1). If up to two mismatches were allowed, a further candidate CIRCE sequence was found upstream of cpn60.2, although two other potential matches were also found upstream of genes that are not usually part of the heat shock regulon (data not shown). It is thus likely that heat shock regulation of cpn10, cpn60.1 and cpn60.2 is mediated by the HrcA protein binding at CIRCE sequences, BKM120 mw but this remains to be proven. No CIRCE sequence was found upstream of cpn60.3, consistent with the observation that it is not induced by heat shock.

In M. tuberculosis, although cpn10 and cpn60.1 are adjacent on the chromosome, two putative transcriptional start sites have been proposed (Kong et al., 1993). One of these is upstream of cpn10, in the region containing the CIRCE sequence

that binds HrcA to regulate the heat shock response (Zuber & Schumann, 1994; Stewart et al., 2002). A second was identified 29 bp upstream of the cpn60.1 gene. However, a more recent report showed no promoter activity in this intergenic region (Aravindhan et al., 2009), raising the possibility that there is Cisplatin mouse a post-transcriptional cleavage of the mRNA for this operon. Because of this, and because our results showed that in M. smegmatis the adjacent cpn10 and cpn60.1 genes are expressed at significantly different levels under similar conditions, we used 5′RACE with the primers cpn60.1 gsp1, cpn60.1 gsp2 and cpn10 gsp1 to determine the transcriptional start sites of the cpn10 and cpn60.1 genes. The results showed two potential

transcriptional start sites, one 133 bp upstream from the cpn10 gene and the second in the intergenic region 31 bp upstream of the cpn60.1 gene (Fig. 1), similar to earlier findings with M. tuberculosis. To investigate whether the intergenic region did indeed contain a promoter, varying lengths of upstream regions of the chaperonin genes and the http://www.selleck.co.jp/products/Fludarabine(Fludara).html cpn10–cpn60.1 intergenic region (Fig. 1) were cloned into the pSD5B reporter plasmid, and LacZ activity was measured following the transformation of these plasmids into M. smegmatis mc2155. Only the regions upstream of cpn10 and cpn60.2 exhibited promoter activity. Neither the shorter nor the longer intergenic fragment reported any promoter activity, as would have been expected had the putative start site identified shortly upstream of the cpn60.1 gene been genuine (Fig. 1). We therefore conclude that the mRNA 5′-end observed between cpn10 and cpn60.1 is likely to arise from a specific post-transcriptional cleavage event, similar to the situation reported in M. tuberculosis. The lower levels of expression of cpn60.1 compared with cpn10 may thus result from differential stabilities of the mRNAs for these two genes. This may have evolved from a need to match the levels of expression of the essential cpn10 and cpn60.2 genes, despite cpn10 being in an operon with the nonessential cpn60.1.

1). If up to two mismatches were allowed, a further candidate CIRCE sequence was found upstream of cpn60.2, although two other potential matches were also found upstream of genes that are not usually part of the heat shock regulon (data not shown). It is thus likely that heat shock regulation of cpn10, cpn60.1 and cpn60.2 is mediated by the HrcA protein binding at CIRCE sequences, Navitoclax datasheet but this remains to be proven. No CIRCE sequence was found upstream of cpn60.3, consistent with the observation that it is not induced by heat shock.

In M. tuberculosis, although cpn10 and cpn60.1 are adjacent on the chromosome, two putative transcriptional start sites have been proposed (Kong et al., 1993). One of these is upstream of cpn10, in the region containing the CIRCE sequence

that binds HrcA to regulate the heat shock response (Zuber & Schumann, 1994; Stewart et al., 2002). A second was identified 29 bp upstream of the cpn60.1 gene. However, a more recent report showed no promoter activity in this intergenic region (Aravindhan et al., 2009), raising the possibility that there is LDK378 supplier a post-transcriptional cleavage of the mRNA for this operon. Because of this, and because our results showed that in M. smegmatis the adjacent cpn10 and cpn60.1 genes are expressed at significantly different levels under similar conditions, we used 5′RACE with the primers cpn60.1 gsp1, cpn60.1 gsp2 and cpn10 gsp1 to determine the transcriptional start sites of the cpn10 and cpn60.1 genes. The results showed two potential

transcriptional start sites, one 133 bp upstream from the cpn10 gene and the second in the intergenic region 31 bp upstream of the cpn60.1 gene (Fig. 1), similar to earlier findings with M. tuberculosis. To investigate whether the intergenic region did indeed contain a promoter, varying lengths of upstream regions of the chaperonin genes and the Adenosine cpn10–cpn60.1 intergenic region (Fig. 1) were cloned into the pSD5B reporter plasmid, and LacZ activity was measured following the transformation of these plasmids into M. smegmatis mc2155. Only the regions upstream of cpn10 and cpn60.2 exhibited promoter activity. Neither the shorter nor the longer intergenic fragment reported any promoter activity, as would have been expected had the putative start site identified shortly upstream of the cpn60.1 gene been genuine (Fig. 1). We therefore conclude that the mRNA 5′-end observed between cpn10 and cpn60.1 is likely to arise from a specific post-transcriptional cleavage event, similar to the situation reported in M. tuberculosis. The lower levels of expression of cpn60.1 compared with cpn10 may thus result from differential stabilities of the mRNAs for these two genes. This may have evolved from a need to match the levels of expression of the essential cpn10 and cpn60.2 genes, despite cpn10 being in an operon with the nonessential cpn60.1.

The bacterial colony counts of the pathogen were performed as described above. Results are expressed as the mean±SEM. Statistical comparisons

and Student’s t-test were performed, with P<0.01 considered statistically significant. Cultures of L. johnsonii NCC533 and L. gasseri KS120.1 were tested for their killing activity against the UPEC CFT073, Silmitasertib in vitro G. vaginalis DSM 4944 and S. typhimurium SL1344. The results reported in Fig. 1 show that the 24-h cultures of both the Lactobacillus strains reduced the viability of the pathogens, but with different efficacies. Our results show that the killing activity of Lactobacillus cultures results from substances present in CFCS, and that isolated bacteria display no killing activity. We next investigated the characteristics of the killing activity of L. johnsonii NCC533 and L. gasseri KS120.1 CFCSs. Part of the killing activity

was attributable to heat-stable components (Fig. 2), which were not sensitive to protease treatment (not shown). The killing activity was not attributable to a pH effect, because MRS at pH PLX4032 supplier 4.5 shows no activity (Fig. 2). Fayol-Messaoudi et al. (2005) have previously demonstrated that the killing activity of lactic acid against S. Typhimurium was inhibited after adding DMEM to the LB culture medium. Here, when DMEM was added to each appropriate pathogen culture medium, the killing activity of Lactobacillus CFCSs was slightly decreased compared with that without DMEM (Fig. 2). In order to investigate the role played by hydrogen peroxide in the killing activity of Lactobacillus CFCS against the three pathogens, the CFCSs were exposed to catalase treatment. As shown in Fig. 3, the killing activity of the CFCS of L. johnsonii NCC533 and of L. gasseri KS120.1 was considerably reduced after catalase treatment. Collectively, Bay 11-7085 these findings indicate that the killing activity of L. johnsonii NCC533 and L. gasseri KS120.1 strains against S. typhimurium SL1344,

UPEC CFT073 and G. vaginalis DSM 4944 was mainly attributable to heat-stable secreted molecules and hydrogen peroxide. Lactic acid was present in culture media of the hydrogen peroxide-producing strain (L. johnsonii NCC533: 61±16 mM, and L. gasseri KS120.1: 63±12 mM). Makras et al. (2006) and De Keersmaecker et al. (2006) concluded that the antibacterial effect of probiotic L. johnsonii NCC533 and Lactobacillus rhamnosus GG strains against serovar Typhimurium results from the accumulation of their main metabolite, lactic acid. The above results (Fig. 2) show that the killing activity of L. johnsonii NCC533 and L. gasseri KS120.1 CFCSs in the presence of DMEM is slightly decreased, which prompted us to investigate the concentration-dependent killing activity of MRS at pH 4.5 containing increasing concentrations of lactic acid. We found that lactic acid alone displayed significant killing activity at concentrations from 100 mM that was greater against G.

The relationship between BI p38 MAPK pathway and BI-WWHAM and each belief were also explored. Spearman’s rank correlation was used to assess the relationship between each belief and BI. Mann–Whitney tests were used to compare the distribution of the beliefs between information givers and non-givers. The overall evaluable response rate was 31.7% (927/2924)

comprising 481/1456 (33.0%) from those sent the direct measures only questionnaire and 446/1468 (30.4%) from those sent the direct measures plus salient beliefs questionnaire. The respondents’ demographics are presented in Table 1. Most were female (73%), married or living with partner (69%) and of white ethnic origin (99%). The mean age was 53.2 years (standard deviation, 16.1). No significant differences in demographics were seen for the two questionnaire versions. Behavioural intention (TPB BI) (3) Q2.5 Strongly agree–strongly disagree (1–7) Reverse coded Behavioural intention (BI –WWHAM) (5) Q2.6 The next time I buy a pharmacy medicine, I intend to tell the MCA the following information. .Who the medicine is for; what symptoms it will be used to treat; how long the symptoms have been present; if any other medicines have been tried already to treat the problem; about other medications that I am currently using Strongly agree–strongly disagree (1–7) Reverse coded and score across the five items summed Attitude (ATT) (4) Q2.8.1, 2.8.3, 2.8.4, 2.8.6 Perceived behavioural

control (PBC) (2) Q2.8.2, 2.8.5 Subjective norm (SN) (2) Q2.9.4, In total, 764/927 (82.4%) respondents provided responses about IWR1 their most recent purchase of a pharmacy medicine that allowed them to be categorised as information givers (n = 289) or non-givers (n = 475). Of these 764 respondents, 164 (21.5%) reported telling the MCA what their health problem had been, 62.2% (n = 475) reported stating which product they had wanted (which was slightly lower than the 75% anticipated), and 16.4% (n = 125) reported

giving information about both. The cumulative percentage of agreement of respondents intending to give each type of WWHAM information was assessed (Figure 2). Between Rucaparib order 70% and 80% of respondents generally agreed (scoring 1–3) that they intended to provide each type of information next time they buy a pharmacy medicine, with highest intention for saying ‘who’ the medicine was for and lowest intentions for saying ‘how long’ or ‘what symptoms’. TPB BI correlated highly with BI-WWHAM (rs = 0.735). Information givers had stronger intentions on each measure than those non-givers (Table 2). Table 2 shows the summary statistics for each of TPB measure. Cronbach’s alpha was acceptable except for subjective norm items (α = 0.372) and so only one of the two original items was used for subsequent analysis i.e. ‘People who are important to me will think I should give information to the MCA’. The correlation coefficients between measures of TPB variables are also shown in Table 2.

Error trials due to breaks in fixation, blinks, and releases of the lever before the offset of the stimulus (in the delayed match-to-sample task) were excluded. There were two types of error trials in

the reaction-time task: miss trials in which the target was present (and should have been Go trials) but the monkeys did not release the lever, and false alarms in which the target was absent (and should have been NoGo trials) but the monkeys released the lever. We computed the choice probabilities for these error types separately: (i) correct detection of target in Go trials vs. miss trials and (ii) false detection of target (false alarm) vs. correct rejection in NoGo trials. The choice probabilities were computed in the same fashion, based on 0.3 s of the fixation period or 0.3 s of the cue period, in the reaction-time task. Choice probabilities were computed for each neuron and distributions Akt inhibitor of values across neurons were then compared for neurons recorded from PPC and dlPFC. The variability of a neuron’s firing rate across trials was expressed as the Fano factor, defined as the variance of spike counts divided by the mean. The Fano factor was computed based on the algorithm developed by Churchland et al. (2010). First,

the variance and mean of the spike count were computed in each trial type, and then a regression of the variance to the mean was performed. The Fano factor reported here was the slope of this regression. Spike counts were computed Rucaparib in a 150-ms sliding window moving in 10-ms steps. The Fano factor was computed in three separate task periods in the delayed match-to-sample task, the fixation period (0.5 s), the cue period (0.5 s) and the delay period (1.0 s). We computed the Fano factor for correct and error trials separately for target in the receptive

field and target outside the receptive field conditions. Neurons with at least five trials per condition were used for this analysis. To evaluate the relationship between the trial-to-trial neuronal activity and behavioral reaction time, we computed a correlation coefficient between firing rate and reaction time using data from the standard version of the reaction-time task (Fig. 1C). however Firing rate when the stimulus appeared at the best location for each neuron was calculated for each 100-ms window, sliding in 20-ms intervals for each trial. A correlation coefficient was computed for each bin between the firing rates and corresponding reaction times. A correlation coefficient was also calculated for the fixation period (0.3 s) or the cue period (0.3 s). A correlation value was determined thus for each neuron. The distributions of correlation values were then compared across areas. Neurophysiological data were collected from areas 8 and 46 of the dlPFC and LIP of the PPC in two monkeys (Fig.

Cel5M thus possessed the typical properties of a cold active cellulase and is the first cold-active cellulase in the newly established subfamily of GH5 (Fig. 1). The effects of metal ions, detergents and chelating agents on Cel5M were

examined (Table 2). CuSO4, SDS and EDTA significantly reduced the activity of Cel5M, indicating that these agents may be inhibitors of Cel5M. CoCl2, FeCl2 and dithiothreitol increased the cellulolytic activity of Cel5M. Most other agents did not significantly influence BAY 57-1293 research buy the cellulolytic activity of Cel5M. Previous studies showed that ferrous and ferric ions may interfere with the activity of most cellulases (Tejirian & Xu, 2010). Cel5M exhibits a novel adaptation to the ferrous ion and may therefore may have a broader application in biofuel and chemical industries. The hydrolytic activity toward different substrates was assayed at 30 °C in phosphate-buffered saline (pH 4.5). Cel5M exhibited high activity toward CMC (26.9 ± 1.35 U mg−1 protein), low activity

toward p-nitrophenyl-β-d-galactopyranoside (0.56 ± 0.03 U mg−1 protein), and no activity toward microcrystalline cellulose or avicel (specific cellulolytic activity was not detectable). These results are consistent with a previous study showing that the CBM is necessary for efficient hydrolysis of crystalline celluloses (Takashima et al., 1998). The check details present work was supported by the China National Natural Science Foundation triclocarban (Grant Nos. 91028011 and 41076091), the China Ocean Mineral Resources R&D Association (Grant Nos. DYXM-115-02-2-20 and DYXM-115-02-2-6), the Fundamental Research Funds for the Central Universities of China (Grant No. 09CX05005A), the National Basic Research Program of China (grant No. 2009CB219506), the Hi-Tech Research and Development Program of China (Grant No. 2007AA091903), the Key Scientific and Technological Development Program of the National Qingdao Economic & Technical Development Zone (Grant No. 2009-2-34), and the Foundation of

the State Key Laboratory of Heavy Oil Processing in China University of Petroleum (Grant No. SKL2010-02). We thank Baosheng Ge for his help in data analysis. “
“FMRP – University of São Paulo, Ribeirao Preto, SP, Brazil Environmental plasmids often expand the metabolic repertoire of bacteria that carry them, but they also interfere with the biochemical and genetic network of the host. The pWW0 plasmid born by Pseudomonas putida mt-2 encodes the TOL pathway for degradation of toluene/m-xylene through production of intermediate compounds benzoate/3-methylbenzoate. These can be also recognized as substrates by the chromosomally encoded ben and cat gene products, thereby creating a manifest regulatory and biochemical conflict. In this context, we have investigated how the introduction of the pWW0 plasmid into P. putida affects behaviour of the promoter of the ben pathway (Pb) in single cells.

Some felt that nurses might not be capable of prescribing

even with extra education (32%), and 10% felt more strongly, saying “nurses aren’t doctors. Logistic regression was used to analyze how feeling competent relates to experience as a travel health nurse, registry as travel health nurse, amount of advice/malaria chemoprophylaxis given per month, and aspiration for prescribing rights. Only aspiration for prescribing rights appeared to be a significant predictor for the travel health nurses who feel competent for prescriptive authority (OR: 6.8; 95% CI: 3.5–13.3). Figure 1 shows that 95% of travel health nurses have one or more educational needs to fill before prescribing. More than half expressed the need for further education in the areas of pharmacology, medication in general, and immunology; more knowledge about malaria chemoprophylaxis was desired by 33% and about diseases in general by 25%. Entries in the open-text Ceritinib fields expressed interest in knowing more about diseases/medication related to immune suppression, altitude disease and acetazolamide, antibiotics, contra-indications and interactions (especially in combination with malaria chemoprophylaxis), and the special needs of pregnant travelers as well as children. Following the United States, the first country to introduce nurse prescribing in 1969,[7] and seven Western European/Anglo-Saxon countries (UK, Canada, New

Zealand, Australia, IWR-1 Sweden, Ireland, and Finland),[8] Oxaprozin the Netherlands has recently introduced prescribing by nurses. The results of our questionnaire survey indicate that most Dutch travel health nurses are prepared to prescribe. Advice and prescription by these nurses is already provided according to highly protocolized criteria; 82% of the travel health nurses aspire to the expanded responsibility and 77% feel competent to undertake it. An interesting

finding was that many positive respondents indicated that ongoing access to a doctor would remain important. This implies that they are not yet completely aware that access to a doctor is a requirement for the designation of supplementary nurse prescribing in travel medicine. There is thus a need to raise awareness among travel health nurses concerning the responsibilities and restrictions associated with their future privileges. Further education is likewise needed before nurse prescribing is implemented in travel medicine. We found that 95% of the travel health nurses have one or more educational needs; they most often mentioned pharmacology. This result is in line with other studies, although comparison among countries is difficult. Differences among their legislative procedures and their regulation of nursing practice have led to different models of prescribing worldwide. A questionnaire survey was performed among UK nurses who prescribe medicine for diabetic patients, in which participants were asked if they had needs for the current 12 months, the following 12 months, or not at all.

This is a more balanced observation than the previous transcriptomic study of P starvation of MED4 (Martiny et al., 2006) that reported 30 upregulated genes and just four downregulated GSK-3 signaling pathway under P starvation conditions.

This difference is understandable as the earlier study monitored healthy cells subjected to a P-depleted medium over a 2-day period, whereas this study focused on the response of a longer term (10 day) exposure to P depletion, and so can be regarded more of an acclimation strategy rather than an immediate stress response. This characteristic of stress against longer term acclimation has been observed recently by comparing the response to varying levels of salt-infused media of two other cyanobacteria: Synechocystis

sp. PCC6803 and Euhalothece sp. BAA001 (Pandhal et al., 2009). Moreover, as later sections will show, the see more cell responds to prolonged P starvation by regulating the abundance of proteins across the proteome, and not just from limited specific areas (Fig. 1, where all identified proteins are depicted with respect to their chromosomal location), as opposed to an immediate shock response (Martiny et al., 2006). It is important to briefly consider the fundamental methodological differences when introducing comparisons between transcriptomic and proteomic data. The half-lives of both mRNA and its encoded protein differ by up to an order of magnitude, and so any direct quantitative correlation between transcript levels and protein abundance is, at the time of writing, very difficult to assert. There are issues with the quantitative nature of both techniques; indeed, microarray experiments have been observed to underestimate the relative change in gene expression (Yuen et al., 2002), and recently iTRAQ has also been shown to potentially underestimate the relative changes in

protein abundances (Ow et al., 2009b). However, qualitative comparisons between the two methodologies are invaluable, and inferences into the physiological state of the cell when stressed are emphasized through the comparison of both transcriptomic and proteomic data. Here, only four proteins from those gene clusters identified previously as responding to P starvation (Martiny et al., 2006) were assessed as significantly more Methocarbamol abundant than the P-replete control: PhoA, the alkaline phosphatase; PhoE, the putative orthophosphate membrane transporter; PstS, the periplasmic P-binding protein; and one protein from the genomic island operon, PMM1416 (Fig. 2a). The first three are part of the phoB region with the pstABCS orthophosphate transport system, and the last one is from the genomic island group PMM1403-1416. In agreement with the transcriptomic data (Martiny et al., 2006), PhoE, PhoA and PMM1416 demonstrate the greatest fold change in response to P deprivation (Fig.

Using PCR on these strains, we also found that short, 2.4 kb, and long, 2.7 kb, versions of both the MTT1 and the MAL31 genes are present (Dietvorst et al., 2005). We have extended these studies by cloning and sequencing long and short versions from two more lager strains and testing their ability to restore the growth of the A15 lager strain on maltotriose with antimycin A. Escherichia coli XL1-Blue (Bullock et al., 1987) was used for plasmid amplification. Standard methods were used for E. coli transformation (Inoue et al., 1990). Four lager

strains, A15 (Dr J. Londesborough, VTT Biotechnology, Finland), WS34/70 (Weihenstephan brewery, Freising, Germany), BS01 and BS07 (Heineken Supply Chain), were used in this study. Standard methods were used for yeast transformation (Gietz et al., 1995). Plasmid pRUL409 was constructed www.selleckchem.com/products/jq1.html by inserting ARS1 from plasmid pNatCre (Steensma

& Ter Linde, 2001) into the XbaI and EcoRI sites of vector pHSS6 (Seifert et al., 1986), thereby introducing a NotI site next to the EcoRI site flanking the ARS. Plasmid pRUL409 was provided with a KanMX cassette (Wach et al., 1994) from pUG6 by inserting it as a BamHI–BglII fragment into its BamHI site yielding pRUL409(KanMX). This plasmid was digested with BamHI and LY294002 in vitro XbaI to clone the various MALx1 and MTT1 genes. To convert the resulting plasmids from a multicopy plasmid into a single copy plasmid, CEN4 from plasmid YCplac33 (Gietz & Sugino, 1988) was amplified using the primers CEN4SacI and CEN4EcoRV and inserted into the EcoRV and SacI sites of pRUL409(KanMX) containing one of the various

MALx1 and MTT1 genes. Yeast cells were grown in YP (Difco peptone 2%, Difco yeast extract 1%) containing 2%d(+)−glucose (Merck), maltose (Merck) or maltotriose (Fluka, 96% pure HPLC). When required, G418 (Duchefa) 17-DMAG (Alvespimycin) HCl was added to the medium at a concentration of 200 μg mL−1, and antimycin A (Fluka) at a concentration of 3 mg L−1. Escherichia coli (XL1-Blue) was grown in Luria–Bertani broth and, if necessary, kanamycin was added to a concentration of 40 μg mL−1. For solid medium, 15 g L−1select agar (Gibco) was added to the liquid media. The primers used for PCR are listed in Table 1. PCR amplifications were performed with 50–100 ng genomic DNA. The amplification conditions were as follows: 5 min at 94 °C, 30 cycles of 1 min at 94 °C, 1 min at 5 °C below Tm of the primers and 1 min kb−1 to be amplified at 74 °C, followed by 10 min at 74 °C. The reactions were performed in a total volume of 50 μL and, per reaction, 0.5 μL Vent polymerase (New England BioLabs) was used with the buffer supplied. All PCR products were cloned into the pCR®-Blunt II-TOPO® vector (Invitrogen) before they were recloned into pRUL409(KanMX). For each of the four strains, 11 independent PCRs were performed. The sequences of the MTT1 and MAL31 gene isolates from strains BS01, BS07, WS34/70 and A15 cloned into vector pRUL409(KanMX) were determined commercially (Baseclear).

This was digested with ApaI and NotI, and then the DNA fragment containing the truncated ndvB fragment and the spectinomycin resistance Ω interposon was transferred to the suicide vector pJQ200SK (Quandt & Hynes, 1993) using the same restriction sites, generating pGF03. Finally, a tri-parental mating procedure with the helper plasmid pRK2013 (Figurski & Helinski, 1979) was used to transfer pGF03 into NGR234. Growth on TY agar plates supplemented with sucrose (5% w/v), and spectinomycin allowed selection for the ndvB mutant (named NGRΔndvB). The ndvB promoter region was amplified using the following primer pair: 5′-GCGAATTCATCAGCGAGCAGGT-3′ and 5′-TTTCTAGACACGGTCATGTGTCCC-3′. The resulting

fragment was digested with EcoRI and XbaI to enable cloning into pBluescript Sorafenib pSK+ resulting in pALQ09. The ndvB promoter region of pALQ09 was then transferred into the PstI and ClaI sites of pBDG116 creating pALQ12. In turn, ndvB promoter was inserted into the HindIII restriction site of pPROBE-GT′ (generating

pALQ27). The flaC promoter region was amplified by PCR using the following primer pair: 5′-CGGAATTCTGGTGCGCTCCTTC-3′ and 5′-GGTCTAGATGCGGTTCTGCG-3′, digested using EcoRI–XbaI and cloned into pBluescript selleck products pSK+ generating pALQ24. The insert was transferred into the KpnI-SacI sites of pPROBE-GT-producing pALQ28. All constructed plasmids were sequenced to confirm PCR fidelity. The final constructs containing the ndvB and

the flaC promoters fused to the GFP-encoding gene (pALQ27 and pALQ28, respectively), or empty vectors were mobilized into recipient strains using tri-parental mating as described previously. To generate GFP-tagged strains, the broad host-range vector pHC60 (Cheng & Walker, 1998) which constitutively expresses GFP was mobilized Tau-protein kinase into NGR234 and the ndvB mutant by tri-parental mating. Extractions of CβGs were performed using the following protocol, based on a method developed by Inon de Iannino et al. (1998). Briefly, strains were cultivated in 50 mL TY for 2 days to a stationary growth phase (i.e., a final OD600 nm of 2.0–2.5). Cells were centrifuged for 10 min at 10 000 g, 10 °C and washed twice with water. Pellets were resuspended in 1 mL of 70% ethanol, incubated for 1 h at 37 °C, and further centrifuged for 2 min at 9000 g. The supernatants were finally desiccated by speed-vacuum and resuspended in 20 μL of 70% ethanol. Aliquots (5 μL) of each extract were separated by thin-layer chromatography (Cromatofolios AL TLC – Silicagel 60F) using n-butanol–ethanol-dH2O (v/v/v of 5 : 5 : 4), and CβGs were visualized by spraying the plates with 5% sulfuric acid in ethanol, followed by heating at 120 °C 10 min. Swimming plates were produced by adding 0.2% agar to GYM medium supplemented with various amounts of NaCl.