Conclusions The c-di-GMP pathway is used by most bacteria (but not eukaryotes or Archaea) to regulate numerous biological processes [36]. Several lines of evidence have indicated the concentration of c-di-GMP, balanced by diguanylate cyclase (DGC) and phosphodiesterase (PDE), account for the fimbrial regulatory network in some microorganisms. In S. Typhimurium, it has been demonstrated that production of curli fimbriae was inhibited by a PDE STM3611[18]. However, no other type of fimbrial expression in this microorganism has thus far been shown to be controlled by DGC or PDE. The present study revealed that a previously uncharacterized

stm0551 gene, which could encode a PDE, contributes to the down-regulation of type 1 fimbrial expression in S. Typhimurium. Our finding may provide valuable information that may Screening Library research buy help to further elucidate the complicated type 1 fimbrial regulatory circuit in this pathogen. Methods Bacterial strains, plasmids, and culture media The bacterial strains, plasmids, and primers used in the present study are listed in Table 1 and Table 2. The S. Typhimurium strain used was LB5010, an LT2 derivative [21]. This strain produces type 1 fimbriae and has a variable fimbrial phase. Bacteria were cultured in Luria-Bertani (LB) broth (Difco/Becton BGB324 mouse Dickinson, Franklin Lakes, NJ) or plated on LB agar. When required, media

were supplemented with antibiotics at the following concentrations: 100 μg/ml ampicillin, 50 μg/ml kanamycin, and 20 μg/ml chloramphenicol. Antibiotics were obtained from Sigma (St. Louis, MO). To detect gene expression, 1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was used (MDbio, Taipei, Taiwan). Construction of a S. Typhimurium stm0551 mutant A stm0551 mutant was created by one-step gene inactivation method as described previously [20]. Briefly, a kanamycin-resistance Rho gene from pKD13 tagged with a flanking sequence of the stm0551 gene was generated by a polymerase chain reaction (PCR) technique. The designed nucleotide sequence was generated

with Pfu polymerase (Fermentas, St. Leon-Rot, Germany) on a GeneAmp PCR system 2700 thermal cycler (Applied Biosystems, Foster City, CA) and initially incubated at 94 ° C for 3 min, followed by 30 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min. Primers used in this approach are listed in Table 3. Then, the PCR product was introduced by electroporation into S. enterica serotype Typhimurium LB5010 possessing the pKD46 plasmid which expressed λ Red recombinase [20]. All transformants were grown on LB agar containing kanamycin. The constructed mutants were verified by PCR with primers located in the flanking sequence of the stm0551 gene. Yeast agglutination and guinea pig erythrocyte hemagglutination test for type 1 fimbriae Tested bacteria were cultured in static LB broth at 37°C for 48 h or on LB agar at 37°C overnight.

Interestingly, our study validates the findings of a previous study which identified pyruvate dehydrogenase E1 α chain and

the protein encoded by MG_328 as phosphoproteins of M. genitalium[47]. Recently, both these proteins were found on the surface of M. genitalium[48], thus suggesting the possibility that they can play a role in Danusertib purchase M. genitalium-host interaction. What is intriguing in this study, however, is the reduced phosphorylation of a protein (PDH) in a phosphatase (MG208) deficient mutant of M. genitalium. Theoretically, if proteins are dephosphorylated by a specific phosphatase, then the expectation, in the absence of the phosphatase, is no change in the phosphorylation levels or increased levels of phosphorylation of proteins. In fact, the proteins identified in PrpC mutant of M. pneumoniae[49] behave in this manner. The differentially phosphorylated

proteins identified in this mutant included RopE, an adhesion related protein P41, HMW3, MPN256 and MPN474. These proteins showed phosphorylation only in the PrpC mutant but not in the wild type. The contrasting situation in MG_207 tends to speculate that additional protein(s) with possible kinase property is affected in this mutant and this putative protein fails to phosphorylate some proteins in the mutant. However, this is only a hypothesis and requires additional studies for confirmation. Nevertheless, differential phosphorylation

find more of proteins has been shown in the STP mutant strains of L. monocytogenes and S. pneumoniae[44]. In addition to changes in protein phosphorylation, an STP (stp1) mutant of Group B Streptococcus has been shown Chloroambucil to have altered expression of 294 genes which included stk1, the gene encoding a major kinase [24]. Table 1 Phosphorylated proteins identified by Mass spectrometry Spot numbera Protein name Gene symbol Gene Putative function 1 Pyruvate dehydrogenase E1 subunit α pdhA MG_274 Metabolism/surface protein 2 Pyruvate dehydrogenase E1 subunit α pdhA MG_274 Metabolism/surface protein 3 Putative cytoskeletal protein – MG_328 Cytoskeletal involvement 4 Conserved hypothetical protein – MG_281 Unknown 5 Thymidine phosphorylase deoA MG_051 Metabolism a Refers protein spots marked in Figure 3A and C. Ability of TIM207 strain to adhere and invade eukaryotic cells Since TIM207 strain showed differential phosphorylation of proteins, we speculated that this would have some impact on the adherence of this strain with eukaryotic cells. Consistent with this notion, we noticed that TIM207 strain adhered only partially to culture flasks when grown in SP-4 medium. The non-adherent mycoplasmas were seen as floating cell suspensions and this phenotype was highly reproducible.

To investigate the role

of fim2 in virulence, isogenic fim2 mutants were constructed and examined in three murine models, each focussed this website on primary infection of a distinct clinically-relevant anatomical site. Surprisingly, despite many fimbrial systems having been clearly implicated in virulence, we detected no clear evidence of attenuation (murine lung and urinary tract infection models) or reduction in colonizing ability (murine intestinal colonization model) in the fim2-negative strains studied. Intriguingly, examination of bladder CFU count-based CIs for the urinary tract infection experiments hinted at a subtle role for fim2 in the colonization of bladder and kidney tissues. In both tissues, median wildtype CFU counts were approximately ten-fold higher than those of the fim2 mutant, although when performed in a fim negative background this difference was reversed and reduced in bladder and kidney samples, respectively. Nevertheless, the latter conflicting results may due to the markedly lower CFU counts

obtained in the fim negative background. As shown by neutral CI values in the lung tissue but an approximately 100-fold higher median liver CFU count for KR2107 as compared to its isogenic fim2 mutant, the fim2 locus would appear to be involved in systemic dissemination BI-D1870 and/or survival of K. pneumoniae following primary infection of the respiratory tract. However, given the noted lack of statistical significance, low numbers of mice examined and substantial mouse-to-mouse variation for these liver CFU data, no firm conclusions can be derived at present. As an aside, the previously demonstrated Paclitaxel dramatic positive contribution of fim to urovirulence in this murine model was also shown to be the case in the KR2107 background [22, 23]. At an overview level, based on total CFU counts per liver and per kidney for the lung infection and ascending urinary tract infection models, respectively, there was

a suggestion, though not supported statistically, of an ordered gradation amongst the four isogenic strains with the most-to-least virulent as follows: KR2107, KR2107∆fim2, KR2107∆fim and KR2107∆fim∆fim2. We speculate this relates to a Fim2-mediated enhancement of bacterial biofilm-forming-, adhesive- and/or invasive-potential under the in vivo conditions tested. In addition, the predicted influence of Fim2K on the c-di-GMP regulatory circuit, may itself impact on virulence via regulation of Fim2, Fim and/or other virulence factors. The fim2 cluster was also assessed for its ability to contribute to biofilm formation. Gene knock-out experiments in KR2107 failed to reveal a role for fim2 in biofilm formation. However, the function of the product of fim2 may have been masked due to physical interference by the K. pneumoniae capsule, a phenomenon previously observed with type 1 fimbriae [38, 39]. Alternatively, it may be a function of limited fim2 expression under the in vitro conditions examined.

1997). a: Molecular structure of BChl a. b: EPR spectrum in isotropic solution with simulation using the hyperfine couplings from ENDOR. c: A 1H ENDOR spectrum showing 11 line pairs which yield 11 isotropic HFIs. In the low frequency range, three 14N HFI constants could be resolved (HFI constants for all four nitrogens were obtained for an 15N labeled Bchl

\( a^ \bullet + \)). B General TRIPLE experiment selleck chemical yielding the relative signs of all HFI couplings (including 14N) via intensity changes relative to the pumped line pair. C ENDOR of a partially deuterated Bchl \( a^ \bullet + \) that carries protons essentially only at the CH3 groups of rings A and C. The respective 2H ENDOR spectrum at low frequencies is also shown. For further details, see (Lubitz et al. 1997) The radical cation of the primary electron donor \( P_865^ \bullet + \) in bacterial RCs The primary electron donor P 865 is a part of the light-induced electron transfer chain in bacterial RCs. According to the X-ray structure, it consists of a BChl a dimer. In the photosynthetic process, upon absorption of a light quantum by P 865, this species

donates an electron to a nearby acceptor, leaving behind a radical cation selleck \( P_865^ \bullet + . \) This can also be created artificially in the RC by chemical oxidation of P 865. The electronic structure of the primary electron donor and its radical cation is of particular interest, since this species is situated at the interface of exciton and electron transfer and is also of crucial importance for the charge recombination process. The X-band EPR spectrum of \( P_865^ \bullet + \) is a broad unresolved Gaussian line, which Paclitaxel solubility dmso indicates that HFI from many nuclei contribute to the EPR, while

the effect of g-anisotropy is small. To obtain HFI values of individual nuclei, CW ENDOR and TRIPLE spectroscopies were applied to \( P_865^ \bullet + \) in liquid and frozen solution as well as in single crystal of bacterial RCs (Lendzian et al. 1993). About 10 lines were resolved in the 1H Special TRIPLE experiment, and their angular dependence was obtained in three crystallographic planes (Fig. 4), which allowed the determination of the complete HFI tensors, including principal values and principal axes directions, for the most prominent protons. Fig. 4 1H Special TRIPLE spectra of the primary donor radical cation \( P_865^ \bullet + \) at ambient temperature in RC single crystals of Rhodobacter (Rb.) sphaeroides R-26, taken with the external field B 0 along the three crystallographic axes (a, b, c) of the unit cell (space group P212121); a comparison is made with the respective spectrum in isotropic solution. On the right, the angular dependence of the line frequencies in the crystallographic ac-plane is shown. For details, see (Lendzian et al.