Each calorimeter had an outer thermostatic loop provided by a Jul

Each calorimeter had an outer thermostatic loop provided by a Julabo F32-HE device operating in standard mode. 3D sensor protection was provided by a Nitrogen gas purge (99.99% SIAD – TP). The Calisto v1.077 software

package was used for data acquisition and primary signal processing. This included baseline integration end export in Excel with equally spaced time increments. Heat values obtained were further analyzed in Excel and Origin 8.1. Exported baselines were further processed in Peakfit. FG-4592 cell line Peakfit processing of the thermograms Data exported from Calisto were processed in Peakfit by means of previously reported routines [16, 17]. Whenever PPAR agonist inhibitor necessary, Savitsky-Golay smoothing was performed, generally with the “Al Expert” option. Calisto-generated baselines were imported and subtracted from the heat flow (HF, mW) signal. The time zero was changed for each thermogram by means of “Enter Calculation” option in Peakfit, allowing to a left shift of the whole data corresponding to the left intersection of the baseline

and HF. This procedure brings all thermograms to a common X (time) scale, but definitely excludes any analysis of the growth lag time. The resulted data were subjected to “Area normalize” resulting in the “normalized heat flow” (NHF, h-1) [16, 17]. This brings all thermograms to a common Y (NHF) scale, with the advantage that areas of the component peaks represent their fraction to the overall thermal effect. All subsequent peak fitting involved the NHF – time thermograms. Several selleck screening library built-in asymmetric peak functions were tried (EMG, GMG, LogN, Giddings, Pearson IV, HVL, etc.). The best one for the analyzed data proved to be the Haarhof – Van der Linde (HVL) chromatography function. This function resulted in both the best statistical criteria (r2, F-statistic, standard errors,

etc.) and most reliable variations of the fitting parameters among the member of each set. As detailed in section C1, peak parameters were allowed to vary independently learn more through the “Vary Widths” and “Vary Shape” options. The “Medium (Lorentz Err.) Robust Minimization” procedure was applied instead of the classical least-squares one. Bacterial strains The reference strains of Staphylococcus aureus – ATCC 25923 and Escherichia coli – ATCC 25922 were used throughout the present study. Culture media Bacterial culture media were prepared from stock Tryptic Soy Broth (TSB, Oxoid, UK), which is a mixture of Pancreatic digest of casein (17 g), NaCl (5 g), Papaic digest of soybean meal (3 g), K2HPO4 (2.5 g), Glucose (2.5 g) to 1 Liter and a pH of 7.3 ± 0.2 at 25°C. The medium was autoclaved before use and was microbiologically pure. For viability counts, preparation of isolated colonies for inoculation and random sample check of aseptic technique, we used plates with Tryptic Soy Agar (TSA, Oxoid, UK); this solid medium has the same basic composition as TSB.

Yet, our two interacting clones remind of theoretical models like

Yet, our two interacting clones remind of theoretical models like “”hawks-doves”" or “”prisoners dilemma”" (or even interaction of a monoculture with weeds or pathogens). The third possibility – regular patterns of colony ontogeny – allows even genuine convergent development entering the game. Our observations suggest that development of bacterial bodies in Serratia sp. includes both events taking place within a body, and transmission of signals between distinct bodies. Signals act at a distance, i.e. they do not require physical contact (as reported,

e.g., in [16, 35, Androgen Receptor signaling Antagonists 36]). Experiments with conditioned agar show that signals do not require simultaneous presence of living entities; hence, actively emitted light, sound, electrical or even chemical pulses of whatever nature can be excluded as carriers of the signal. We are left with a compound or a cocktail of compounds, emitted by living entities into their environment, persisting there for some time, and being actively interpreted by recipients that happen to be present in their range. While our observations do

not provide any hints yet as to the chemical identity of these signals, they at least point towards some of their properties. Experiments with signaling across the septum suggest that the signal from the macula spreads via the gas phase. For a different bacterial system, indole could be the carrier of a volatile signal ([37]; however, this conclusion was later questioned [38]). Ammonia appeared to be the signal carrier in yeast colonies [39]. As a first step AG-881 price towards characterizing our signal, we demonstrated that it is readily cleared away by non-volatile acid or alkali traps. We Selleck PRIMA-1MET propose a simple model capable of simulating some aspects of our experimentally characterized examples of bacterial body morphogenesis (the F and R colonies).

This model involves two http://www.selleck.co.jp/products/BafilomycinA1.html factors carrying information for both morphogenesis and mutual influencing of neighbors, generated in bodies at certain developmental stages, and diffusible to the environment. One of the signals travels (slowly) through the substrate, the other is transmitted via the gas phase. These bearers of the signals (or even a sign) are perceived by all cells, allowing their orientation and behavior in the developing colony; timing may be the second critical factor at play. While several theoretical models of microbial colony morphogenesis have been published, they mostly focus on such aspects as kinetics of colony expansion controlled by nutrient diffusion through the colony and surrounding medium [40–43], intra-colony spatial organization of cells [44, 45] or fine patterning of the colony margin based on interplay of nutrient and signal diffusion and, in some cases, also swarming behavior of the bacteria [46–48].

Table 8 APC family member representation in Sco and Mxa TC # Fami

Table 8 APC family member representation in Sco and Mxa TC # Family Sco Mxa 2.A.3 Amino Acid-Polyamine-Organocation (APC) Superfamily 17 2 2.A.15 Betaine/PND-1186 mw Carnitine/Choline Transporter (BCCT) Family 1   2.A.18 Amino Acid/Auxin Permease (AAAP) Family     2.A.21 Solute:Sodium

Symporter (SSS) Family 8 4 2.A.22 Neurotransmitter:Sodium Symporter (NSS) Family     2.A.25 Alanine or Glycine:Cation Symporter (AGCS) Family 1   2.A.30 Cation-Chloride Cotransporter (CCC) Family     2.A.39 Nucleobase:Cation Symporter-1 (NCS1) Family 5   2.A.42 Hydroxy/Aromatic Amino Acid Permease (HAAAP) Family     Numbers of APC Superfamily proteins in Sco and Mxa are arranged by family. The SSS Family of solute:Na+ symporters, a constituent BACE inhibitor member of the APC Superfamily [59], transports a wide variety of solutes. Of the eight SSS family members in Sco, five probably transport short monocarboxylic acids (acetate, lactate, pyruvate, etc.), while three probably transport sugars. Of the four hits in Mxa, two may be monocarboxylate transporters while the other two are probably

non-transporting signal transduction proteins with C-terminal sensor kinase domains. Only one of them is homologous to SSS transporters in its transmembrane domain. Heavy metal carriers Both Sco and Mxa have members (five and three members, respectively) of the heavy metal efflux Cation Diffusion Facilitator (CDF) Family 2.A.4; [60], but only Mxa has members (two) of the metal uptake Zinc-Iron Permease (ZIP) Family 2.A.5; [61]. Only Sco has a member of the Nramp Family Proteases inhibitor of divalent cation transporters. These proteins exhibit varying specificities for heavy metals and are involved in metal ion homeostasis. Heavy metal transporters are also found in other families such as the RND very Superfamily. The RND superfamily The RND Superfamily 2.A.6; [62, 63] is well represented in both Sco and Mxa with 16 members in Sco and 20 in Mxa. Family 1 (Heavy Metal Efflux (HME)) is prevalent in Mxa with six members (see TCDB; 2.A.6.1.7-11 and 2.A.6.3.2), but absent in Sco. Based on induction properties, one may export

Zn2+, two may export heavy metals (one of these is induced under starvation conditions), and three may export copper [64]. Similarly, the (largely Gram-negative bacterial) Hydrophobe/Amphiphile Efflux-1 (HAE1) Family (Family 2), usually considered to be concerned with drug export, is found in Mxa (four members) but not Sco. Surprisingly, the lipooligosaccharide Nodulation Factor Exporter (NFE) Family (Family 3) is represented in both organisms, but with six members in Mxa and only one in Sco. These proteins may transport substrates resembling rhizobial nodulation factor lipooligosaccharides, which are the substrates of the only characterized member of the NFE Family [65]. Such substrates are not known to be present in myxobacteria or actinobacteria.

pini (P < 0 0001), and 0 6 for P tropicalis (P = 0 0004), respec

pini (P < 0.0001), and 0.6 for P. tropicalis (P = 0.0004), respectively. The only exceptions were observed in P. megasperma at 24 h, P. nicotianae Ruboxistaurin in vitro at 10 min and 24 h, as well as P. pini at 10 min. These results indicated that zoospore survival in runoff water containment basins is subjected to fluctuations of dissolved oxygen concentration in particular of hyperoxia conditions although there are slightly differences among the four species assessed in this study.

P. megasperma was least affected by elevated concentrations of dissolved oxygen as was by a range of pH in a previous study [7]. Differences in oxygen response were previously observed among oomycetes and fungi. By their oxygen response, these fungi and oomycetes can be grouped into three categories. First, mycelial growth is directly proportional to atmospheric oxygen level with the optimum at 21.0%. This pattern is exemplified by P. GW786034 mw nicotianae (syn. P. parasitica), P. citrophthora and T. basicola[17] and P. cactorum[15]. Second, mycelial growth has a clear optimal oxygen level typically well below 21.0%, which distinguishes this

group from those of the first pattern. Examples of this group included A. euteiches that had optimal growth at 5.0% [24]. Third, mycelial growth increases with increasing atmospheric oxygen only to a concentration, above which results in no further growth benefits. This pattern is illustrated by P. ultimum, of which mycelial growth was reduced at oxygen concentration of 1.3% but was the same for all oxygen levels from 4.0%

to 21.0% [25]. It is unclear how the elevated concentrations of dissolved oxygen affected zoospore survival of different species. In this study we did observe that zoospores of P. nicotianae, P. pini and P. tropicalis remained motile for more than 2 h after their release from sporangia while Mirabegron the most zoospores of P. megasperma had NCT-501 already encysted before they were added to the 500-ml volume at the various dissolved oxygen concentrations. It is reasonable to assume that motile zoospores are more vulnerable to environmental stresses including elevated concentrations of dissolved oxygen or hyperoxia than those encycled ones with cell wall. It is worth of noting that zoospores of P. nicotianae died instantly in a 9.5-L fish tank being bubbled with oxygen at 0.5 L min-1 for 20 min under a separate experiment [22]. The dissolved oxygen concentration in this fish tank was estimated to be over 27.3 mg L-1 according to the formula developed above. It also was previously reported that hyperoxia suppressed fungi and bacteria [29, 30]. Artificial oxygenation of irrigation water for pathogen mitigation may not be economically feasible. However, dissolved oxygen concentration in irrigation reservoirs can naturally rise up to 26.5 mg L-1 due to phytosynthetic activities [13]. Zoospores are the principal, if not sole, dispersal and infective propagules of Phytophthora and Pythium species in recycling irrigation systems [31–35].

(a) Photocurrent density-voltage characteristics

of NF- a

(a) Photocurrent density-voltage characteristics

of NF- and HNF-based ssDSC measured under one sun illumination. IPCE spectra of the above-mentioned cells are given in the inset. (b) UV–vis absorption EPZ015666 mouse spectra of the amount of dye desorbed from the respective photoanodes. The electrochemical impedance spectroscopy measurements are further performed to elucidate the enhancement of V oc in the HNF cell. Figure  5a depicts the Nyquist plots of the two cells under open circuit voltage condition. The line connecting the first semicircle at higher frequencies and the semicircle at intermediate frequencies denote the charge transport resistance within the TiO2 film. By fitting the EIS spectra using the transmission line model of DSC [28, 29], it is observed that the resistance to transport of charge within plain nanofiber is higher because the SB525334 charge has to encounter more number of grain boundaries as each nanofiber is composed of several nanofibrils. Whereas in the case of HNF cell, each nanofiber is covered with single crystalline nanorods in which the transport of electron is less inhibited. Since the nanofiber acts as a seeding layer for the growth of nanorods and the nanorods grow at the expense of the nanofiber, the nanofiber is reduced in size leading to less number of defects (as in Figure  3c). The second semicircle at the intermediate frequencies in the Nyquist plot denotes the charge recombination resistance

between TiO2 and HTM layer. It is observed that the semicircle of HNF cell is larger than the semicircle of the NF cell, implying that the HNF cell exhibited higher resistance to charge recombination

as compared to that of NF cell. The plot of charge recombination resistance (Rct) vs. chemical capacitance (Cμ) is shown in Figure  5b. It was reported that this approach provides information analogous to that obtained from the approach of comparing lifetimes or dark current at constant charge density [30]. So at a particular Cμ which is a measure of density of states at quasi-Fermi level, Rct of HNF is higher than the Rct of NF (Figure  Vildagliptin 5b). This indicates that the HNF exhibited higher resistance to recombination of injected charge with holes in spiro-OMeTAD. As a result of the higher charge recombination resistance, HNF cell exhibited higher V oc. The densely populated nanorods with higher dye loading provide greater screening between the injected electrons in TiO2 film and holes in HTM, thereby suppressing the recombination of electrons at the TiO2 and spiro-OMeTAD interface [31]. In the case of NF-based cell, the pores between the nanofibers are big and the dye Thiazovivin cost coverage is relatively lower, ensuing the recombination of electron hole pair at TiO2/spiro-OMeTAD. This is also supported by the delayed onset of dark current in the case of HNF-based DSC, which is suggestive of the good blocking property of HNF (as seen in Figure  4a).

The fluorescence intensity of each measurement is represented as

The fluorescence intensity of each measurement is represented as a percentage of the initial acridine orange fluorescence signal prior to addition of lactate. The control vesicles (Figure 6; grey traces) exhibited negligible Na+/H+ or K+/H+ activities at pH values of 9.0 to 9.75. This was expected because the TO114 cells from which the inverted vesicles were generated are devoid of the major antiporters NhaA, NhaB and ChaA that function primarily in monovalent metal cation/H+ exchange at alkaline pH [12, 26]. However, at pH 8.5 the controls exhibited some degree of exchange activity; this activity was more pronounced upon addition of K+ ions and resulted in ~30% www.selleckchem.com/products/Cediranib.html dequenching of the initial lactate-induced

fluorescence quench (Figure 6B, top panel). It is conceivable that this dequenching was due to the activity learn more of other, chromosomally-encoded antiporters that operate in the same pH range and that have a greater affinity for K+ than Na+ ions. In all control experiments, addition of 100 μM CCCP at the time indicated resulted

in dissipation of the ΔpH, as revealed by an instantaneous dequenching check details of the fluorescence signal. This confirmed that the inverted vesicles had maintained integrity over the lifetime of the assay. In contrast to the controls, addition of Na+ or K+ to inverted vesicles containing recombinant wild-type MdtM resulted in a rapid and significant dequenching of the lactate-induced, acridine orange steady state fluorescence at all the alkaline pH values tested (Figure 6; black traces), thus indicating that MdtM was responsible for catalysing both Na+/H+ and K+/H+ exchange reactions. The magnitude of the dequenching at each pH value, however, varied depending upon the pH and the metal cation added;

in the case of added Na+ the most pronounced dequenching was observed at pH 9.25 (Figure 6A; black traces) whereas the maximal K+-induced dequenching occurred at pH 9.0 (Figure 6B; black traces). As observed from the assays performed on control vesicles, the addition of CCCP to the reaction mixtures resulted in a further dequenching of the fluorescence signal, confirming Ribociclib in vivo that the MdtM-containing inverted vesicles had also maintained integrity for the lifetime of the assay. pH profiles of MdtM-catalysed K+/H+ and Na+/H+ exchange activities Measurements of the acridine orange fluorescence dequenching enabled a plot of the K+/H+ and Na+/H+ exchange activities (expressed as the percentage dequenching of the lactate-induced fluorescence quenching) as a function of pH to be constructed, and this revealed a clear pH-dependence for both (Figure 7A). At pH ≤6.5, no transport of the probed K+ and Na+ cations was detected, providing further evidence that MdtM does not operate as a monovalent metal cation/H+ antiporter at acidic pH. However, as the pH increased and became more alkaline, a significant exchange activity was recorded. From no detectable activity at pH 6.

They create link between innate and adaptive immunity TLRs are a

They create link between innate and adaptive immunity. TLRs are abundant on cells of the immune system but have been also demonstrated on cells of other origin such as various epithelia. We were HMPL-504 purchase able to show the expression of three TLRs (TLR2, 3 and 4) on tumor cells of human laryngeal carcinoma by means of immunohistochemistry. This study was followed by the demonstration of most TLRs on cell lines of this

cancer both, on protein and molecular level. On the current study we wished to see the impact of respective TLR ligands on TLR expression in the cells mentioned. Six larynx carcinoma cell lines obtained from Pittsburgh Cancer Institute, USA, (courtesy of prof. Theresa Whiteside), have been used. They were cultured for 24 hrs in the presence of respective TLR ligands. Following culture cells were harvested and subjected to flow cytometry both on intact and permeabilized PLX3397 chemical structure cells, using fluorochrome labelled anti TLR1-10 monoclonal Moabs. The cells were evaluated in FacsCanto (BD) flow cytometer for mean fluorescence intensity (MFI) of membrane and cytoplasmic

cell staining, using FacsDiva software. Results: Each cell line exhibited distinct pattern of expression of individual TLRs following interaction with respective ligand. Unexpectedly, cell culture with ligand resulted in the decrease of TLR expression in some cell lines. Cytoplasmic TLR staining had usually Molecular motor higher MFI value than membrane one. TLRs 5, 7 and 9 showed the highest expression in the majority of tumor cells tested. In general, cytoplasmic TLR protein product formation seems to exceed cell membrane expression. In conclusion, culture of TLR expressing tumor cells with respective ligand has ambiguous effect on TLR expression but

points out for potential reactivity of tumor cells with TLR agonists. O104 Functional Assessment of the Inflammatory Tumor Microenvironment during Spontaneous Breast Cancer Progression and Metastasis Formation Metamia Ciampricotti1, Tisee Hau1, Ewoud Speksnijder1, Jos Jonkers1, Karin de Visser 1 1 Department of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands Interactions between cancer cells and normal, healthy cells present cells are one of the most abundant cell types recruited to the microenvironment of many tumors. The role of the immune system during tumorigenesis is this website rather controversial; both tumor-protective and tumor-promoting properties of the immune system have been described. It is currently unclear which tumor types and which tumor stages are either positively or negatively regulated by specific components of the immune system.

In Mexico, one out of 20 Mexican men and one out of 12 Mexican wo

In Mexico, one out of 20 Mexican men and one out of 12 Mexican women older than 50 years of age will sustain a hip fracture [5]. The rate of radiographically defined vertebral fractures in Mexican women is

high [6] (overall rate of 19.2%), but no data regarding the epidemiology of vertebral fractures in men have been published. The aim of the present study was to determine the prevalence of radiographically defined vertebral fractures by age in a random sample of Mexican men over 50 years and to identify potential selleck inhibitor conventional risk factors associated with vertebral fractures in this group. Methods Study design A radiographical survey was designed for this study; 413 Mexican men were included from a population-based random sample in the city of Puebla. The random probability sample was generated with the advice of

the National GSK2118436 order Institute of Geography and Statistics (INEGI) in Mexico. We used the last census available to build a stratified sample of 100 men for the following age groupings: 50–59, 60–69, 70–79, and ≥80. We used the demographic information available from every district and group of households blocks within the city and we used the maps and cartography provided by INEGI during the survey. Before the study began, a training workshop was held with the interviewers to review the questionnaire and survey methods. Eligible men participated in a face-to-face interview in their homes; Chloroambucil after the interview, they were asked to have lateral X-rays of the 4SC-202 cell line thoracic and lumbar spine taken. If a man was unable or unwilling to participate, he was replaced by the first man available of the same age stratum, making home visits to houses from right to left of the first assigned house in the same block of households until a man who fulfilled the criteria was found. The protocol was submitted to and approved by the Institutional Review Board, and a written consent of all participants

was obtained before the interview. Questionnaire The questionnaire used was originally designed for Latin American Vertebral Osteoporosis Study (LAVOS) [6]. All questions were developed based on the questionnaires of two large studies, the European Prospective Osteoporosis Study (EPOS) and the Study of Osteoporotic Fractures (SOF) [7, 8], and collected self-reported data on demographics, lifestyle factors, and conventional risk factors for osteoporosis [9]. To assess dietary calcium, we included a semi-quantitative food questionnaire validated in Spanish for the Mexican population [10]. The Sistema de Nutrimientos (SNUT) program was used to compute dietary calcium in milligram per day. Commercial calcium supplement intake was calculated according to names and daily doses reported by participants.

Coyle EF: Timing and method of increased carbohydrate intake to c

References 1. Coyle EF: Timing and method of increased carbohydrate intake to cope with heavy training, competition and

recovery. J Sports Sci 1991,9(Suppl 1):29–52.PubMed 2. Ivy JL, Goforth HW Jr, Damon BM, McCauley TR, Parsons EC, Price TB: Early postexercise muscle glycogen recovery is enhanced with a carbohydrate-protein supplement. J Appl Physiol 2002, 93:1337–1344.PubMed 3. Tsintzas K, Williams C: Human muscle glycogen metabolism during exercise. Effect of carbohydrate supplementation. Sports Med 1998, 25:7–23.CrossRefPubMed”
“Background Long-term dieting has been reported to learn more reduce https://www.selleckchem.com/products/lxh254.html resting energy expenditure (REE) leading to weight regain once the diet has been curtailed. Diets are also difficult to follow for a significant length

of time. The purpose of this preliminary proof of concept study was to examine the effects of short-term intermittent dieting during exercise training on REE and weight loss in overweight women. Methods 16 sedentary women (37 ± 7 yrs, 162 ± 6 cm; 89 ± 17 kg; 42.5 ± 3% body fat) were assigned to an exercise & normal diet group (E, n = 6) or an click here exercise and diet intervention group (ED, n = 10). Diets were maintained for 30 days and consisted of 1,200 kcals/d for 1-wk followed by ingesting 1,500 kcals/d for 3-wks. Subjects then followed a 2,200 kcals/d maintenance diet for 4 wks and repeated the cycle each month for 6-months. Diets were either 45% CHO, 30% PRO, and 25% F or 45% PRO, 30% Orotic acid CHO, and 25% F. Subjects participated in a supervised Curves circuit training program 3-d per wk and walked for 30-min 3-d per wk. Body weight, DEXA body composition,

and REE measurements were obtained at 0, 1, 2, 3, 4, and 5 months and were analyzed by repeated measures ANOVA. Data are presented as means ± SD changes from baseline for the E and ED groups, respectively, at 1, 2, 3, 4, and 5 months. Results Preliminary results revealed that subjects in the ED group lost significantly more weight (E 0.4 ± 2.9, -2.9 ± 2.5; -1.8 ± 4.1, -1.9 ± 5.1; ED -6.7 ± 3.0; -8.7 ± 4.5, -10.8 ± 6.7; -11.3 ± 7.3 lbs, p = 0.03) and tended to lose more fat mass (E 0.83.0, -3.0 ± 3.8; -1.0 ± 4.5, -1.5 ± 3.7; ED -4.4 ± 3.6; -6.4 ± 3.5, -7.5 ± 5.2; -7.5 ± 6.6 lbs, p = 0.11) than subjects in the E groups. REE rebounded after dieting during each maintenance phase in the ED group (E 19.4 ± 2.2, 19.1 ± 1.6, 18.4 ± 1.7, 18.4 ± 1.9; 18.2 ± 1.6; ED 19.0 ± 1.3, 18.1 ± 1.6, 19.3 ± 2.2, 18.2 ± 1.7, 18.6 ± 1.5, kcal/kg, O4 p = 0.004). Conclusion Preliminary results indicate that following 30 day cycles of dieting/maintenance can promote gradual weight loss while allowing for a rebound in REE during the maintenance phase. This strategy may be an effective way to promote weight loss without concomitant reductions in resting metabolism.

CmR This study pAL18 2133 bp fragment of approximately 1 kb upstr




CTATTAGGCGTGAAAGCAGTT   PilT_B TAGTAATTTTCTTATCATATCAAG   PilT_C ATGATGCGAGATTTAGGGTA   PilT_D CAGCAGGTGGAAATACAGAT   PilT_E TACATTGTAGTAAGGTAATCA   PilT_F GGTAGAGTTGAATCAGCGTTTA   Construction of deletion mutants of pilA, pilC, pilQ, and pilT in FSC237 Left and right flanking regions of pilA (FTT0890c, SCHU S4 nomenclature) were PCR amplified using the primer pairs pilA_LFF/pilA_LFR and pilA_RFF/pilA_RFR, and cloned into pGEMT-easy (Promega). The left flank was excised with EcoRI and PstI and the right flank was excised with BamHI and PstI. The fragments were ligated into an EcoRI/BamHI digested pBluescript KS+ vector (Stratagene), giving rise to pSMP47. A chloramphenicol resistance gene was PCR amplified from pDM4 with the primer pair CAM_PstIF/CAM_PstIR, digested with PstI, and cloned into 3-Methyladenine in vitro pSMP47, generating pSMP48 containing the left and right flanks of pilA disrupted by a chloramphenicol cassette. The mutated allele of pilA was excised from pSMP48 with SphI and MluI, cloned into pSMP22, and the resulting plasmid pSMP50CAM (Table 2) was introduced into strain FSC237 by conjugal mating as previously described [7].