The cold shock response is highly conserved amongst bacteria with Csps as well as PNPase also contributing to the cold http://www.selleckchem.com/products/PF-2341066.html shock response in other species such as Yersinia and Bacillus (Palonen et al., 2010). The enteric pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is closely related to E. coli. It is a successful pathogen capable of infecting both warm-blooded and poikilothermic animals

including fish, nematodes, amoebas and plants (Lewis, 1975; Van der Walt et al., 1997; Charkowski et al., 2002; Cooley et al., 2003; Tenor et al., 2004; Doyel & Beuchat, 2007; Onyango et al., 2009). Hence, S. Typhimurium is also expected to experience wide fluctuations in environmental temperature. Both pnp and csdA (the latter also termed deaD) are closely linked on the genome of S. Typhimurium and only separated by nlpI that encodes for a membrane lipoprotein (Blattner et al., 1997; Ohara et al., 1999; McClelland et al., 2001; Parkhill et al., 2001; Nie et al., 2006). We have previously shown that pnp and nlpI have opposing effects on biofilm formation at decreased growth temperatures with PNPase and NlpI, respectively, enhancing and suppressing biofilm formation (Rouf et al., 2011). As nlpI is positioned between pnp and csdA, we have here investigated the contribution of pnp, nlpI and csdA (the latter hereafter referred to as deaD) in the cold

acclimatization response in S. Typhimurium. Our data show that pnp and nlpI constitute an operon that is transcriptionally separate from deaD and that PNPase, NlpI and DeaD individually selleck kinase inhibitor contribute to the growth of S. Typhimurium at 15 °C. Our findings thereby define a new role for NlpI in bacterial cold acclimatization. Bacterial strains and plasmids are listed in Table 1. Bacteria were grown in Luria–Bertani medium (LB). Antibiotics (Sigma) Immune system were used where appropriate including ampicillin, 100 μg mL−1; chloramphenicol, 10 μg mL−1; kanamycin, 30 μg mL−1; and tetracycline, 10 μg mL−1. For induction of recombinant NlpI, media were supplemented with 0.1% L (+)-Arabinose (Sigma). Salmonella

enterica serovar Typhimurium SR-11 mutants (∆pnp, ∆nlpI and ∆deaD) were created by the one-step gene inactivation technique described previously (Datsenko & Wanner, 2000; Rouf et al., 2011). Mutated genes were transferred into S. Typhimurium SR-11 by phage P22 int transduction from S. Typhimurium ATCC 14028 background (Schmieger, 1972). The pnp–nlpI double mutant was constructed in succession. First, the PCR-amplified tetracycline resistance gene from pACYC184 was cloned into the KpnI site at codon 201 of pnp in vector pSU41. Then, the pnp::tet mutation was cloned into the pCVD442 suicide plasmid (Donnenberg & Kaper, 1991) and introduced into S. Typhimurium SR-11. The integrated vector was excised through sucrose selection to generate the pnp* mutant strain MC55.

The cold shock response is highly conserved amongst bacteria with Csps as well as PNPase also contributing to the cold DAPT shock response in other species such as Yersinia and Bacillus (Palonen et al., 2010). The enteric pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is closely related to E. coli. It is a successful pathogen capable of infecting both warm-blooded and poikilothermic animals

including fish, nematodes, amoebas and plants (Lewis, 1975; Van der Walt et al., 1997; Charkowski et al., 2002; Cooley et al., 2003; Tenor et al., 2004; Doyel & Beuchat, 2007; Onyango et al., 2009). Hence, S. Typhimurium is also expected to experience wide fluctuations in environmental temperature. Both pnp and csdA (the latter also termed deaD) are closely linked on the genome of S. Typhimurium and only separated by nlpI that encodes for a membrane lipoprotein (Blattner et al., 1997; Ohara et al., 1999; McClelland et al., 2001; Parkhill et al., 2001; Nie et al., 2006). We have previously shown that pnp and nlpI have opposing effects on biofilm formation at decreased growth temperatures with PNPase and NlpI, respectively, enhancing and suppressing biofilm formation (Rouf et al., 2011). As nlpI is positioned between pnp and csdA, we have here investigated the contribution of pnp, nlpI and csdA (the latter hereafter referred to as deaD) in the cold

acclimatization response in S. Typhimurium. Our data show that pnp and nlpI constitute an operon that is transcriptionally separate from deaD and that PNPase, NlpI and DeaD individually Selleckchem Lumacaftor contribute to the growth of S. Typhimurium at 15 °C. Our findings thereby define a new role for NlpI in bacterial cold acclimatization. Bacterial strains and plasmids are listed in Table 1. Bacteria were grown in Luria–Bertani medium (LB). Antibiotics (Sigma) PAK6 were used where appropriate including ampicillin, 100 μg mL−1; chloramphenicol, 10 μg mL−1; kanamycin, 30 μg mL−1; and tetracycline, 10 μg mL−1. For induction of recombinant NlpI, media were supplemented with 0.1% L (+)-Arabinose (Sigma). Salmonella

enterica serovar Typhimurium SR-11 mutants (∆pnp, ∆nlpI and ∆deaD) were created by the one-step gene inactivation technique described previously (Datsenko & Wanner, 2000; Rouf et al., 2011). Mutated genes were transferred into S. Typhimurium SR-11 by phage P22 int transduction from S. Typhimurium ATCC 14028 background (Schmieger, 1972). The pnp–nlpI double mutant was constructed in succession. First, the PCR-amplified tetracycline resistance gene from pACYC184 was cloned into the KpnI site at codon 201 of pnp in vector pSU41. Then, the pnp::tet mutation was cloned into the pCVD442 suicide plasmid (Donnenberg & Kaper, 1991) and introduced into S. Typhimurium SR-11. The integrated vector was excised through sucrose selection to generate the pnp* mutant strain MC55.

Similar to what was observed previously, a single

mutation at H94 strikingly decreased the repression activity of IrrAt (pIRR94, 61% β-Gal activity) compared with single mutations at H45, H65 or H127 (pIRR45, pIRR65 and pIRR127: 11%, 14% and 30% β-Gal activity, respectively) (Fig. 3a). H94, which lies in the HHH motif, seemed to play the most influential role in the function of IrrAt, whereas H45, H65 and H127 played less of a role. H45 and H65 were essential for maintaining the repressor activity of IrrAt when H94 was lost. This notion was supported by the observation that double mutations at H94 in combination with H45 or H65 caused complete loss of IrrAt repressor activity (pIRR45/94 and pIRR65/94: 104% and 102% β-Gal activity, respectively) (Fig. 3a). Triple mutation in the HHH motif (pIRRHHH) SAHA HDAC in vitro and a double mutation at residues H94 and H127 (pIRR94/127) caused a severe defect in the repressor activity of IrrAt (93% and 92% β-Gal activity, respectively) (Fig. 3a). An additional mutation at D86 could fully reverse the defect caused by

the HHH mutation (pIRRHHH86, 1% β-Gal activity) (Fig. 3a). It has been shown previously that FK506 the hyper-resistant phenotype of WK074 to H2O2 was partly due to the high expression of mbfA (Ruangkiattikul et al., 2012). Analysis of the mutant IrrAt proteins showed that the proteins proved to have differential abilities to reverse the H2O2-hyper-resistant phenotype of WK074. The cells exhibiting a higher expression of mbfA-lacZ (Fig. 3a) showed higher resistance to H2O2 (Fig. 3b), consistent with the notion that the high expression of mbfA partly contributes to H2O2 resistance in WK074 cells (Ruangkiattikul et al., 2012). As expected, the mutant WK074/pBBR cells were more resistant to 350 μM H2O2 than were wild-type NTL4/pBBR cells. WK074 cells complemented with pIRR (WK074/pIRR) were hypersensitive to H2O2 (Fig. 3b) in accordance with the observation that mbfA-lacZ was strongly repressed in this strain (Fig. 3a). Expression of mbfA-lacZ from WK074/pIRR45, WK074/pIRR65, WK074/pIRR127 cells was slightly higher oxyclozanide than in WK074/pIRR (Fig. 3a) and these cells exhibited slightly higher resistance to H2O2 than WK074/pIRR

(Fig. 3b). The IrrAt mutant proteins expressed from pIRR94, pIRR45/94, pIRR65/94, pIRR94/127 and pIRRHHH demonstrated a severe defect in mbfA-lacZ repression (Fig. 3a) and were unable to reverse the H2O2-hyper-resistant phenotype of WK074 cells (Fig. 3b). A possible explanation for this result is that the expression level of mbfA in the WK074 cells complemented with these plasmids was high enough to allow the bacteria to survive the 350 μM H2O2 treatment. However, it is possible that other mechanisms of Irr-mediated H2O2 resistance may be involved. WK074/pIRRHHH86 cells exhibited low levels of mbfA-lacZ expression (Fig. 3a) and were hypersensitive to H2O2 (Fig. 3b). The expression of the A. tumefaciens mbfA gene is responsive to iron levels (Ruangkiattikul et al., 2012).

When PPi is hydrolyzed to Pi by a cytosolic pyrophosphatase, the free-energy change is not preserved, but dissipated as heat. However, the involvement of a membrane-bound H+-translocating pyrophosphatase makes it possible to use the energy released CHIR-99021 molecular weight upon PPi hydrolysis by establishing a proton motive force (McIntosh & Vaidya, 2002;

Serrano et al., 2004). The energy in PPi also drives the PPi-dependent reactions in the glycolytic pathway of C. saccharolyticus. This has also been shown for some other organisms, which conserve the free energy using a PPi-PFK (Reeves et al., 1974; Desantis et al., 1989; Alves et al., 2001) or a PPDK (Saavedra et al., 2005; Tjaden et al., 2006; Feng

et al., 2008) in their central carbon catabolism. Mertens (1991) argued that a PPi-dependent glycolysis could be a way for fermentative bacteria to cope with a lower ATP yield. Overall, the results presented indicate that PPi plays a central role in the metabolism of the hydrogen-producing C. saccharolyticus. This type of metabolism agrees well with the observed physiology with respect to its sugar utilization (VanFossen et al., 2009). The wide range of high-affinity sugar uptake systems and the absence of carbon catabolite repression suggest that C. saccharolyticus is not fastidious, but rather has evolved to conserve energy in many different ways. The research of A.A.M.B. was supported by the IP/OP program ‘Systems Biology,’ subsidized by Wageningen ABT-737 ic50 University. The work of K.W. was supported by the EU FP6-SES IP HYVOLUTION (contract no. 019825). A.A.M.B. and K.W. contributed equally to this work. “
“Genome analysis of the Gram-positive cellulolytic bacterium Clostridium thermocellum revealed the presence of multiple negative regulators of alternative σ factors. Nine of the deduced proteins share a strong similarity in their N-terminal sequences

Non-specific serine/threonine protein kinase to the Bacillus subtilis membrane-associated anti-σI factor RsgI and have an unusual domain organization. In six RsgI-like proteins, the C-terminal sequences contain predicted carbohydrate-binding modules. Three of these modules were overexpressed and shown to bind specifically to cellulose and/or pectin. Bioinformatic analysis of >1200 bacterial genomes revealed that the C. thermocellum RsgI-like proteins are unique to this species and are not present in other cellulolytic clostridial species (e.g. Clostridium cellulolyticum and Clostridium papyrosolvens). Eight of the nine genes encoding putative C. thermocellum RsgI-like anti-σ factors form predicted bicistronic operons, in which the first gene encodes a putative alternative σ factor, similar to B. subtilisσI, but lacking in one of its domains. These observations suggest a novel carbohydrate-sensing mechanism in C.

21 and 0.31, respectively. Moreover, being located at a distance of 570 kbp in the R. grylli genome, the simultaneous use of both markers will make it likely that possible LGT events will not have affected both genes at a time. In particular, on the basis of the above analysis and within the range of infra-generic diversity covered by the present study,

these markers’ reliability and resolution potential for taxonomic studies within the genus Rickettsiella appear higher than those of the corresponding 16S rRNA-encoding sequences. The currently accepted view that the MK0683 in vitro Rickettsiella pathotypes ‘R. melolonthae’ and ‘R. tipulae’ are synonyms of the species R. popilliae and should therefore be more distantly related to R. grylli than to each other is strengthened by the results from phylogenetic reconstruction and significance testing for these two markers. In addition to gidA and sucB, four further genetic markers, namely the 16S and 23S rRNA-encoding as well as the rpsA and ftsY gene sequences, were found to be sufficiently phylogeny informative to produce Ixazomib mw a significant genus-level classification of Rickettsiella-like bacteria. Whereas the 23S rRNA and rpsA genes appear uninformative at the infra-generic level, the 16S rRNA and the ftsY sequences, even if inappropriate markers in view of the generation of significantly supported

results, might be useful heuristic indicators for studies dealing with the internal taxonomic or phylogenetic structure of the genus Rickettsiella. However, for supra-generic studies within the order Legionellales, both ribosomal RNA markers, and particularly so the 16S rRNA gene, are likely to produce superior

results when compared to the investigated protein-encoding markers. We are highly indebted to Helga Radke (JKI) for excellent technical assistance. “
“The Cpx-envelope Branched chain aminotransferase stress system coordinates the expression and assembly of surface structures important for the virulence of Gram-negative pathogenic bacteria. It is comprised of the membrane-anchored sensor kinase CpxA, the cytosolic response regulator CpxR and the accessory protein CpxP. Characteristic of the group of two-component systems, the Cpx system responds to a broad range of stimuli including pH, salt, metals, lipids and misfolded proteins that cause perturbation in the envelope. Moreover, the Cpx system has been linked to inter-kingdom signalling and bacterial cell death. However, although signal specificity has been assumed, for most signals the mechanism of signal integration is not understood. Recent structural and functional studies provide the first insights into how CpxP inhibits CpxA and serves as sensor for misfolded pilus subunits, pH and salt. Here, we summarize and reflect on the current knowledge on signal integration by the Cpx-envelope stress system.

21 and 0.31, respectively. Moreover, being located at a distance of 570 kbp in the R. grylli genome, the simultaneous use of both markers will make it likely that possible LGT events will not have affected both genes at a time. In particular, on the basis of the above analysis and within the range of infra-generic diversity covered by the present study,

these markers’ reliability and resolution potential for taxonomic studies within the genus Rickettsiella appear higher than those of the corresponding 16S rRNA-encoding sequences. The currently accepted view that the MK-1775 Rickettsiella pathotypes ‘R. melolonthae’ and ‘R. tipulae’ are synonyms of the species R. popilliae and should therefore be more distantly related to R. grylli than to each other is strengthened by the results from phylogenetic reconstruction and significance testing for these two markers. In addition to gidA and sucB, four further genetic markers, namely the 16S and 23S rRNA-encoding as well as the rpsA and ftsY gene sequences, were found to be sufficiently phylogeny informative to produce www.selleckchem.com/products/Deforolimus.html a significant genus-level classification of Rickettsiella-like bacteria. Whereas the 23S rRNA and rpsA genes appear uninformative at the infra-generic level, the 16S rRNA and the ftsY sequences, even if inappropriate markers in view of the generation of significantly supported

results, might be useful heuristic indicators for studies dealing with the internal taxonomic or phylogenetic structure of the genus Rickettsiella. However, for supra-generic studies within the order Legionellales, both ribosomal RNA markers, and particularly so the 16S rRNA gene, are likely to produce superior

results when compared to the investigated protein-encoding markers. We are highly indebted to Helga Radke (JKI) for excellent technical assistance. “
“The Cpx-envelope Tobramycin stress system coordinates the expression and assembly of surface structures important for the virulence of Gram-negative pathogenic bacteria. It is comprised of the membrane-anchored sensor kinase CpxA, the cytosolic response regulator CpxR and the accessory protein CpxP. Characteristic of the group of two-component systems, the Cpx system responds to a broad range of stimuli including pH, salt, metals, lipids and misfolded proteins that cause perturbation in the envelope. Moreover, the Cpx system has been linked to inter-kingdom signalling and bacterial cell death. However, although signal specificity has been assumed, for most signals the mechanism of signal integration is not understood. Recent structural and functional studies provide the first insights into how CpxP inhibits CpxA and serves as sensor for misfolded pilus subunits, pH and salt. Here, we summarize and reflect on the current knowledge on signal integration by the Cpx-envelope stress system.

3a). It was important to verify that the C-terminal HemA truncations encode functional enzymes and exhibit normal regulation. Plasmid-encoded, truncated, and tagged S. enterica hemA complemented an E. coli hemA mutant. Regulation in response to heme was tested by Western blot (Fig. 3b). To eliminate the possibility that a partial defect in the enzyme activity of the truncated proteins could affect the results of the test, an E. coli host that is wild type for hemA was used, and the plasmid-encoded proteins were specifically detected by an additional C-terminal FLAG tag. Truncated HemA exhibited

normal regulation in response to heme limitation. His-tagged C-terminally truncated HemA was selleck screening library purified by Ni-NTA affinity chromatography. The purified protein was red in color, suggesting the presence of bound heme. The absorption spectrum of purified HemA protein (Fig. 1a) contains features characteristic of heme, including a prominent peak at 424 nm (the NVP-BKM120 cost Soret band). Upon reduction with Na-dithionite, the peak at 424 nm became sharper and shifted toward a longer wavelength (426 nm), and two other peaks appeared: one at 530 nm and another at 560 nm. The spectrum of reduced heme (hemin), which was used as a control, was very similar to that of the purified protein (data not shown). Three separate protein preparations

averaged 0.055 mol heme mol−1 protein monomer as determined by the pyridine hemochromogen assay. HemA1−412 [C170A]-His6 was purified according to the Edoxaban same protocol as that used for HemA1−412-His6. The C170A mutant protein was colorless, suggesting that it is unable to bind heme. The absence of heme was also demonstrated by its absorption spectrum, which lacks the peaks characteristic of heme-containing proteins (Fig. 1b). The HemA spectrum is that of a b-type heme; this class of molecules is attached noncovalently. Treatment with the strong denaturant, 6 M guanidine-HCl, removed a maximum of 7% of heme from HemA, and in two trials, failed

to remove any (Supporting Information). The ability to retain noncovalently bound heme in the presence of strong denaturants has been documented for other proteins (Hargrove & Olson, 1996; Wójtowicz et al., 2009). Although these results demonstrate a strong association between heme and HemA, covalent binding cannot be inferred from this assay. Thiol reagents, which have been used to distinguish covalent heme-protein bonds, are incompatible with Ni-NTA. The nature of the association between heme and HemA was further examined using a different method. Heme-associated peroxidase activity, which can be measured by standard ECL reagents (a Western blot without the antibody; Dorward, 1993), detects heme-binding proteins (such as cytochrome c). Purified proteins were separated by SDS-PAGE and then assessed for heme-associated peroxidase activity.

, 2001). KirP contains all three conserved sequence motifs described by Lambalot et al. (1996) and Sanchez et al. (2001). Based on the presence of a conserved FSxKESLxK in motif P3 and its phylogenetic relationship to other PPTases, KirP can be assigned to the F/KES subfamily (Copp & Neilan, 2006) of Sfp-type PPTases. To analyze the role of KirP in vivo, kirP was inactivated by gene replacement. The gene replacement plasmid pEP10 was introduced into the wild-type strain S. collinus Tü 365. Homologous recombination resulted in the replacement of kirP with the thiostrepton resistance cassette of pEP10. The genotype of the resulting mutant strain, EP-P1, was confirmed

by Southern analysis with a kirP probe (Fig. 1a and b). Extracts from wild-type XAV-939 supplier and EP-P1 cultures

were analyzed for kirromycin production by HPLC. The mutant strain showed a substantial reduction in kirromycin yield of approximately 90%. The identity of kirromycin was confirmed by comparison with an HPLC-UV/Vis spectra library (Fiedler, 1993) and by MS (m/z of kirromycin=795 [M-H]−). To prove that GSK126 chemical structure the significant reduction in kirromycin yield is due to the inactivation of kirP, plasmid pEP11 expressing the intact wild-type kirP gene under control of the consitutive ermE* promoter was used to complement the inactivated kirP gene. The pEP11 construct was introduced into the mutant strain EP-P1. In the complemented strain, kirromycin production was partially restored, increasing by a factor of 3 compared with the mutant and reaching approximately 30% of the wild-type production level. Observations that gene replacement mutations in streptomycetes can

be only partially complemented have been made in many pathways, for example daptomycin biosynthesis (Coeffet-Le Gal et al., 2006) when genes are deleted and subsequently reintroduced in a different context (for a review, also see Baltz, 1998). The partial complementation of the kirP deletion in mutant EP-P1 indicated that Rebamipide the loss of kirP activity was responsible for the large decrease in kirromycin production and thus that kirP plays an important role in the biosynthesis of kirromycin. However, the kirP gene replacement mutant was viable and produced low amounts of kirromycin. This finding implies that the genome of the producer strain S. collinus Tü 365 includes additional PPTase genes. Indeed, analysis of preliminary data of an ongoing whole genome sequencing project of S. collinus enabled the identification of at least six additional Sfp-type PPTase genes and one ACPS-type PPTase gene in the genome of the kirromycin producer strain. Thus, one or more of these enzymes might provide some phosphopantetheinylation of the kirromycin PKS/NRPS enzyme, albeit with a much lower efficiency than KirP, as indicated by the 90% drop in kirromycin yield in the kirP deletion mutant EP-P1.

Inspired by these studies we used a reinnervation model of synaptogenesis to analyze neuromuscular function in mice lacking neural cell adhesion molecule (NCAM), the Fasciclin II vertebrate homolog. Our results showed that the recovery of contractile force was the same Angiogenesis inhibitor in wild-type and NCAM−/− mice at 1 month after nerve injury, indicating that endplates were appropriately

reformed. This normality was only transient because the contractile force and myofiber number decreased at 3 months after injury in NCAM−/− mice. Both declined further 3 months later. Myofibers degenerated, not because motoneurons died but because synapses were withdrawn. Although neurotransmission was initially normal at reinnervated NCAM−/− NMJs, it was significantly compromised 3 months later. Interestingly, the selective ablation of NCAM from motoneurons, or muscle fibers, did not mimic the deficits observed in reinnervated NCAM−/− mice. Taken together, these results indicate that NCAM is required to maintain normal synaptic function at reinnervated NMJs, although its loss pre-synaptically or post-synaptically is not sufficient to induce synaptic destabilization.

Consideration is given to the role of AZD0530 molecular weight NCAM in terminal Schwann cells for maintaining synaptic integrity and how NCAM dysfunction may contribute to motoneuron disorders. “
“GABA transporter subtype 1 (GAT-1) and GABA transporter subtype 3 (GAT-3) are the main transporters that regulate inhibitory GABAergic transmission in the mammalian brain through GABA reuptake. In this study, we characterized the ultrastructural localizations and determined the respective roles of these transporters in regulating evoked inhibitory postsynaptic currents (eIPSCs) in globus pallidus (GP) neurons after striatal stimulation. In the young and adult rat GP, GAT-1 was preferentially expressed

aminophylline in unmyelinated axons, whereas GAT-3 was almost exclusively found in glial processes. Except for rare instances of GAT-1 localization, neither of the two transporters was significantly expressed in GABAergic terminals in the rat GP. 1-(4,4-Diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride (SKF 89976A) (10 μm), a GAT-1 inhibitor, significantly prolonged the decay time, but did not affect the amplitude, of eIPSCs induced by striatal stimulation (15–20 V). On the other hand, the semi-selective GAT-3 inhibitor 1-(2-[tris(4-methoxyphenyl)methoxy]ethyl)-(S)-3-piperidinecarboxylic acid (SNAP 5114) (10 μm) increased the amplitude and prolonged the decay time of eIPSCs. The effects of transporter blockade on the decay time and amplitude of eIPSCs were further increased when both inhibitors were applied together. Furthermore, SKF 89976A or SNAP 5114 blockade also increased the amplitude and frequency of spontaneous IPSCs, but did not affect miniature IPSCs.

This work was supported by a Grant-in-aid

for Scientific Research (C) from The Japan Society for the Promotion of Science (No. 19592135 to K.S.). Fig. S1. Verification of inner membrane fractions (IM) and outer membrane Stem Cell Compound Library fractions (OM) prepared from W83 (WT), 83K5 (Sov-His), and 83K3 (Δsov). Outer membrane fractions were verified by immnoblot analysis using anti-Pgm6/7 atiserum raised against Pgm6/7 proteins from Porphyromonas gingivalis ATCC33277 [Nagano K, Read EK, Murakami Y, Masuda T, Noguchi T & Yoshimura Y (2005) J Bacteriol187: 902-911]. Inner membrane fractions were verified by checking the NADH-ferricyanide oxidoreductase activity [Futai M (1974) J Membr Biol15: 15-28]. The reduction of ferricyanide was determined in 80 mM Tris-HCl, pH 7.4, 9.0 mM KCN, 1.0 mM NADH, and 0.7 mM ferricyanide, and measured at 420 nm. We thank Fuminobu Yoshimura for the anti-Pgm6/7 antiserum. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“It has been demonstrated previously that Enterococcus faecalis produces secreted endoglycosidases that enable the bacteria to remove N-linked glycans from glycoproteins. One enzyme potentially responsible for this activity is EF0114, comprising

a typical GH18 endoglycosidase domain and a GH20 domain. We have analyzed the other candidate, EF2863, selleck chemical and show that this predicted single domain GH18 protein is an endo-β-N-acetylglucosaminidase. EF2863 hydrolyzes the glycosidic bond between two N-acetylglucosamines (GlcNAc) in N-linked glycans of the high-mannose and hybrid type, releasing the glycan and leaving one GlcNAc attached to the protein. The activity

of EF2863 is similar to that of the well known deglycosylating enzyme EndoH from Streptomyces plicatus. According to the CAZy nomenclature, the enzyme is designated EfEndo18A. Enterococci are Gram-positive lactic acid bacteria that traditionally have been selleck compound considered harmless inhabitants of the intestinal tract of mammals. However, in recent years, enterococci have emerged as nosocomial pathogens, causing urinary tract infections, bacteraemia and infective endocarditis. Most clinical infections caused by enterococci are due to Enterococcus faecalis (Tendolkar et al., 2003; Fisher & Phillips, 2009). Enterococcus faecalis V583 was the first clinical vancomycin-resistant enterococcal isolate reported in the USA (Sahm et al., 1989) and sequencing of the genome was completed in 2003 (Paulsen et al., 2003). Due to increasing problems with antibiotic resistance, treatment of enterococcal infections is difficult, and there is a need for alternative strategies for overcoming pathogen-related diseases.