Among the Rhizobiaceae, the best studied species regarding osmoad

Among the Rhizobiaceae, the best studied species regarding osmoadaptation is Sinorhizobium meliloti one of the most common alfalfa microsymbionts. Specific Belnacasan molecular weight concomitant accumulation of potassium and glutamate was found to be the primary response in Selleck Selumetinib S. meliloti to hyperosmotic stress [9]. Out of four potassium uptake systems found within the S. meliloti genome, Trk was shown to be the most important K+ importer involved in the osmoadaptation of this bacterium [10]. By using 13C nuclear magnetic resonance spectroscopy (a particularly useful

technique for osmoadaptation studies because all types of organic compounds can be detected at once), it was shown selleck that S. meliloti long term response to hyperosmotic stress involves the synthesis and

accumulation of the dipeptide N-acetylglutaminylglutamine amide and the disaccharide trehalose, the latter one specially when cells are subjected to severe osmotic stress [3, 11]. Trehalose is a non-reducing glucose disaccharide that is widespread in nature. It protects numerous biological structures against abiotic stresses including desiccation, oxidation, heat, cold, dehydration, and hyperosmotic conditions [6]. Recently, the importance of trehalose in osmotolerance and nodulation of their legume hosts by S. meliloti [12] and Bradyrhizobium japonicum [13] has been firmly established. Trehalose

has shown to play also a major role in desiccation tolerance of R. leguminosarum bv. trifolii [14]. Common bean (Phaseolus vulgaris) is an important staple crop in the diets of people of Latin America, Asia, Africa, and other regions of the developing world. Paradoxically, despite common bean is a promiscuous legume able to form symbioses with a number of rhizobial species including R. tropici, R. etli, R. gallicum, R. leguminosarum bv. phaseoli or R. giardinii [15–17], it is considered as a poor nitrogen fixer, if compared to other grain legumes [18, 19]. This problem has been attributed to the ineffectiveness of indigenous rhizobia [20] or to adverse abiotic Cyclin-dependent kinase 3 conditions [21]. In a recent work, Suarez et al. [22] reported an increase in root nodule number and nitrogen fixation by P. vulgaris cv. Negro Jamapa (a Mesoamerican cultivar) inoculated with a trehalose-6-phosphate synthase-overexpressing strain of R. etli. Thus, manipulating trehalose metabolism in P. vulgaris looks a promising strategy to improve plant tolerance to osmotic stress and grain yield. Compared to this body of knowledge on the osmoadaptation of these agronomically important rhizobacteria, little is known about the osmostress responses of rhizobial strains nodulating common bean in Africa. The purpose of the work described here was threefold.

Arthritis Res Ther 2010, 12:R25 PubMedCrossRef Competing interest

Arthritis Res Ther 2010, 12:R25.PubMedCrossRef Competing interests Curves International (Waco, TX, USA) provided funding for this project through an unrestricted research grant to Baylor University when the Principal Investigator and the Exercise & Sport Nutrition Lab were affiliated with that institution and currently provides funding

to Texas A&M University to conduct exercise and nutrition related research. All researchers involved independently collected, analyzed, and interpreted the results from this study and have no financial interests concerning the outcome of this investigation. Data from this study have been presented at the Federation of American Societies of Experimental Biology annual meeting. Publication of these findings should not be viewed as endorsement by the investigators or their institutions of the programs or materials investigated. Authors’ contributions TMC served as the study supervisor, oversaw all testing, and assisted in writing of the

Dinaciclib manuscript. CW assisted in data collection and manuscript preparation. CR, MF, LG, BC, CMK, KD, RL, EN, MI and MC assisted in data collection, data analysis, and/or manuscript preparation. DW oversaw Ilomastat purchase analysis of blood work. LS provided input on study design and results. RBK served as Principal Investigator and contributed to the design of the study, statistical analysis, manuscript preparation, and procurement of external funding. All authors read and approved the final manuscript.”
“Background The International Association of Athletic Sorafenib price Federations (IAAF) Consensus Statement on Nutrition for athletics published in 2007 states: “”Well chosen foods will help athletes train hard, reduce risk of illness and injury, and achieve performance goals,

regardless of the diversity of events, environments, nationality and level of competitors.”" [1]. Specific nutritional recommendations for optimal performance, particularly for endurance athletes, include a daily carbohydrate (CHO) intake ranging from 6 to 10 g/kg body mass (BM) considered essential for replacing liver and muscle glycogen stores [2]. A significant protein intake ranging between 1.2 to 1.7 g/kg BM per day is required for optimal health and performance of endurance athletes [2]. Studies examining protein intake in athletes have shown an increased requirement for protein in endurance trained athletes [3–5] as opposed to healthy adult males (i.e., 0.8 g/kg) due to increased amino acid oxidation during exercise and for growth and repair of muscle tissue [6]. Maintenance of normal body water during strenuous training and minimising the level of dehydration (i.e., preventing a BM loss of > 2%) during endurance exercise achieved by consuming fluids at a rate of 0.4 to 0.8 L/h ad libitum is now recommended [7].

Lung Cancer 2008, 59: 155–163 CrossRefPubMed 19 Zhuo W, Wang Y,

Lung Cancer 2008, 59: 155–163.CrossRefPubMed 19. Zhuo W, Wang Y, Zhuo X, Zhu Y,

Wang W, Zhu B, Li D, Chen Z: CYP1A1 and GSTM1 Polymorphisms and Oral Cancer Risk: Association Studies Via Evidence-Based Meta-Analyses. Cancer Invest 2009, 27: 86–95.CrossRefPubMed 20. White DL, Li D, Nurgalieva Z, El-Serag HB: Genetic variants of glutathione S-transferase as possible risk factors for hepatocellular carcinoma: a HuGE systematic review and meta-analysis. Am J Epidemiol 2008, 167: 377–389.CrossRefPubMed 21. Lai R, Crevier L, Thabane L: Genetic polymorphisms Torin 2 nmr of glutathione S-transferases and the risk of adult brain tumors: a meta-analysis. Cancer Epidemiol Biomarkers Prev 2005, 14: 1784–1790.CrossRefPubMed 22. La Torre G, Boccia S, Ricciardi G: Glutathione S-transferase M1 status and gastric cancer risk: a meta-analysis. Cancer Lett 2005, 217: 53–60.CrossRefPubMed 23. Yang CX, Matsuo K, Wang ZM, Tajima K: Phase I/II enzyme gene polymorphisms and esophageal

cancer risk: a selleckchem meta-analysis of the literature. World J Gastroenterol 2005, 11: 2531–2538.PubMed 24. Ntais C, Polycarpou A, Ioannidis JP: Association of GSTM1, GSTT1, and GSTP1 gene polymorphisms with the risk of prostate cancer: a meta-analysis. Cancer Epidemiol Biomarkers Prev 2005, 14: 176–181.PubMed 25. Hosgood HD, Berndt SI, Lan Q: GST genotypes and lung cancer susceptibility in Asian populations with indoor air pollution exposures: a meta-analysis. Mutat Res 2007, 636: 134–143.CrossRefPubMed 26. Saadat M: Genetic polymorphisms of glutathione check details S-transferase T1 (GSTT1) and susceptibility to gastric cancer: a meta-analysis. Cancer Sci 2006, 97: Fenbendazole 505–509.CrossRefPubMed 27. Chen K, Jiang QT, He HQ: Relationship between metabolic enzyme polymorphism and colorectal cancer. World J Gastroenterol 2005, 11: 331–335.PubMed 28. Ye Z, Song H: Glutathione s-transferase polymorphisms (GSTM1, GSTP1 and GSTT1) and the risk of acute

leukaemia: a systematic review and meta-analysis. Eur J Cancer 2005, 41: 980–989.CrossRefPubMed 29. Hashibe M, Brennan P, Strange RC, Bhisey R, Cascorbi I, Lazarus P, Oude Ophuis MB, Benhamou S, Foulkes WD, Katoh T, Coutelle C, Romkes M, Gaspari L, Taioli E, Boffetta P: Meta- and pooled analyses of GSTM1, GSTT1, GSTP1, and CYP1A1 genotypes and risk of head and neck cancer. Cancer Epidemiol Biomarkers Prev 2003, 12: 1509–1517.PubMed 30. Vogl FD, Taioli E, Maugard C, Zheng W, Pinto LF, Ambrosone C, Parl FF, Nedelcheva-Kristensen V, Rebbeck TR, Brennan P, Boffetta P: Glutathione S-transferases M1, T1, and P1 and breast cancer: a pooled analysis. Cancer Epidemiol Biomarkers Prev 2004, 13: 1473–1479.PubMed 31. Bolt HM, Thier R: Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology. Curr Drug Metab 2006, 7: 613–628.CrossRefPubMed 32.

This is important because some

This is important because some C188-9 price of the risk factors affect the risk of death as well as the fracture risk. Examples include increasing age, sex, low BMI, low BMD, use of glucocorticoids and smoking. Fig. 10 The risk of hip fracture with age in a model that considers 10-year fracture risk alone (the Garvan tool) and FRAX which computes the probability of hip fracture from the fracture and death hazards (FRAX). The T-scores are set differently in the two models so that the risks

are approximately equal at the age of 60 years. Data are computed from the respective websites [127]. With kind permission from Springer Science and Business Media General management Mobility and falls Immobilisation is an important cause of bone loss. Immobilised patients may lose as much bone in a week when confined to bed than they would otherwise lose in a year. For this reason, immobility

should, wherever possible, be avoided. The amount of weight-bearing exercise that is optimal for skeletal health in patients with osteoporosis is not known, but exercise forms an integral component of management [128–130]. Physiotherapy is an important component of rehabilitation after fracture. At all times, increased strength may prevent falls by improving confidence and coordination as well as maintaining bone mass by stimulating bone formation and by decreasing bone resorption, 17DMAG chemical structure and by preserving muscle strength. Such Pitavastatin in vivo measures together can be coupled with a programme to reduce the likelihood of falls in those at high risk. Risk factors for falling are shown in Table 10 [131]. Modifiable factors such as correcting decreased visual acuity, reducing consumption of medication that alters alertness and balance and improving the home environment (slippery floors, obstacles, insufficient lighting, handrails) are important measures aimed at preventing falls [132, 133]. Although large trials have shown that it is possible NADPH-cytochrome-c2 reductase to reduce falls [134, 135], randomised studies have not shown any significant decrease in fracture risk. Some randomised trials have shown that wearing hip protectors can markedly reduce hip fracture risk, particularly in the elderly

living in nursing homes. A meta-analysis of well-conducted randomised controlled trials has, however, cast some doubt about the anti-fracture efficacy of this preventive measure [136–139]. Table 10 Risk factors associated with falls (adapted from [131] with permission from Elsevier) 1. Impaired mobility, disability 2. Impaired gait and balance 3. Neuromuscular or musculoskeletal disorders 4. Age 5. Impaired vision 6. Neurological, heart disorders 7. History of falls 8. Medication 9. Cognitive impairment Nutrition At every stage of life, adequate dietary intakes of key bone nutrients such as calcium, vitamin D and protein contribute to bone health and reduce thereby the risk of osteoporosis and of fracture later in life [140].

Yasumitsu et al [33] determined gelatinase activity in human sch

Yasumitsu et al. [33] determined gelatinase activity in human schwannoma YST-3 cell lines using zymography, and found that MMP-9 activity in degrading collagen was about 25 times that of MMP−2. Previous studies suggested that MMP-9 expression were closely related to tumour angiogenesis than MMP-2 [34, 35]. Conclusion Obviously, tumour cells and stromal cells can expression high MMP levels, which are closely related to poor prognosis. In exploring

ColIV expression, we also found that tumour expressions of MMP-2 and MMP-9 showed certain variations. The MMP-9 expression may be closely related to proliferation, invasion, and metastasis of tumour cells, and even to tumour angiogenesis. This may

be related to the activity of MMP-9; C646 concentration however, its specific mechanism of action merits further research. In addition, which specific stromal cell (e.g. macrophages, URMC-099 molecular weight fibroblasts, etc.) and which cell subtype (e.g. M1 and M2 macrophages) interact with tumour cells also remains unknown. Nevertheless, clinical application of agents that may inhibit MMP-9 secretion by stromal cells may be a key to achieving clinical control of invasion and metastasis of oral tumours. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (305400083). Electronic supplementary material Additional file 1: Immunofluorescence staining for ColIV, MMP-9 and PCNA in OTSCC. Figure S1 Immunofluorescence staining for ColIV in normal group, dysplastic oral mucosa group and OTSCC group. Comparative immunolocalization of ColIV in normal group, dysplastic oral mucosa group and OTSCC (T and S indicate the tumour and NSC 683864 cost stroma respectively) by immunofluorescence. (A) The expression of ColIV in the BM of normal group showing linear and continuous marking (red arrow). (B)

The expression of ColIV in the BM of normal group showing interrupted (red arrow). (C) In the OTSCC, the expression of ColIV are showed fragmented or collapsed (red arrow). Original Terminal deoxynucleotidyl transferase magnification, 200×. Figure S2 Double immunofluorescence staining for PCNA and MMP-9 in the stromal of OTSCC. Expression of PCNA and MMP-9 proteins detected by double immunofluorescence staining in the stromal of OTSCC (S indicate the stroma). (A) The expression of PCNA in the stromal cells (red). (B) The expression of MMP-9 in the stromal cells (green). (C) Double-labeled cells of PCNA/MMP-9 in the OTSCC. Original magnification, 200×. (PPT 3 MB) Additional file 2: Table S1. Association between MMP-2 and MMP-9 expression and PCNA in OTSCC patients. (DOC 24 KB) References 1. Regezi JA, Sciubba JJ, Jordan RCK: Oral pathology : clinical pathologic correlations. St. Louis, Mo: Saunders/Elsevier; 2008. 2.

, Corning NY) using an Affymetrix

, Corning NY) using an Affymetrix HSP mutation GeneChip instrument at the MSU Research Technology Support Facility (RTSF). Each strain was streaked from frozen stock on two tryptose soya agar plates containing 5% defibrinated sheeps’ blood; plates were incubated for 48 hours at 37°C in 5% CO2. A single isolated colony from each plate was chosen and streaked onto another plate (biological replicates). Growth from each of the second plates was harvested separately and genomic DNA was isolated using the CTAB procedure described in Ausubel et al. [69]. One μg DNA was sheared by sonication to 0.5–2.0 kbp and labeled with aminoallyl-dUTP using the BioPrime random priming

DNA labeling kit® (Invitrogen, Carlsbad, CA). Unreacted components were removed using a Qiagen PCR Purification kit® (Qiagen, Valencia, CA). Aliquots of the purified aminoallyl-dU-containing DNA were then reacted with Cy5 or Cy3 dye (Amersham, Piscataway, NJ). Unreacted dye was removed using a Qiagen PCR Purification kit®. The

two separate DNA extractions done for each strain were used in separate hybridizations (technical replicates). The experiment was repeated with the dyes reversed; thus a total of four chips were hybridized and compared for each strain. The spots for each gene are duplicated three times on each chip, for a total of 12 comparisons for each strain. For hybridization, Ambion SlideHyb solution (Ambion, Austin, TX) was preheated to 54°C. The combined Cy3/Cy5 labeled DNA samples were resuspended in 4 Selonsertib research buy μl 10 mM EDTA. The sample was then check details denatured at 95°C for 10 minutes. During this time the cover slip was washed in 95% ETOH, 0.1% SDS and sterile ddH2O. Cover slips were dried with filtered air. After denaturation of sample, 30 μl of prewarmed

Ambion SlideHyb solution was added. The slides were Cyclin-dependent kinase 3 centered on a warmed hybridization cassette and a cleaned cover slip was placed face down and centered over the spots. The denatured sample was then gently pipetted using capillary action to fill the area underneath the cover slip. Sixteen μl of ddH2O was added to the grooved edge of each hybridization chamber. The top of the hybridization chamber was then secured; the slides were placed on a rack in a 54°C water bath overnight. All steps were performed in the dark. Post-hybridization washes were performed as follows. In the dark, the opened cassette was gently moved up and down in warmed 1 × SSC, 0.2% SDS until the cover slip fell off. The slide was placed on an orbital platform shaker at low speed for 4 minutes in the dark. The slide was transferred to 0.1 × SSC containing 0.2% SDS and incubated on the platform shaker at low speed for 4 min. The process was repeated twice with 0.1 × SSC. The slide was placed in a 50 ml conical tube covered with aluminum foil and centrifuged at 1000 rpm in a clinical centrifuge in a swinging bucket rotor for 5 min.

8% in our control subjects This frequency is also similar to the

8% in our control subjects. This frequency is also similar to the frequencies Pifithrin-�� manufacturer found in other studies that analyzed GSTP1 polymorphism [18–20]. Some studies have reported a relationship between GST variants and risk of prostate cancer [9, 10, 12, 13, 21]. Investigation of the GSTP1 gene did not reveal any significant association between heterozygous GSTP1 genotype (Ile/Val) and prostate cancer. However, our results Eltanexor solubility dmso suggest that Val/Val genotype of GSTP1

gene could modulate the risk of prostate cancer, even if this association did not reach statistical significance. It should be kept in mind that the inability to reject the null hypothesis could be due to low power of the test because of a relatively buy AZD7762 small sample size. Therefore, the lack of significance does not necessarily mean equality of the distributions. It is plausible that polymorphism at the GSTP1 locus can play an important role in the susceptibility to different types of cancer. Association of the GSTP1 Val allele with cancer could be expected since the conversion of the amino acid at codon 105 from isoleucine to valine substantially lowers activity of the altered enzyme. It has been predicted

from molecular modelling that the amino acid at this site lies in a hydrophobic binding site for electrophile substrates and thus affects the substrate binding [22]. On the other hand, there are also studies which did not prove any independent effect of this type of polymorphism on the susceptibility for prostate cancer [23–25]. In the present study, we did not observe significantly different crude rates of the GSTM1 and GSTT1 null genotypes in the men diagnosed with prostate cancer and those in the control group. Our

data and the data published by other research groups suggest that differences in the GST frequencies between prostate cancer patients and the control group are relatively small, which therefore makes it difficult to separate the groups from each other Masitinib (AB1010) based on statistical data analysis. Once again, the high variability in the groups could mask statistical differences due to low power. The easiest way to improve precision is to increase the number of subjects and patients in the experimental design. However, this may not be applicable to all research conditions due to such factors as additional costs, poorer availability of resources, lower population, which compromises the number of subjects eligible for investigation. In order to achieve a power of at least 80%, we have to identify other explanatory variables and the control for them, and/or apply meta-analysis in order to increase sample size.

In our study, according to the outcome explored, the CPRD data we

In our study, according to the outcome explored, the CPRD data were linked to the Hospital Episode Statistics (HES) and the Office of National Statistics see more (ONS) databases to obtain additional information on hospitalisations and fatalities, respectively. The study protocol was approved by the Independent Scientific Advisory Committee of the Medicines and Healthcare Products Regulatory

Agency (MHRA). We identified all male and female patients who had received a prescription for osteoporosis treatment or a medical record of primary osteoporosis between 1 January 2002 and 30 April 2012. The cohort entry date was fixed as the date of the first prescription of osteoporosis treatment during the study period. Patients were excluded if they had had a prescription for an osteoporosis treatment

in the previous year or had received a prescription for bisphosphonate for indications other than osteoporosis (e.g., Paget’s disease, hypercalcaemia, breast cancer, or myeloma). Patients could also be excluded if they came from a practice with less than 1 year of UTS CPRD data at their cohort entry date. From this population, we then excluded successively patients who had never received a treatment for their osteoporosis, and then all male patients, to reach a population of women with treated osteoporosis. The follow-up period extended from the cohort entry date to the date of the last data collection from Lazertinib cell line the practice, the date of transfer if the patient left the practice, or the date of death. Outcomes and selection of controls Benzatropine The primary

outcomes of our nested case–control study were first definite MI (fatal or nonfatal), hospitalisation with MI (fatal or nonfatal, first or Salubrinal datasheet subsequent), and cardiovascular death occurring after the cohort entry date. The index date for cases was defined as the date of event. Cases of MI were qualified as definite [13] if there was a CPRD record of MI, and the patient either (1) died within 30 days, or (2) was initiated on relevant treatment (e.g., statins, nitrates, and/or beta-blockers), and had other supporting evidence (e.g., location of infarct, coronary artery revascularisation, and/or elevated cardiac enzymes) within 2 months of the MI. Analyses on first definite MI excluded patients with previous MI. Cases of hospitalisation with MI were identified in the HES dataset in patients eligible for linkage, which ensured detection of cases not otherwise apparent in the GP record. Analyses of cases of hospitalisation with MI did not exclude patients with previous MI. Cases of cardiovascular death were identified in the ONS death dataset in patients eligible for linkage. This dataset provides information on cause and date of death, which may be missing in the general practice-based CPRD. Three case–control analyses were performed successively.

The three

The three promoters were all induced in a concentration dependent manner, with induction lag times becoming shorter and induction rates steeper as oxacillin concentrations increased. This was mirrored by a corresponding stepwise decrease in growth rates. Induction rates generally began to slow after 60 min, upon the onset of oxacillin induced lysis [28], but again this was concentration dependent with induction rates beginning to decrease earlier in cultures with higher oxacillin concentrations. Figure 2 Induction kinetics

of three CWSS promoters in response to Dactolisib nmr varying concentrations of oxacillin. Luciferase Entospletinib price activities and growth curves of strain BB255 containing: A, p sas016-luc+; B, p sa0908-luc+; and C, p tcaA-luc+; after addition of 0, 0.5, 1, 2 or 5-fold the MIC of oxacillin at time point zero. Previous findings, using Northern blots to measure oxacillin induction levels of sas016 after 30 min, indicated that inhibitory concentrations of oxacillin

were required for induction [20]. Figure 2 confirmed that the sub-inhibitory concentration of 0.5x MIC did not noticeably induce promoter activity after 30 min, however, luciferase activity from all three promoters began to increase sharply after 60 min and find more continued to rise up to the final sampling point of 120 min. Although all three promoters displayed similar relative concentration- and time-dependent induction kinetics,

the sas016 promoter produced the highest levels of luciferase activity, resulting in greater fold-changes between samples and making it the most sensitive of the three reporters. Therefore we chose the sas016 promoter-luciferase fusion construct as the best indicator to compare induction characteristics of different cell wall active antibiotics. Correlation between sas016 transcript induction and luciferase activity from p sas016 p -luc+ To confirm that levels of luciferase activity from p sas016 p- luc + accurately represented levels of sas016 gene expression, Northern blots were performed on BB255 p sas016 p- luc + RNA samples extracted from cultures grown using the same conditions and oxacillin concentrations used for luciferase assays. Samples were harvested 20 min and 60 min after antibiotic induction and hybridized with sas016 and luc + specific DIG probes (Figure 3). Northern blots showed identical patterns of transcriptional induction for both the chromosomal sas016 gene and the luciferase gene under the control of the sas016 promoter in p sas016 p- luc +. Induction of both transcripts was highly oxacillin-concentration dependent and transcript intensities increased over time becoming stronger after 60 min than after 20 min, correlating very well with concentration-specific induction curves from luciferase assays (Figure 2).

On day 5, 30 μL of the culture was transferred into LB broth cont

On day 5, 30 μL of the culture was transferred into LB broth containing tigecycline at 16× the MIC (passage 3), and the cultures

were again incubated at 37°C with shaking (220 rpm). This passaging was repeated on day 7 (passage 4). On day 9, aliquots (3 mL) of the cultures were mixed with 10% glycerol and stored at -80°C until use. Daily passaging in tigecycline-free LB was conducted for 30 days for both ATCC 17978 and the clinical strain. Construction of baeR deletion mutants and baeR reconstituted strains To assess the contribution of BaeR to the regulation of tigecycline resistance, baeR deletion mutants of P5091 datasheet A. baumannii ATCC 17978 were constructed as previously described [23] with some modifications. The suicide vector pEX18Tc [40] was first cloned with a 953-bp DNA fragment carrying a kanamycin resistance cassette, which was PCR-amplified from the pSFS2A plasmid [41], to generate pEX18Tc-kan r . DNA fragments carrying the upstream and downstream regions of the baeR gene, referred to as baeR-up and baeR-dw, were independently amplified by PCR using the primer pairs baeR-up-SalI-F and baeR-up-BamHI-R or baeR-dw-KpnI-F and baeR-dw-SacI-R (Table  1). The baeR-up fragment (1,119 bp) was digested with SalI and BamHI enzymes, whereas SB-715992 the baeR-dw fragment (1,120 bp) was digested with KpnI and SacI enzymes (Additional file 4: Figure S4A). Both enzyme-digested DNA fragments

were then independently cloned into the corresponding restriction sites of pEX18Tc-kan r , generating pEX18Tc-kan r -baeR-flanking. The resultant plasmid was then transformed into the E. coli S17-1 strain using the standard CaCl2/heat shock method [38]. Then, trans-conjugation was performed between E. coli S17-1 donor cells and A. baumannii ATCC

17978 recipient cells Tobramycin to transfer and integrate pEX18Tc-kan r -baeR-flanking into the chromosome of ATCC 17978 (Additional file 4: Figure S4B). By growing the ATCC 17978 conjugate cells on LB agar containing 10% sucrose, the cells were able to resolve the suicide plasmid pEX18Tc (Additional file 4: Figure S4C). Sucrose-resistant colonies were examined to verify that they had the kanamycin-resistant phenotype as a Natural Product Library clinical trial result of plasmid eviction. The absence of the baeR gene sequence in the genome was verified by PCR and RT-PCR and further confirmed by Southern blot hybridization. To reconstitute the baeR gene in the baeR-deleted mutants, a DNA fragment carrying the entire baeR gene sequence was generated by PCR using the genomic DNA of A. baumannii ATCC 17978 as the template. Briefly, a kanamycin resistance cassette was first amplified from the pC2HP vector [42] and cloned into the E. coli/Acinetobacter shuttle vector pWH1266 [43, 44] (Additional file 5: Figure S5A and S5B). Subsequently, the baeR DNA fragment was cloned into the XbaI/XhoI restriction sites (Additional file 5: Figure S5C).