However, Hongyo et al, claimed that H. pylori infection was more common in patients without any mutation in p53 [22]. The development of an enzyme-linked immunosorbent assay (ELISA) for mutant p53 protein makes it possible to determine most mutant p53 proteins in humans and other mammals [23]. This test has been used to determine mutant p53 protein in the serum of apparently healthy persons with H. pylori infection, detected as the presence of antibodies to specific IgG [24], beacuse most patients infected with H. pylori

produce an easily LCZ696 in vitro identified systemic humoral immune responde, composed primarily of IgG. Circulating H. pylori antibodies persist at constant levels for years during infection. Mutant p53 proteins have a half-life of Erastin purchase approximately 24 h, whereas normal proteins have a half-life of about 20 min. It is this prolonged half-life which leads to the accumulation of detectable amounts of p53 protein [25]. Reactive oxygen species (ROS) are a group of highly reactive oxidative molecules implicated in the aging process, in several chronic inflammatory disorders, and in carcinogenic pathways in different epithelial districts [26]. An increase in cell ROS, be it due to overproduction

and/or scavenging inability, may result in severe damage to various cell components, including membranes, mitochondria, and YAP-TEAD Inhibitor 1 order nuclear as well as mitochondrial DNA [27]. Ceruloplasmin (CP) is a 132 kd cuproprotein which, together with transferrin, provides the majority of anti-oxidant capacity in serum. Cp is a serum ferroxidase that contains greater than 95% of the copper found in plasma. This protein is a member of the multicopper oxidase family, an evolutionarily conserved group of proteins that utilize copper to couple substrate oxidation with the four-electron reduction of oxygen to water. Despite the need for copper in ceruloplasmin function, this protein plays no essential role in the transport or metabolism of this metal [28, 29]. In this study, we sought to compare the relation between serum levels of mutant p53

protein and H. pylori infection in two populations of similar socioeconomic status, but with very different mortality rates for gastric cancer. A second objective was examine indirectly by measuring Immune system the serum concentration of the antioxidant ceruloplasmin in patients with evidence of H. pylori infection. Serum levels of ceruloplasmin usually vary inversely with serum nitrite levels [30–32]. Materials and methods Type of study This was a comparative, cross-sectional, case-control study of two populations with different rates of mortality from gastric cancer. This study has been ongoing since March 2002 to October 2005. Serum ceruloplasmin levels were also compared in patients with and without H. pylori infection, and in patients with and without mutant forms of p53. The investigators did not know whether the subject was positive or negative for H. pylori antibodies when they tested p53 status.

The positive reaction

located in cytosol was stained in brown. The color of the stain is positively correlated to the protein expression. The IOD of each group revealed that in the SHG44 -DDK-1 the expression of bax and caspase-3 increased, whereas JIB04 clinical trial the expression of bcl-2 decreased (Table 1). Figure 6 Bax, bcl-2 and caspase-3 protein expression inthree groups cell (×400). (A) Bax normal SHG44;(B)Bax SHG44-EV; (C)Bax SHG44-DKK-1;(D) Bcl-2 normal SHG44 (E)Bcl-2 SHG44-EV; (F)Bcl-2 SHG44-DKK-1; (G)Caspase-3 normal SHG44; (H)Caspase-3 SHG44-EV; (I)Caspase-3 SHG44-DKK-1 Table 1 Bax, bcl-2 and caspase-3 expression (in IOD) in normal SHG44, SHG44-EV and SHG44-DKK-1 cells.   Bax protein expression Bcl-2 protein express Caspase-3 protein express   n = 6 IOD n = 6 IOD

n = 6 IOD normal SHG44 2323 ± 305 5046 ± 521 1845 ± 126 SHG44-EV 2623 ± 420 6417 ± 462 1920 ± 231 SHG44-DKK-1 4567 ± 598* 2900 ± 302* 3944 ± 511* *P < 0.05 Discussion The family of DKK genes is a small, but conservative gene family, which is composed of DKK-1, DKK-2, DKK-3, DKK-4 and DKKL-1 (also called Soggy), a DKK-3 related gene. DKK proteins possess different structure and function, but many of them play important roles in various human EPZ-6438 nmr diseases [2]. DKK-1 is the most well-studied gene in the DKK gene family. It is mapped to chromosome 10q11.2 [11] and encodes a secretory glucoprotein, which contains 266 amino acids with a molecular weight of 35KD. The glucoprotein contains a N-terminal signal peptide of 31 amino acids, two conserved cysteine-rich domains and a C-terminus with glycosylation function. DKK-1 acts as a wnt antagonist by forming a complex with the transmembrane proteins

Kremen1 and 2 (Krm1/2) and low- density-lipoprotein 5/6(LRP5/6). The complex is then removed through endocytosis, resulting in the removal of LRP5/6 from the cell surface [12, 13] Recent studies revealed that DKK-1 is not only an antagonist of classic Wnt/β-cantenin signaling many pathway but also a direct regulator of transcription of its target genes [14]. The function of DKK-1 in tumor progression has been shown to be complicated and even controversial. A number of studies showed that DKK-1 induces apoptosis and inhibits tumor growth [15–17] DKK-1 expression in primary medulloblastoma cells is significantly down-regulated relative to normal cerebellum and Eltanexor transfection of a DKK-1 gene construct into D283 cell line suppresses medulloblastoma tumor growth [18]. In addition, adenoviral vector-mediated expression of DKK-1 in medulloblastoma cells significantly increases the apoptosis rate. DKK-1, however, is also reported to be overexpressed in tissues and serum of lung cancers and esophageal squamous cell carcinoma, suggesting that DKK-1 may act as pro-oncogene [19].

This is consistent with our results, in which we did not detect any activity from promoters other than those upstream of the dksA gene (Figure 3). This unusual arrangement suggests that

gluQ-rs expression is dependent on dksA-regulated conditions. Because DksA is a key member of the Selumetinib order stringent response in bacteria and regulates a number of processes in the cell, including its own expression [25, 28], the data suggest that there is coordinate regulation of tRNA modification and other DksA targets. Although we could not detect any promoter activity specific for gluQ-rs in the growth conditions tested (i.e. altering the pH, presence of glutamate), we cannot Selleckchem PD0325901 discount the possibility that the gene is specifically regulated under some other conditions. The regulon database (http://​regulondb.​ccg.​unam.​mx/​index.​jsp) indicates that the E. coli gluQ-rs gene has a recognition site for the σ24 subunit of RNA polymerase. From our analysis, this sequence 8-Bromo-cAMP molecular weight is identical to S. flexneri, but there is no experimental evidence of this recognition. Interestingly, when the gluQ-rs gene was deleted in S. flexneri, the mutant showed impaired growth in the presence of osmolytes (Figure 6). A recent publication demonstrated that σ24 and σS proteins from Salmonella enterica serovar Typhi are important

for the expression of several genes induced by osmotic stress in this bacterium [29]. Moreover, the expression of the gene encoding σ24 in E. coli is regulated by the stringent response [30]. The possible role of σ24 on the expression of gluQ-rs under osmotic stress might be interesting to study. GluQ-RS is an enzyme responsible for the formation of the GluQ tRNA modification, and two

independent groups [10, 11] have shown that this enzyme required a high concentration of glutamate to be activated and transferred to the queuosine base present on the tRNAAsp. Interestingly, one of the first events to occur when bacteria are subject to high osmolyte stress is an increase in glutamate levels within the cytoplasm [31]. Our observation indicates an important role of the tRNA modification for the growth of S. flexneri in the presence of osmolytes (Figure 6). Other tRNA modifications might play a similar role in this stress condition. In E. coli, inactivation of the through yfiC gene, responsible for the modification at the adenosine 37 present on the tRNAVal, leads to a high sensitivity to osmotic stress [32]. Transcription of gluQ-rs is regulated by a terminator The results obtained in the present work show the presence of a terminator and suggested the functionality of this structure (Figure 3 and Figure 4). To our knowledge, there are few examples of bacterial genes that have similar structures. There is a terminator structure upstream of the DNA primase gene, dnaG, which also has an unusual Shine Dalgarno sequence [33]. Another example is the recX gene in E.

According to these authors, the salivarius group is composed of three species: (1) S. salivarius, a pioneer colonizer of the human oral mucosa that is isolated mainly from the dorsum of the tongue, the cheeks, and the palate [3],

(2) S. vestibularis, a mutualistic bacterium that is present on the vestibulum of the human oral mucosa [4], and (3) S. thermophilus, a thermophilic species [5] that is part of starter cultures used in the production of yogurt and Swiss- or Italian-type cooked cheeses. Unlike S. salivarius and S. vestibularis, S. thermophilus is not a natural inhabitant of the human oral mucosa and is commonly found on the mammary mucosa of bovines, its natural ecosystem, as inferred from its presence and that of thermophilus-specific bacteriophages selleck screening library in raw milk isolates [6–8]. The common www.selleckchem.com/products/ly3039478.html ecosystem is not the only feature shared by S. salivarius and S. vestibularis. Biochemical

investigations of functional metabolic pathways have VX-689 cost revealed that these two species share a high level of physiological resemblance. For example, S. salivarius and S. vestibularis are capable of hydrolyzing esculin and generating acidic compounds from maltose and N-acetyl-glucosamine, while S. thermophilus is not ([9] and references therein). Both S. salivarius and S. vestibularis are also opportunistic pathogens that can cause mild to severe infective endocarditis [10–12], whereas S. thermophilus has never been implicated in such infections. Given the home environments of the organisms, the high level of metabolic similarity between S. salivarius and S. vestibularis, and the more restricted

spectrum of Endonuclease carbon sources that can be used by S. thermophilus [13], one would assume that S. salivarius and S. vestibularis would be more related to each other than to S. thermophilus. However the few phylogenetic trees published so far that include all three species, as inferred from 16S rRNA-encoding gene sequences [2] and the housekeeping gene sodA that encodes the manganese-dependent superoxide dismutase [14], suggest that a schism generated S. vestibularis and S. thermophilus subsequent to the early divergence of S. salivarius. However, since these two phylogenetic studies [2, 14] were limited to only one taxon for each species, the inferred relationships between these three species might be inaccurate. To investigate the evolutionary relationships between the three species making up the salivarius group, we performed phylogenetic inferences based on the 16S rRNA-encoding, secA and secY housekeeping genes and the important yet non-essential recA gene using an identical distribution of streptococcal strains among the various markers to facilitate direct comparisons and allow the concatenation of the individual sequences into a single matrix. These four ubiquitous genes are widely distributed and have homologues in all three kingdoms, i.e., Bacteria, Archaea, and Eukarya (for reviews see [15–17]).

2007, H. Pexidartinib clinical trial Voglmayr & W. Jaklitsch, W.J. 3158 (WU 29479, culture C.P.K. 3150). Norfolk, Thetford, Emilys Wood, near Brandon, MTB 35-31/2, 52° 28′ 08″ N, 00° 38′ 20″ E, elev. 20 m, on partly decorticated branch of Fagus sylvatica 3.5 cm thick on the ground, present as anamorph, soc. Hypocrea neorufoides, 13 Sep. 2004, H. Voglmayr & W. Jaklitsch (deposited as H. neorufoides WU 29300; culture C.P.K. 1978). Thetford, close to the town on the right side of the road from Elveden, at a parking place, 52° 24′ 00″ N, 00° 42′ 43″ E, elev. 30 m, on branches of Fagus sylvatica 10 cm thick in a small pile on the ground, holomorph, teleomorph immature, culture from conidia, 12 Selleck CH5183284 Sep. 2004, H. Voglmayr & W. Jaklitsch,

W.J. selleck kinase inhibitor 2704 (WU 29477, culture C.P.K. 1977). Notes: Hypocrea stilbohypoxyli is a typical species of the section Trichoderma with low tendency to form pulvinate stromata, i.e. often maturing when effused. It produces the largest stromata of the section in Europe apart from H. ochroleuca and H. subeffusa. The anamorph

of H. stilbohypoxyli may attract attention in nature due to its abundance under favourable conditions and its bright blue-green colour. In culture, T. stilbohypoxyli is conspicuous particularly on CMD at 25°C, due to pustules with a yellow reverse that consist of a dense core of curved conidiophores and phialides reminiscent of H. rufa/T. viride, surrounded by regularly tree-like conidiophores. Characteristic are also the irregularly thickened cells in surface hyphae around pustules, and notable the abundant chlamydospores on SNA at 30°C that are sometimes reminiscent of ascospores. These cultural traits have not been ascertained in non-European strains.

According to Samuels et al. (2006a) H. stilbohypoxyli has a remarkably wide geographic Nintedanib (BIBF 1120) distribution. Whether or not all these specimens and cultures represent a single species is not clear. In fact, although clustering together, the European isolates differ from others consistently in gene sequences, one nucleotide in ITS, five in rpb2 and 21 nucleotides in tef1 introns four and five. Other differences deduced from the description in Samuels et al. (2006a) are smaller stroma size, slightly smaller ascospores, faster growth, distinctly zonate, green colonies on PDA, and infrequent chlamydospores in non-European strains. Hypocrea subeffusa Jaklitsch, sp. nov. Fig. 22 Fig. 22 Teleomorph of Hypocrea subeffusa. a–i. Dry stromata (a. habit, nearly fresh; b. stroma initial; c–e. immature). j. Rehydrated mature stroma. k. Stroma of j in 3% KOH. l. Hairs on stroma surface. m. Perithecium in section. n. Rehydrated stroma surface. o. Stroma surface in face view. p. Cortical and subcortical tissue in section. q. Subperithecial tissue in section. r. Stroma base in section. s–u. Asci with ascospores (t, u. in cotton blue/lactic acid). a, c, e, j–t. holotype WU 29487. b, d, g–i. WU 29488.

TRX in general regulates protein-nucleic acid interactions through the redox regulation of cystein residues

[34]. In addition, cellular redox status is pivotal to regulation of apoptosis. TRX has been shown to bind and inactivate learn more apoptosis signal-regulating kinase 1 (ASK1), with the latter to be released upon oxidative stress [35]. Apart from its cellular functions, TRX can be secreted as an autocrine growth factor by a yet unknown mechanism. It is then stimulating the proliferation of cells derived from a variety of solid tumors [36]. In addition, the cytochrom P450 subtype 1B1 (CYP1B1) converts 17β-estradiol (abbreviated as E2) into the carcinogenic 4-hydroxyestradiol (4-OHE2). A study conducted in ER-positive MCF-7 breast cancer cells suggested TRX to be involved in the constitutive expression of CYP1B1 and the dioxin mediated induction of CYP1B1 [37]. It may, thus, be a potent co-factor of mammary carcinogenesis at least in estradiol responsive tumours. Like other thiol-containing proteins, thioredoxin overexpression was suspected triggering chemotherapy resistance [24]. Hence, TRX overexpression in several tumour derived

cell lines Ralimetinib mw is associated with resistance to Cisplatin [38]. However, TRX effects on anti-cancer drug resistance are complex and depend strictly on the tissue type. For instance, hepatocellular carcinoma cells with elevated thioredoxin levels are resistant to Cisplatin, but not to the antracyclin Doxorubicin [39]. However, bladder- and prostate cancer cell lines Non-specific serine/threonine protein kinase with TRX

overexpression are Cisplatin resistant and cross-resistant to Doxorubicin [40]. Cisplatin resistance in ovarian cancer cell lines is associated with high TRX levels, but recombinant TRX overexpression in non-resistant cells does not confer resistance to Cisplatin or Doxorubicin [41]. Thus, Cisplatin-responsiveness of a given tumour entity overexpressing TRX is unpredictable at present. Breast cancer For midaged women in the industrialized countries, breast cancer is the second most common cause of cancer-death [10]. Carcinomas of the mammary gland comprise rather different diseases referring to divergent cell types found in the female breast. Breast cancers are divided into ductal, medullary, lobar, papillary, tubular, apocrine and adeno-carcinomas, respectively [42]. Breast cancer is not a purely gynecological disorder: approximately 1% of breast cancer cases are male patients. Apart from histological classification, breast cancers are biochemically categorized independent of the tissue origin with respect to their receptor status: 1. HER-2 positive tumours   2. triple-negative breast cancer (TNBC), which are ER, PR, and HER-2 negative   3. endocrine-responsive tumours   HER-2 positive tumours are selleck compound characterized by constitutive overexpression of the HER-2 receptor subtype of the epidermal growth factor receptor family.

​mbio.​ncsu.​edu/​bioedit/​bioedit.​html 20. Felsenstein J: Distance methods for inferring phylogenies: a justification. Evolution 1984, 38:16–24.CrossRef 21. Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003, 52:696–704.PubMedCrossRef 22. Posada D: jModelTest: phylogenetic model averaging. Mol Biol Evol 2008, 25:1253–1256.PubMedCrossRef 23. Feil EJ, Li BC, www.selleckchem.com/products/SB-202190.html Aanensen DM, Hanage WP, Spratt BG: eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol 2004, 186:1518–1530.PubMedCrossRef 24. Haubold B, Hudson RR: LIAN 3.0: detecting

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EJ, Chan MS, Maiden Akt inhibitor MC: Sequence type analysis and recombinational tests (START). Bioinformatics 2001, 17:1230–1231.PubMedCrossRef 26. Huson DH, Bryant D: Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 2006, 23:254–267.PubMedCrossRef 27. Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P: RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics 2010, 26:2462–2463.PubMedCrossRef 28. Silver AC, Williams D, Faucher J, Horneman AJ, Gogarten JP, Graf J: Complex evolutionary history of the Aeromonas veronii group revealed by host interaction and DNA sequence data. PLoS One 2011, 6:e16751.PubMedCrossRef KU55933 price 29. Khan NH, Ahsan M, Yoshizawa S, Hosoya S, Yokota A, Kogure K: Multilocus sequence typing and phylogenetic analyses of Pseudomonas aeruginosa isolates from the ocean. Appl Env Microbiol 2008, 74:6194–6205.CrossRef 30. Jolley KA, Maiden MC: BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics Tenofovir 2010, 11:595.PubMedCrossRef 31. Beatson SA, das Graças de Luna M, Bachmann NL, Alikhan N-F, Hanks KR,

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PLD expression is uncommon among other bacterial pathogens and these PLDs are exclusively of the HKD superfamily. However, most of the pathogens that do express PLD have obligate or facultative intracellular lifestyles and expression of this enzyme is thought to be involved in disease pathogenesis [31–35]. Specifically in Neisseria gonorrhoeae and Rickettsia spp., PLDs are required for invasion of host cells [32, 35]. This work characterizes the effects of A. haemolyticum Cell Cycle inhibitor PLD on host cells, with an aim to elucidating the role of this toxic enzyme in disease pathogenesis. We report that PLD is required for optimal adhesion to host cells, via remodeling

of lipid rafts. Furthermore, PLD expressed inside host cells is directly toxic, leading to cell death via necrosis. These findings provide the first conclusive evidence that PLD may be required for A. haemolyticum disease pathogenesis. Results Analysis of the pld gene region A draft genome sequence of A. haemolyticum ATCC9345 was determined (B.H. Jost and S.J. Billington, unpublished

data), and this data was used to identify sequences flanking the pld gene (GenBank Accession Number L16583). The pld gene was found in a region resembling a 1.9-kb genomic island of lower %G + C than the rest of the A. haemolyticum genome (53.1%). This region consists of pld (47.2% G + C), and orf489 (50.3% G + C) which lacks a signal sequence and is of unknown Selleckchem GSK621 function (Figure 1). 43-bp downstream Depsipeptide cell line of pld and 17-bp upstream of orf489 is a stem-loop structure with a ΔG = -20.8 kcal/mol, which may act as a transcriptional terminator or attenuator. There does not appear to be any BAY 11-7082 mw direct or indirect repeats flanking this region. The pld region is flanked upstream by three tRNA genes and gluRS, encoding a glutamyl-tRNA synthetase (EC 6.1.1.17), and downstream by dcp, encoding a peptidyl-dipeptidase (EC 3.4.15.5), which is divergently transcribed

compared to pld (Figure 1). The %G + C of the surrounding housekeeping genes (Figure 1) more closely resembles the %G + C of the A. haemolyticum genome. Figure 1 Map of the pld gene region. The open arrows indicate genes and the direction of transcription. Triangles below the sequence indicate the location of stem-loop structures, with the ΔG (kcal/mol) shown inside the triangle. Gene names are given above or below the arrows and the number below the name indicates the %G + C of the gene. A bar indicating 1-kb is shown on the right. Given the variation in %G + C of the pld gene and the presence of adjacent tRNA genes, which often act as sites of foreign gene insertion [36], it is possible that the A. haemolyticum pld gene was acquired by horizontal gene transfer. It would appear that orf489 is also part of the transferred DNA, and while it is not translationally coupled to pld, its transcription may be linked to that of pld despite the presence of a transcriptional terminator/attenuator between the two genes.

It seems to be a freak of nature that in M. hominis, OppA has gained an additional ATPase click here activity which raises the question as to its function. To date ecto-ATPase activity of OppA is unique to M. hominis among substrate-binding proteins of ABC-transporters of all three kingdoms. Thus it seems illogical that the ecto-ATPase is required for optimized peptide import. The findings of this study clearly demonstrate

that the OppA ecto-ATPase is essential for maximal cytoadhesion of M. hominis. In studying bacterial adhesion to polymer surfaces selleck products Stollenwerk and coworkers found that under conditions of starvation – by incubation in nutrient-poor buffer – the ATP content of adherent bacteria decreased after 24 h to 96 h whereas that of planktonic bacteria remained stable for up to 20 days [28]. This suggests that cytoadhesion is an energy-consuming process. Similar to our results presented here an ecto-ATPase-dependent cytoadherence has already been suggested

for Trypanosoma cruzi whose ATPase activity was strongly inhibited by using DIDS or suramin attended by a reduced adhesion to mouse resident macrophages [25]. Early work of Bredt and coworkers in the 1980′s demonstrated that cytoadhesion of the cell wall-less mollicutes is modulated by ATP. By monitoring the ATP content in the supernatant attachment of M. pneumoniae to glass surfaces was shown to depend on an intact energy metabolism [29]. In using a glucose-inhibitor, the ATP content declined and attachment was abrogated. In using STK38 an ATPase inhibitor, ATP content accumulates leading

Erastin to a decreased cytoadherence. Bredt and coworkers hypothesized that the first step of colonization is energy dependent either to energize the membrane thus increasing some binding sites on the surface, or to modulate the contractile cytoskeleton [29]. The free energy of ATP hydrolysis by P-loop NTPases is typically utilized to introduce conformational changes in other molecules [30]. As adhesion of mycoplasmal cytoadhesins does not depend on ATP-hydrolysis at all, as demonstrated in this study for the P60/P80 membrane complex of M. hominis, ATPase dependent adhesion of OppA is predicted to play a special role in M. hominis. In 2008 OppA was shown to mediate apoptosis, to induce ATP-efflux and a concomitant ATP-depletion of the M. hominis-colonized host cell [15]. This is in accordance to the recent findings that the cytoadherence of M. pneumoniae induces an ATP-efflux from the colonized host [31]. ATP- efflux was considered as a stress-associated danger signal as it stimulates P2X7-receptors of the host leading to the expression of pro-inflammatory cytokines. It is well known that extracellular ATP signals through P2 receptors to modulate the immune and inflammatory response in a variety of host cells, including immune and non-immune cells, sometimes leading to apoptosis or necrosis of the cells [32].

In contrast, in case of GI5 we were not able to detect a circular intermediate neither with the originally predicted borders nor with the additional genes suggested by the microarray experiments (Bpet3771–3779), although the microarray data of the phenotypic learn more variants f, g, and k definitely revealed the deletion of this element from their genomes. As shown above, we were

able to detect circular intermediates of most genomic islands by PCR amplification, although the microarray experiments with the phenotypic variants clearly demonstrated the deletion events. Possible explanations for this fact could be that the excised islands are diluted during growth of the bacteria since they cannot replicate. Moreover, the experimental protocols for the two methods are different and PCR amplification is much more sensitive as compared to cy3/cy5 labeling by

Klenow polymerisation. Stability of genomic island GI3 The frequent appearance of phenotypic variants involving the genomic islands AG-120 order present in the B. petrii genome and the detection of circular intermediates of these islands under standard growth conditions indicates that these genomic islands are rather unstable and active at least in terms of excision. To assess the stability of one of these islands (GI3) by homologous recombination we integrated a tetracycline resistance cassette in GI3 between the genes Bpet1523 and Bpet1524 coding for a putative transposase and a glycosyltransferase, respectively. Under standard growth conditions, the resulting strain B. petrii GI3::tetR

did not show any change in its maximum specific growth rate as compared to the wild type (data not shown). This strain was then used for KPT-8602 order growth experiments without selective pressure in which the bacteria were cultivated for about 150 consecutive generations. Exponentially growing B. petrii before has a generation time of about 90 min (data not shown). Figure 5 shows the time course of loss of GI3::tetR determined by differential counting of tetracycline resistant and sensitive bacteria plated out on the respective agar plates. GI3 was stably present in the B. petrii population for about 40 generations, then the proportion of tetracycline resistant bacteria declined steadily and virtually no tetracycline resistant bacteria were found in the population after about 100 generations. Lack of the entire GI3 was confirmed by Southern blotting in representatives of these bacteria (data not shown). Although we cannot exclude a destabilizing effect of the tetracycline cassette on the island, it is likely that GI3 is highly unstable and gets lost with a high incidence when no selective pressure for its persistence is present. Figure 5 Stability of the genomic island GI3 in the genome of B. petrii during culture grown without selective pressure. On the x-axis the number of consecutive generations of the bacteria culture and on the y-axis the proportion of tetracycline resistant bacteria in the culture is shown.