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In addition, it has been shown that the Bp alternative sigma factor RpoS, which is involved in genome-wide regulation of bacterial adaptation to environmental stress (i.e. nutrient limitation), plays a role in Bp induced MNGC formation selleck [59].

Recently, the molecular mechanism of Bp MNGC formation was revealed by Toesca et al.[60]. The T6SS-1 valine-glycine repeat tail spike protein (VgrG1) possesses a novel fusogenic domain at its C-terminus that mediates cell fusion and allows Bp cell to cell spread. Automated high content imaging (HCI) microscopy is a powerful technique to quantitatively characterize cellular phenotypes at the single cell level in response to bacterial and viral infection, exposure to drug agonists and antagonists and for drug mechanism of action determination [61–69]. This work describes the development of a cell-based HCI immunofluorescence assay

to quantitatively characterize MK-8931 the MNGC phenotype induced in murine macrophages upon infection with Bp K96243. As a proof of principle for its applicability in a relevant biological setting, this assay was validated using mutants of Bp that were previously described to be defective for MNGC formation in mouse macrophages [58, 70]. Furthermore, we used the MNGC HCI assay to screen a focused small molecule library to identify compounds that interfere with MNGC formation induced by Bp. Together, the results of these experiments indicated that the HCI MNGC assay can be used in a medium-throughput format to identify and characterize Bp mutants that are defective in their ability to induce MNGCs and to identify small molecules that inhibit this phenotype. Results & discussion Optimization of the MNGC assay To develop an automated high-throughput method for quantitating

MNGCs, RAW264.7 macrophages were either not infected (Figure  1A, Top panel-mock) or infected at an MOI Bcl-w of 30 with wild-type Bp K96243 (Figure  1A, bottom panel-wild-type Bp). After 2 h excess extracellular bacteria were then eliminated by sequential washes in PBS and cells were further incubated in medium containing kanamycin. At 10 h post-infection macrophages were first fixed, and then immunofluorescence (IF) staining was performed to detect bacteria and cellular structures. Finally, samples were imaged by high-throughput confocal fluorescence microscopy. Cell nuclei were stained with the DNA dye Hoechst 33342 and the cell body with the CellMask DeepRed dye. Bacteria associated with or internalized by macrophages were detected by staining cells with an anti-Burkholderia pseudomallei monoclonal antibody. Figure 1 Quantitative analysis of B. pseudomallei K96243 induced murine macrophage MNGC formation. (A) Representative 20X magnification confocal images of RAW264.7 macrophages that were not infected (mock) or infected with wild-type B. pseudomallei K96243 at a MOI of 30 at 10 h post-infection.

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S., Melville, NY). β-glucuronidase expression by bacteria on LBMC plates was detected by streaking bacteria to plates that had been spread with

40 μL of X-gluc solution (100 mM 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, cyclohexylammonium salt solution in dimethylformamide). Table 3 Expression of β-glucuronidase (GUS) fusions ORF strain % of nodules with GUS expression Strength of nodule GUS expression Staining time Pattern of nodule GUS expression Free-living GUS expression N/A S. meliloti 1021 Selleckchem Fludarabine wild type (negative control) 0/39 = 0% − variable none − SMc00911 SMc00911.original 18/20 = 90% ++++ 1.5–3.75 hr whole nodule +   SMc00911.Xsd1 18/18 = 100% ++++ 1.5–3.75 hr whole nodule n.d.   SMc00911.original2 n.d. n.d. N/A N/A + SMb20360 SMb20360.original 8/13 = 62% ++ 3–5 hr invasion zone-fixation zone −   SMb20360.Xsd1

13/16 = 81% ++ 3–5 hr invasion zone-fixation zone − SMc00135 B104.3A 6/8 = 75% + 2–3 hr LY3039478 mouse invasion zone-interzone +   B104.4B 8/8 = 100% + 2–3 hr invasion zone-interzone ++   B104.2 C 6/8 = 75% ++ 2–3 hr invasion zone-interzone ++ SMc01562 A104U.original 7/8 = 88% + 4–6 hr interzone −   A104U.Xsd1 3/7 = 43% +/− 4–6 hr interzone-fixation zone n.d.   A104U.Xsd6 8/8 = 100% + 4–6 hr interzone-fixation zone n.d.   A104U.Xsd25 3/8 = 38% +/− 4–6 hr interzone-fixation zone n.d.   A104U.Xs100 4/9 = 44% + 4–6 hr fixation zone n.d. SMc01266 SMc01266.original 13/18 = 72% + 3 hr invasion zone-fixation zone +/−   SMc01266.Xsd1 13/18 = 72% ++ 3 hr invasion zone − SMc03964 SMc03964.original 8/15 = 53% ++ 3–5 hr interzone +/−   SMc03964.Xsd6 9/19 = 47% ++ 3–5 hr interzone-fixation zone − SMc01424-22 D104.2A 0/8 = 0% − 4–6 hr N/A +/−   D104.3B 7/8 = 88% ++ 4–6 hr invasion zone-interzone +/−   D104.1 C 6/8 = 75% + 4–6 hr invasion zone-fixation zone +/− SMa0044 SMa0044.104.1A 4/8 = 50% +/− 6–7 hr invasion zone-interzone Idoxuridine +++   SMa0044.104.1B 4/8 = 50% +/− 6–7 hr interzone

+++   SMa0044.104.4 C 4/8% 50% +/− 6–7 hr interzone +++ SMb20431 SMb20431.original 10/16 = 63% + 5–12 hr invasion zone-fixation zone −   SMb20431.Xsd1 11/15 = 73% + 5–12 hr interzone − SMc01986 C104.1A.Xsd1 0/6 = 0% − 24 hr N/A n.d.   C104.1A.original n.d. n.d. 24 hr n.d. +/−   C104.2B.Xsd100 2/18 = 11% +/− 24 hr fixation zone n.d. SMa1334 SMa1334.original 0/11 = 0% − 5–24 hr N/A −   SMa1334.Xsd1 0/13 = 0% − 5–24 hr N/A − Results Comparisons of Sinorhizobium meliloti open reading frames with those of other rhizobia and with non-nitrogen fixing α-proteobacteria Rhizobial functions required for symbiotic nitrogen fixation with legume plants have typically been discovered through the classical bacterial genetic technique of transposon mutagenesis, followed by screening mutants for loss of symbiotic function. We have used an alternative comparative genomics strategy to search for rhizobial genes involved in symbiosis.

On the left side of the integration side an inverted repeat (IR) is indicated. Upstream of the IR a gene encoding a tRNACys is located. In B. bronchiseptica GI3::tetR is once more integrated in a gene encoding a tRNAGly (tRNA45) leading to a 18 bp duplication of its 3′-end. Much alike in B. petrii the direct repeat

sequence is followed by an inverted repeat (IR). Below the schematic presentations of the integration regions the respective DNA sequences of the integration sites are shown. The selleck kinase inhibitor start points of the tRNA genes are indicated by horizontal arrows indicating transcriptional polarity of the genes followed by a bar marked with a star which indicates the end of the tRNA gene. Vertical arrows indicate the integration sites of the GIs in the tRNA genes. Related inverted repeat sequences (IR) present in both species are boxed. In the case of B. bronchiseptica the sequence position indicated is taken from the genome sequence PF477736 mw of strain RB50 [13]. Conclusion The data presented here underline the previous notion of a highly mosaic genome of B. petrii. By microarray analysis of spontaneous phenotypic variants of B. petrii and by direct detection of excised circular intermediates of the B. petrii GIs we show that all of them are active at least in terms of excision. We provide evidence that the adjacent integration of highly related elements may enable these elements to pick up additional

genomic material placed between the integration sites thereby leading to

an increase in the size of the islands. Moreover, the adjacent placement of islands encoding highly similar integrases and attachment sites may also lead to the formation of novel huge composite islands. For ICE-GI3 we show that without selective pressure this island is lost from the bacterial population. Moreover, 3-mercaptopyruvate sulfurtransferase we show that this island is self transmissible and can be transferred to another Bordetella species, B. bronchiseptica. Therefore, the evolution of B. petrii involved massive horiztonal gene transfer, while in the classical pathogenic Bordetella species only very few examples of HGT have been reported, e.g. the horizontal transfer of insertion elements, the acquisition of an genomic region encoding an iron uptake system in B. holmesii and, possibly, the inactivation of the genes encoding adenylate cyclase toxin in a specific B. bronchiseptica lineage by a horizontally acquired gene cluster encoding peptide transport genes [12, 23, 24]. This may indicate that their unique habitat due to an obligate host association has dramatically limited the impact on horizontal gene transfer for the pathogenic Bordetellae once they had acquired their capacity to infect and to persist exclusively in vertebrate hosts. Methods Bacterial strains and growth conditions In this study B. petrii DSM12804, the type strain of the species [5], B. bronchiseptica BB7866 [25], and B.

In this model, cells exist in two states, normal and persister. During antibiotic treatment, normal cells die at a rate μ and switch to a persister state at rate α. Persister cells

do not die or grow, and switch to a normal state selleck at rate β (see Additional file 1). The advantage of using a this model is that the parameters that we infer, such as the fraction of persister cells, do not depend on experimental idiosyncrasies, for example, the time at which cell numbers are measured. It has been difficult to compare the results of many previous experiments on persisters for this reason. Persister fractions differ between environmental isolates We selected 11 E. coli isolates from a collection of more than 450 environmental isolates sampled over a period of 12 months from two sites approximately 2m apart near a watershed of Lake Superior (46°42’04′N, find more and 92°12’26′W) [26]. Despite the nearly identical geographical provenance of these isolates, partial genomic sequencing of a subset of these 450 strains has shown that while all are Escherichia species, they encompass a genetic diversity greater than the standard panel of E. coli strain diversity, the ECOR collection. This initial genomic data show that isolates from this location are spread across the E. coli phylogeny, with members in clades A, B1, B2, D, E, F, and C-V [27] (Bertels et al., in prep). Although

the strains in this collection harbor considerable genetic diversity, for the most part, they are not pathogenic, typing negatively for most common virulence loci (M. Sadowsky, personal communication).

We selected the subset of 11 environmental isolates on the basis of their differential levels of survival in ampicillin after 24 hours of treatment (using CFU counts; see Methods). In doing so, we aimed to find strains that differed to the greatest extent in the fraction of persisters that were formed in ampicillin, such that we would have the greatest power to discern whether these differences were paralleled in other antibiotics. In addition to these isolates, we used the standard laboratory strain 5-Fluoracil chemical structure E. coli K12 MG1655, for a total of 12 strains in which we quantified persister fractions. For each of these strains, we first determined the MIC for ampicillin (see Methods), and found that the MICs for these strains differed by less than two-fold (Additional file 2: Table S1). This suggested that the differences in survival did not arise simply from differences in growth and killing dynamics, and may instead have resulted from differences in persister formation. We then quantified, for each strain, survival curves over 48 hours during treatment with 100 mg/ml of ampicillin (Figure 1). In the vast majority of cases, the curves that we observed were clearly not characterized by a single exponential decrease, as would be expected if all individuals in the population had equal susceptibility to the antibiotic.