator) and Mip-like proteins have been demonstrated to be involved in virulence of several animal pathogens, but as yet none of their native bacterial targets has been identified. Our previous work demonstrated that the Mip-like protein found in the plant pathogen Xanthomonas campestris pv. campestris (Xcc) (hereafter called

MipXcc) is also involved in virulence. Inactivation of the mipXcc gene leads to a significant reduction in exopolysaccharide production and extracellular protease activity via an unknown mechanism. The Xcc genome encodes six extracellular proteases, all of which are secreted via the type II secretion system. The serine protease PrtA makes the largest contribution to Xcc’s Selleck MK0683 total extracellular proteolytic activity. In this study, Western blotting analysis demonstrated that MipXcc was located in the periplasm. Bacterial two-hybrid and far-Western analysis indicated that MipXcc interacted with PrtA directly. Purified MipXcc was found to be able to rescue the protease activity of periplasmic proteins extracted from the mipXcc mutant. These findings show that MipXcc plays a role in

the maturation of PrtA, which is the novel native target for at least one Mip or Mip-like protein. Mip (macrophage infectivity potentiator) and Mip-like proteins make up a family of bacterial proteins that comprises two domains: Aspartate an N-terminal dimerization region and a C-terminal PPIase (peptidyl prolyl cis/trans isomerase) region exhibiting similarity to the human FK506-binding protein (Riboldi-Tunnicliffe et al., 2001). In 1989, Mip was first identified as an important virulence factor in Legionella pneumophila (Cianciotto et al., 1989). Since then, Mip and Mip-like proteins have been found to be associated with the virulence of several other animal pathogens, such as Chlamydia trachomatis, Trypanosoma cruzi, Neisseria gonorrhoeae, and Chlamydophila pneumoniae, as well as the plant pathogen Xanthomonas campestris pv. campestris (Xcc) (Lundemose et al., 1993; Moro et al., 1995; Leuzzi et al., 2005; Herrmann et al., 2006; Zang et al., 2007).

Here we revisit the Hering-versus-Helmholtz controversy on binocular coordination from the psychophysician’s description of combined saccade-vergence eye movements to the neurophysiological recording

of motor and premotor neurons of the oculomotor neural circuitry. Whilst neo-Heringian psychophysicians and physiologists have accumulated arguments for separate saccade and vergence systems, at both the behavioral and the Atezolizumab price neural premotor levels, neo-Helmholtzians have also provided evidence for monocular programmed eye movements and commands at the premotor level. Bridging the two, we conclude that Hering and Helmholtz were both right. Importantly, the latter’s viewpoint brings to the fore the importance of adaptive processes throughout life, in view of the neurobiological constraints emphasized by the former. “
“AMPA-type glutamate receptors (AMPARs), as well as most other transmembrane proteins, are not stable in the postsynaptic density as was previously thought, but undergo constant trafficking in and out of synapses by a combination of endo/exocytosis and lateral diffusion. The respective

contributions of membrane recycling events and surface trafficking to setting AMPAR numbers at synapses have been the subject of intense debate. Although this discussion is not yet settled, it is safe to state that both categories of processes participate in receptor exchange at synapses at rest and during various forms of plasticity. More unexpectedly, AMPARs can diffuse at such high check details rates within the postsynaptic density itself that their surface trafficking could participate not only in setting receptor numbers at individual synapses but also in tuning synaptic transmission during short-term plasticity. I here review recent results that characterize the activity-dependent properties of AMPAR surface trafficking and their possible links to fast synaptic transmission. “
“Olfactory and visual sensory mechanisms seem to play a

critical role in migratory orientation and navigation. How these two mechanisms fantofarone are functionally linked with other migratory processes is unknown. We investigated this, in relation to the profound behavioural shift that occurs during migration in the night-migratory blackheaded bunting (Emberiza melanocephala). Photosensitive unstimulated birds singly housed in activity cages were subjected to long days (LD 16/8). The activity of each bird was continuously monitored. Daily activity pattern defined the nonmigratory phase (no nocturnal activity) and migratory phase (intense nocturnal activity, Zugunruhe). Body mass and testis size were measured at the beginning and end of the experiment. Long days induced the migratory phenotype (body fattening and Zugunruhe) and testis maturation.

volcanii in microtiter

plates has been developed (Blaby et al., 2010). The advantages and disadvantages of the two approaches are compared. Three H. volcanii strains used in this study were obtained from Thorsten Allers (University of Nottingham, UK). H26 is a pyrE deletion strain and is thus auxotrophic for uracil, but has wild-type characteristics for all the features analyzed in this study. The other two strains were derived from H26. H53 is a trpA deletion strain and is auxotrophic for tryptophan; H66 is a leuB deletion strain and is auxotrophic for leucine (Allers et al., 2004). In addition, deletion mutants of the following eight sRNA genes were used: sRNA63, sRNA132, sRNA168, sRNA194, sRNA235, sRNA288, sRNA308 and sRNA500. The identification of the sRNA genes and the generation of two deletion mutants including LY2606368 concentration a deletion mutant of sRNA63 have already been described (Straub et al., 2009); the remaining seven mutants were constructed using the same protocol. The sequences of the oligonucleotides used for mutant construction are available upon request. Haloferax volcanii was grown in a complex medium and a synthetic medium as described

previously (Dambeck & Soppa, 2008). Cultures were grown in Erlenmeyer flasks in a rotary shaker at 42 °C and 250 r.p.m. During BGB324 research buy the first trials to grow H. volcanii cultures in 96-well microtiter plates, pelleting of the cells was a problem. This problem was solved using an orbital shaker (1.5 mm orbit) and increasing the shaking velocity to 1100 r.p.m. A further problem was the evaporation of water during the incubation at the optimal growth temperature of 42 °C over several days, which led to an uneven loss of volume in the inner and outer wells and precipitation of NaCl in some wells. This problem was solved using the outer wells not for cell growth, but for the creation of an ‘evaporation barrier’. However, filling them with water led to a very fast evaporation

Meloxicam in the outer wells and to a volume increase in the inner, medium-containing wells. Therefore, salt solutions of different concentrations were tested, and a solution of 150 μL of 1 M NaCl turned out to be optimal. The evaporated water in the outmost wells was replaced daily; the volume of the inner wells remained constant throughout the experiments. Using the outmost wells for the ‘evaporation barrier’ 60 wells remained for cell culturing, which enables to test, for example, 20 conditions simultaneously using triplicate cultures, or to compare many mutants with the wild type under few conditions. Precultures were grown in Erlenmeyer flasks in a synthetic medium with glucose as described above to the early exponential growth phase (OD600 nm=0.3±0.1). The vitamin solution contained nine different vitamins (Sigma, Taufkirchen, Germany; order no. B6891) and was diluted 1 : 1000 into the medium.

For example, and as we documented earlier (Hafed et al., 2011), our two monkeys showed different patterns

of microsaccades in the early cue-induced analysis intervals of Figs 8 and 9. The fact that the monkeys behaved similarly later in the trials (Fig. 10) might hint at some possible reasons for the earlier differences. One such reason could be related to the task design, in which the monkeys knew with 100% certainty that no perceptual discrimination stimuli could appear before ~1500 ms after cue onset. Thus, it may be the case that each monkey adopted a different strategy of ‘covertly’ inspecting the stimulus array at the Decitabine cell line beginning of a trial, and that the patterns of microsaccades that we observed in this epoch revealed this difference. As a particular strategy was not reinforced this early in the trials, individual differences between the two monkeys in

the initial stages of the trial are conceivable. In contrast, at the ends of the trials (Fig. 10), when paying attention to the relevant locations was behaviorally reinforced in both monkeys, both of them showed selleck products similar patterns of microsaccade directions, and this was true for both the normal behavior without SC inactivation (Fig. 10A) (Hafed et al., 2011) and during SC inactivation (Fig. 10B). More importantly, the fact that SC inactivation resulted in a repulsion of microsaccades away from the affected region in both monkeys, despite their individual differences, supports the view that it is activity modulations in the peripheral SC that may be sufficient to bias the overall representation in the SC map and alter the triggering of microsaccades. This result may be interesting in the light of recent behavioral observations of a clear dissociation between microsaccade rate and microsaccade directions during covert visual attention tasks (Pastukhov & Braun, 2010; Pastukhov et al., 2012). It would be interesting to further test such a dissociation in the light of our results, especially because we also saw clear differences between the effects of peripheral SC inactivation on microsaccade rate and those on microsaccade direction. Finally, our results indicate that the multifaceted role

of the SC Doxacurium chloride in vision, cognition and oculomotor control contributes to the correlations between attentional cueing and microsaccades. In addition, these results can help to explain the reproducible, almost machine-like, manner in which stimulus transients, such as attentional cues, induce microsaccades (Hafed et al., 2011): this arises because of the sensitivity of the SC to such transients as well as its proximity to the motor output. However, these results also raise the question of why such a relationship exists in the first place. Given that microsaccades cause transient extra-retinal changes in vision (Zuber & Stark, 1966; Beeler, 1967; Hafed & Krauzlis, 2010) and concomitant changes in visual responses in the brain, including at the level of the SC (Martinez-Conde et al.

A kanamycin resistance cassette from pACYC177 was amplified using primers kana1 and kana2

(Table 1) and then cloned into the ApaI-XbaI site of the pYG1 to generate pYG2. The sacB gene of pYLTAC7 was removed by EcoRI-restriction, generating a 1.7-kb check details fragment. Then, the sacB-containing fragment was cloned into the EcoRI site of the pYG2 resulting in pYG3. Finally, the vector pYG3 was digested by ApalI to remove the ampicillin resistance and was self-ligated to create the final plasmid pYG4. As described by Link et al. (1997), the 2067-bp in-frame deletion of the yncD gene was constructed by cross-over PCR with primers k1, k2, k3 and k4 (Table 1). The product was ligated directly to the pMD18-T vector (Takara Co., Dalian, China)

and confirmed by sequencing. The recombinant plasmid was digested by NdeI and the fragment containing the deletion copy of the yncD gene was ligated to pYG4. The resulting vector was introduced into E. coli S17-1/λpir by electroporation. The hybrid plasmid was transferred into YGC101 (wild type) by electroporation to perform mutagenesis. Selleckchem FK866 Integrons were selected from the LB plates containing kanamycin and were confirmed through PCR analysis. Overnight cultures of the identified integron grown in the absence of antibiotics were streaked onto LB agar containing 5% sucrose. Selected colonies with normal colony phenotypes were patched onto LB agar with and without kanamycin. The colonies that were sensitive to kanamycin were analyzed for the deletion by PCR with the primers O1 and O2, as well as I1 and I2 (Table 1). The strain carrying the desired deletion was selected

and designated as YGC102. The gene yncD was PCR-amplified from the wild-type strain using the primers C1 and C2 (Table 1), which were designed based on sequences external to the yncD coding region. After amplification, the DNA fragment was digested by EcoRI and HindIII and ligated to the pBR322 to obtain PYN plasmid. The resulting vector was introduced into the mutant strain YGC102 by electroporation to produce the strain YGC103. To determine the involvement of yncD in virulence, 3-mercaptopyruvate sulfurtransferase the median lethal dose (LD50) of YGC101, YGC102 and YGC103 was determined as described by Wang et al. (2001) with minor modifications. Female BALB/c mice aged 6–8 weeks (three mice per group, three groups per strain) were injected intraperitoneally with various dilutions of the different strains mixed with 7% (w/v) mucin from porcine stomach (Sigma) at a final volume of 0.5 mL in phosphate-buffered saline (PBS). The number of deaths that occurred within 72 h after inoculation was counted. The LD50 was calculated as described by Reed & Muench (1938). To evaluate the effect of yncD gene deletion on the survival capability in vivo, we performed bacterial competition experiments in the mouse model.