The treatment of MGC803 and

HGC27 cells with SPARC siRNA

The treatment of MGC803 and

HGC27 cells with SPARC siRNA increased early apoptotic cells as well as late apoptotic cells, compared with negative control siRNA treatment (Figure 4A) as measured by the Annexin V assay. As expected, the decreased survival #PI3K activator randurls[1|1|,|CHEM1|]# of the cells transfected with SPARC siRNA was associated with increased rates of apoptosis by 91% in MGC803 and 92% in HGC27 cells (Figure 4B). These findings suggest that SPARC is involved in apoptosis to maintain cellular survival in some gastric cancer cells. Figure 4 SPARC knockdown results in induction of apoptosis in gastric cancer cell lines. For flow cytometric analysis, cells were harvested 96 h after transfection with SPARC siRNA or negative control siRNA, then stained with annexin V-FITC and propidium iodide (PI). the left half data represent data obtained from MGC 803 cells and the right ones represent data obtained from HGC 27 cells. The percentages SAHA HDAC in vivo of annexin V/PI(early apoptotic) and annexin V/PI(late apoptotic) cells is shown

in each panel. Values in bold indicate decreasing SPARC expression increased apoptosis by 65% in MGC803 and 92% in HGC27 compared with negative control siRNA. Apoptotic effect of SPARC siRNA transfected treatment in MGC 803 and HGC27 cells In an effort to elucidate the mechanism of SPARC siRNA induced apoptosis in MGC 803 cells and HGC27 cells, expression levels of apoptotic-regulation proteins such as Bcl-2, Bax and caspase-3 and PARP were evaluated. MGC 803 cells and HGC27 cells were transfected with SPARC siRNA. As shown in Figure 5, There were significant differences in the expressions of Bax

and Bcl-2 in MGC 803 cells and HGC27 cells in comparison with the negative control group (P < 0.05 and P < 0.01). In response to apoptotic stimuli, procaspase-3 is cleaved into a 20 kDa fragment, and the subsequent autocatalytic reaction leads to the formation of the active 17 kDa fragment. When Olopatadine the caspase-3 is activated, PARP is cleaved. Thus cleavage of PARP is used as an indicator of apoptosis. In order to obtain direct evidence showing the relationship of caspase activation and apoptosis, procaspase-3 cleavage and PARP were examined in MGC 803 cells and HGC27 cells after SPARC siRNA transfected. As shown in Figure 5, SPARC SiRNA induced the cleavage of 32 kDa procaspase-3 into its active 17 kDa form and cleavage of PARP appeared in MGC 803 cells and HGC27 cells. Figure 5 The expression of apoptosis proteins in MGC 803 and HGC27 cells after transfection with either control or SPARC siRNA. The cell lysates were separated on 10% SDS-PAGE gel, transferred to nitrocellulose membrane and probed with anti-PARP, anti-caspase-3, anti-Bcl-2, and anti-Bax. Protein contents were normalized by probing the same membrane with anti-β-actin.

sakazakii by API 20E analysis were not confirmed by the other met

sakazakii by API 20E analysis were not confirmed by the other methods used including chromogenic, PCR and the final 16S rRNA sequence analysis. There have been several comparative studies performed to determine the usefulness of biochemical test strips and chromogenic as a diagnostic

tool for the identification of Cronobacter spp. However, these studies have given conflicting results [48, 50, 51] highlighting the need for other methods of confirmation such as molecular and the DNA sequencing methods. PCR analysis using eight different sets of primers from six separate studies [3, 13, 44–47] was used to help ascertain the identity of all the presumptive isolates. Standard ATCC strains (51329 and 29544) were used as a positive selleck compound control. Although eight sets of PCR primers from six different studies each claiming high sensitivity and specificity for detection and confirmation of Cronobacter spp. were used to ascertain the identity of the isolates in this study, only 13 isolates in addition to the ATCC (51329) strain were positive with all the primers (Table 5). The other 16 isolates did not give the predicted PCR product with at least one set of primers although they were identified as

Cronobacter spp. by other biochemical and/or

chromogenic methods. When the isolates were selleck products tested with the PCR primer sets, DNA was not amplified in a high number of strains especially when tested with the zpx (94 bp product) and gluB detecting only 21/31 and 2/5 respectively. The other sets of primers Etofibrate where more reliable detecting 25/31, 26/30, 27/30, 28/31 for gluA, Saka, SI and BAM primer sets respectively while both OmpA and SG appeared to be most reliable among the tested primer sets detecting 28/30 isolates. These observations suggest that there may be some sequence variability in the genes of these strains of Cronobacter spp. that were not observed by the reporting authors [3, 13, 47]. In addition, it is noteworthy to mention that strains Jor149, Jor154, Jor175, Jor 52, Jor170, Jor184, Jor51, Jor153B and Jor151 gave conflicting α-glucosidase activity (on α-MUG or DFI) that did not correspond with PCR results for the presence of gluA. All these strains had expressed α-glucosidase activity on both α-MUG and DFI, but were negative by PCR for the presence of gluA. Because of these results we tested some of the gluA PCR negative strains with primers that targeted gluB by using primers, parameters and PCR reaction conditions described by Lehner et al [47].

(See Shevela et al 2012, for a review ) To me, this discovery, i

(See Shevela et al. 2012, for a review.) To me, this discovery, in addition to its well-known role in carbon fixation, of the unique role of bicarbonate/CO2 on the electron acceptor side of PS II, by Selleck PF2341066 Govindjee and coworkers (including Julian Eaton-Rye, author of this Tribute to Govindjee), is a major discovery, and we owe this

to Govindjee’s ingenuity, persistence, and drive unmatched in the history of photosynthesis research. I marvel at this research and I believe that he will go down in the history of photosynthesis research for this unique finding. John C. Munday, Jr. Professor of Natural Metabolism inhibitor Science and Mathematics Regent University, Virginia Beach, VA Tribute to Dr. Govindjee Graduate study is a special time of life. The opportunity to be immersed in research on a topic of choice, after years of preparatory schooling, is a time of deep intellectual reward. My choice to study photosynthesis was largely because of its biophysical complexity. The research methods enabled “seeing” events at the molecular level and gaining insights that could explain a process basic to all life on earth. Choosing a major professor was a major decision. On reflection about the options in the Photosynthesis Laboratory at the University of Illinois, I concluded that Dr. Govindjee would be a selleck compound wise mentor, a steady hand of guidance, and an

encourager. He had already proven his skill at research and his deep knowledge of the field of photosynthesis. Dr. Govindjee provided a list of problems where he believed that research would bear fruit. This suited my temperament and level at the time. After some investigation I developed a proposal, “owning” the content as my own; but later, looking back, I realized that he had foreseen my proposal exactly as one from his original list. Dr. Govindjee proved to be an exceptionally wise mentor. He was full of patience, manifested fully

a teaching spirit, and with painstaking care instilled a sense of excellence and quality in research. He demonstrated in his own research what he strove to teach. He was ever-present in the laboratory. Always with a cheerful smile, and obviously enjoying research, he made the laboratory a place where students, research associates, and visiting faculty wanted to be. He organized seminars in the lab and at his home. His wife Rajni had the gift of hospitality and we enjoyed her refreshments. Dichloromethane dehalogenase (She also made significant contributions of her own in photosynthesis research, and cared for their young family.) Along the way his comments and critique about my research were the stimulus for pushing forward, solving problems, and thinking creatively. I distinctly remember various points he made about how to do quality research. And in a final exam, he defended this student against a visitor’s mistaken claims about unpublished research from abroad, pointing out the core principle that what counts in scientific advance is peer-reviewed publication.

The results show that

there is a small dispersion in stop

The results show that

there is a small dispersion in stop band width for the different temperatures. Since the stop band width depends basically on the refractive index contrast that can be achieved within a cycle, it can be concluded that the anodization temperature has a small influence in the refractive index contrast. Figure 4 Evolution of central wavelength of the first stop band as function of pore-widening time for different anodization LY333531 in vitro temperatures. Table 1 Average stop band width and corresponding standard deviation as a function of the pore-widening time Pore-widening time (min) Average stop band width (nm) Stop band width standard deviation (nm) 0 103 22 9 68 14 18 50 5 27 46 6 The average and standard deviation have been obtained for all the samples with a given pore-widening time and different temperatures. The small value of the standard deviation selleck as compared with the average stop band width indicates that the temperature has a small influence in the refractive index contrast obtained with the cyclic voltage anodization. Conclusions In this work, we analyzed the influence of the anodization temperature and of the

number of applied voltage cycles on the check details photonic properties of NAA-based DBRs obtained by cyclic voltage anodization. In previous works, it was shown that DBR structures with stop bands can be obtained by the application of an anodization based in the repetition of voltage cycles between 20 and 50 V in 0.3 M oxalic acid. It was also shown that the application of a pore-widening step after anodization is crucial in order to obtain well-defined stop bands with low transmittance and high reflectance. In this work, these nanoporous structures have been obtained in the range of temperatures between 8°C and 11°C, for 50 and 150 applied voltage cycles and pore-widening times up to 27 min. The effect of these parameters

on the morphologic and photonic properties of the nanostructures has been studied by means of SEM and spectroscopic transmittance measurements. The results show that 50 applied voltage cycles are enough to produce stop bands and that increasing the number of cycles has two opposite effects: on one hand, RVX-208 an enhancement of the photonic stop bands is observed, in particular specially for the case of the as-produced samples, which is much better defined for samples with higher number of cycles. On the other hand, scattering losses are observed in the spectra caused by the irregular interfaces between cycles observed in the SEM images. Such losses increase with increasing number cycles and the corresponding interfaces. Increasing the anodization temperature produces a remarkable shift of the photonic stop band central wavelength, with a linear rate of 42.5 nm/°C. On the other hand, a change in anodization temperature does not influence noticeably the obtained stop band widths or the rate of the subsequent pore widening.

The dominant inheritance

The dominant inheritance INCB28060 purchase can be explained by hetero-oligomerization of wild-type/mutant AQP2 proteins and dominant-negative effect of mutant protein on wild-type protein [7]. In a female patient of family 5, a novel

heterozygous 1-nucleotide deletion mutation (750delG) was found. The patient’s sister and father were symptomatic. Her urine osmolality did not respond to vasopressin. This mutation causes a frame shift, with a new amino acids sequence starting from Val251 and ending at codon 334 in the C-terminal of AQP2. In Family 6, a 2-year-old girl was found to have a novel heterozygous 1-nucleotide deletion mutation (775delC) that causes frame shift with a new C-terminus starting at Leu259. The parents did not show NDI symptoms and did not carry the mutation, which indicated that the mutation occurred de novo. selleck The girl showed polyuria and polydipsia and NDI was diagnosed by water deprivation and vasopressin administration tests. These identified two deletion mutations cause frame shifts from Val251 and Leu259 and a new C-terminal tail ending at codon 334 (Table 4). We previously reported three small

deletion mutations in the C-terminus that cause similar frame shifts and show dominant inheritance [12] (Table 4). These frame-shift mutations share the loss of the last tail of the AQP2 protein, the site where PDZ proteins and ubiquitines interact, and the presence of extended C-terminal tails that contain missorting signals. As a result of these effects, these mutant AQP2 proteins making tetramers with wild-type proteins are incorrectly translocated to the basolateral membrane instead of the apical membrane [20, 30, 31]. This missorting is confirmed in knockin mice harboring a human C-terminal deletion mutation (c.763–772del) [32]. It is interesting that these deletion mutations are observed more often that missense mutations in Japanese patients, which is different from the frequencies in a total global

summary [3, 20]. We could not detect mutations in the two genes in seven families (9 %, Table 1). It is said that causative gene mutations cannot be found in oxyclozanide approximately 5 % of all congenital NDI patients [4]. Possibilities such as the presence of mutations in the promoter regions of the AVPR2 or AQP2 genes are a likely explanation [4]. Our mutational analysis does not usually cover the promoter regions; thus, this possibility remains to be examined. To date, no genes other than AVPR2 and AQP2 have been attributed to NDI. However, it is possible that mutations in the genes encoding signaling cascade molecules connecting these two key membrane proteins cause NDI. Progress in gene mutational analysis methods such as whole-exome sequencing will address this possibility. Acknowledgments We thank Mieko Goto for technical assistance and Dr. Daniel selleck chemicals llc Bichet for help in mutation analysis. We thank Drs. M. Asai, A Ashida, T. Aso, T. Hamajima, T. Hasegawa, M. Hayashi, D. Hirano, K. Ichida, E. Ihara, M. Iketani, T. Imanishi, H.

Prolonged retain period and unsuccessful attempts to remove recta

Prolonged retain period and unsuccessful attempts to remove rectal foreign body by the patient are two important factors that reduce transanal achievement. In our series the success rate of transanal extraction is up to 90 percent. It is related to advantages of operating room and short admission time of our patients. Objects larger than 10cm and those located in the proximal rectum are most likely to require surgical intervention SCH727965 datasheet in literature [10]. In our study proximal rectal localization of foreign bodies were more affected laparatomy requirement. When endoscopic or manual transanal extraction

fails or complications are present, laparatomy is necessary [17–19]. Different operative techniques can also be used for the selleck chemicals llc removal of the foreign body and treatment of the complications.

The decision to perform colostomy to primary rectal suturing only depends on various factors such as intraabdominal contamination, grade of rectal injury, extend of perianal trauma and chronicity of the case. On laparatomy milking the objects towards the rectum or anus enables the surgeon to extract FB without colotomy. Laparascopic asistance can be used in transanal extraction of proximally migrated FB. It allows for easy removal and direct visualization of the rectum to evaluate for injury. Laparascopic primary suturing, resection and diverting colostomy could be realised [20]. After difficult extraction procedure rectal and distal colonic mucosa is have to evaluate with rectosigmoidoscopy that determine extend of injury and exclude possible perforation. In postextraction rectosigmoidoscopy most of

the rectal injuries are in grade I and II as in our series [11]. Surgeons must be aware, in patients with chronicity, of serious anorectal injuries, possibility of perirectal sepcis, and important sequelae such as anal incontinence, fistulas and stenosis in the follow-up Venetoclax chemical structure period [21]. Our clinical algorithm was showed in Figure 3. This treatment guide was developed in the light of our clinical experiences. This sequential management system which we use in our clinical practice of colorectal FB, have helped transanal extraction rate to reach over 90%. Figure 3 Management algorithm of colorectal foreign body. All the patients should be evaluated psychologically. Patients presented with foreign bodies in the rectum should be asked for different sexual behaviours such as homosexuality. Most of the patients reject the abnormal sexual activities. Additionally, the patients should be examined for the use of alcohol and narcotic drugs. 50% of our cases reported high level intake of alcoholic beverages before rectal FB introduction. Conclusions Retained rectal foreign bodies are usually related to improper anal sexual behaviour. Patients should be evaluated with a AZD2014 mouse careful physical and rectal examination and plain radiograms for correct diagnosis and localization.

DhMotC, an inhibitor of tumor cell invasion [19], also inhibits s

DhMotC, an inhibitor of tumor cell invasion [19], also inhibits sphingolipid biosynthesis and genes of the sphingolipid biosynthesis pathway show dhMotC-induced haploinsufficiency [6]. Interestingly, suloctidil was recently shown to inhibit acid sphingomyelinase, a lysosomal enzyme catalyzing the degradation of sphingolipids and generating ceramide, which can be metabolised selleck into sphingosine [20]. These results show that the majority of chemicals that inhibit yeast growth do not require functional mitochondria to exert their effect but that 3 Temsirolimus ic50 compounds affecting sphingolipid metabolism all

require functional mitochondria to inhibit growth. We then further explored the requirement for functional mitochondria in the mechanism of action of 1 of these chemicals, dhMotC, using genetic screens and biological assays. Prolonged exposure to dhMotC kills yeast Growth-inhibitory compounds can reversibly prevent cell proliferation (cytostatic activity) or induce death (cytocidal activity). To distinguish between these outcomes, cells this website were exposed to inhibitory concentrations of dhMotC in liquid culture for different times and equal cell numbers were plated onto drug-free agar plates for 2 days at 30°C. Cells exposed to dhMotC for 1, 3

or 6 hours all formed the same number of colonies as untreated cultures. However, exposure to dhMotC for 20 h resulted in no colony growth (Figure 2). These Exoribonuclease observations show that dhMotC exposure initially triggers a reversible growth arrest that eventually leads to cell death after longer exposure. Figure 2 Viability test of FY1679-28C/TDEC yeast strain exposed to dhMotC. Short exposure times result in reversible growth inhibition. There is no observable

growth after long drug exposure. DhMotC sensitivity suppressor screen reveals genes involved in mitochondrial function Screens using increased gene dosage, relying on the assumption that increased levels of a protein targeted by a drug increase resistance to the drug, can help identify specific drug targets [9]. Drug sensitivity suppressor screens can be carried out with pooled genomic library transformants, leading to enrichment of resistant strains and depletion of hypersensitive strains, relative to untreated pools. Analysis of relative strain sensitivity is performed by hybridization of labelled DNA to an oligonucleotide tag array [21]. A pooled collection of yeast strains expressing genes from a random genomic DNA fragment library was exposed to dhMotC and resistant strains were identified. Similar experiments were carried out using 3 close structural analogues (Figure 3). Syntenic regions (i.e.

Since Lüneberg et al analyzed the strain RC1 which had 30 ORFs t

Since Lüneberg et al. analyzed the strain RC1 which had 30 ORFs the numbering of ORFs in other L. pneumophila Sg1 GS-4997 research buy strains with deviating ORF numbers is not continual [21]. The genes iraA (ORF 29) and iraB (ORF 30) were not taken into account as part of the LPS-biosynthesis locus. Both formed a small 2-gene operon responsible for iron assimilation, infection and virulence [60]. The putative coding regions were compared to already known LPS-biosynthesis ORFs of published L pneumophila strains using the SeqMan program. The LPS-biosynthesis clusters of the strains were deposited in the EMBL database under the number [EMBL: HE980447] for strain Camperdown 1 (mAb-subgroup GSK2399872A cost Camperdown), [EMBL: HE980446] for strain

Heysham 1 (mAb-subgroup Heysham), [EMBL: HE980445] for strain Uppsala 3 (mAb-subgroup Knoxville), [EMBL: HF678227] for strain Görlitz 6543 (mAb-subgroup

Bellingham) and [EMBL: HF545881] for strain L10/23 (mAb-subgroup Knoxville) (Table  2). Sequence homologies of single ORFs were calculated based on multiple alignments using BioNumerics 6.0 (Applied Maths NV, Belgium) Pexidartinib concentration and BLASTP [57]. Cluster analysis was performed using the UPGMA method of the BioNumerics 6.0 software package. The sequences of other LPS-biosynthesis loci were obtained from complete genomes of the following strains: Paris (mAb-subgroup Philadelphia) (GenBank: NC_006368.1), Lens (mAb-subgroup Benidorm) (GenBank: NC_006369.1), Philadelphia 1 (mAb-subgroup Philadelphia) (GenBank: NC_002942.5), Alcoy 2300/99 (mAb-subgroup Knoxville) (GenBank: NC_014125.1), Corby (mAb-subgroup Knoxville) (GenBank: NC_009494.2), Lorraine (mAb-subgroup Allentown) (EMBL: FQ958210), HL 06041035 (mAb-subgroup Bellingham) (EMBL: FQ958211), RC1 (mAb-subgroup OLDA) (EMBL: AJ277755) and 130b (mAb-subgroup Benidorm) (EMBL: FR687201.1) (Table  2) [21, 28, Selleckchem Fludarabine 29, 31–34]. Since the genome of 130b is a draft version we closed a sequencing gap in scaffold

4 (position 918107 to 918206) using PCR and sequencing. Availability of supporting data The data sets supporting the results of this article are available in the LabArchives repository, DOI:http://​dx.​doi.​org/​http://​dx.​doi.​org/​10.​6070/​H4WM1BBQ. It includes a list of all primers used for ORF amplification and sequence generation (Additional file 2: Table S1), a spreadsheet containing detailed information about the LPS-biosynthesis locus such as ORF identifier, ORF size and putative size of the translated ORF product (Additional file 1: Table S2) as well as the % GC content of the ORFs of the Sg1-specific region (Additional file 1: Table S3). Acknowledgement We thank Sigrid Gäbler, Kerstin Lück and Ines Wolf for technical assistance. This work was partly supported by the Robert Koch-Institute grant 1369–364 to CL. Dedicated to the memory of Dr. Jürgen Helbig, Dresden, Germany. Electronic supplementary material Additional file 2: Table S1: This document summarizes all primers used for amplification of LPS-biosynthesis ORFs and sequence generation.

Such a “”proof of concept”" has already been provided by mimics o

Such a “”proof of concept”" has already been provided by mimics of the acylated homoserine lactone signalling molecules, such as synthetic derivatives of natural furanones, which are able to inhibit in vivo biofilm development of Pseudomonas aeruginosa [13], and when covalently bound to surfaces BKM120 also those of Staphylococcus epidermidis [14]. Moreover the quorum-sensing RNA III inhibiting peptide was shown to inhibit in vivo biofilm formation of Staphylococcus aureus [15]. However none of the quorum sensing blockers tested in animal models so far was suitable for human use. Our project is aimed at finding new inhibitors

of biofilm formation of the Gram-positive facultative anaerobic bacterium S. mutans. Among more than 500 bacterial species found in dental plaque [16–18]S. mutans is considered to be the principal pathogen causing human dental caries [19]. While metabolising dietary carbohydrates, S. mutans rapidly produces acid endproducts lowering the pH to approximately pH 3.5 resulting in demineralisation

of the dental enamel and caries formation [20–22]. Moreover S. mutans can be a cause of subacute infective endocarditis [19, 23]. We focused in our search for biofilm inhibitors on our ATM/ATR inhibitor clinical trial collection of secondary metabolites from myxobacteria. They are ubiquitous Gram-negative soil bacteria characterized by their ability to glide in swarms, as well as by their complex life cycle that upon starvation culminates in the formation of multicellular fruiting bodies, containing dormant myxospores [24]. The complexity of their social behaviour and morphogenetic potential is reflected in their large genomes Chlormezanone (9-13 Mbp), some of which, e.g. Sorangium cellulosum So ce56 [25] have been sequenced. The overrepresentation of genes involved in secondary metabolism also explains the capability of myxobacteria to produce such a high number of potentially useful low molecular weight compounds. Over the past 25 years, more than 100 secondary metabolites with more than 450 structural variants have been isolated from myxobacteria at the HZI. Most of these compounds turned out to be new and show novel

unrelated structures as well as different biological activities with interesting mechanisms of action [24, 26, 27]. About a third of these myxobacterial compounds however did not show any biological or biochemical effect in our test battery, which until now predominantly focused at killing or inhibiting microbial growth. It should be pointed out that especially these compounds are of interest in exploring new targets. Here we DNA Damage inhibitor report on the efficacy of the secondary metabolite carolacton produced by S. cellulosum to biofilms of the cariogenic bacterium S. mutans. Figure 1 shows the structure of carolacton [28, 29], the elucidation of which as well as its production and isolation have been reported elsewhere [30]. Carolacton induced damage of S.

This allows activation of pigA, carA and rap transcription Rap,

This allows activation of pigA, carA and rap transcription. Rap, which is activated via QS and the phosphate response, can then further activate carA and pigA transcription. This results in upregulation of both Car and Pig production via multiple pathways. Figure learn more 9 The proposed mechanism by P i limitation can upregulate secondary metabolism in Serratia 39006. In response to Pi limitation (or pstS mutation), PhoR activates PhoB by phosphorylation. Active PhoB can then activate click here transcription of smaI, pigA and rap (indicated using

solid arrows). Upregulation of smaI results in activation of the QS regulated genes (pigA, carA and rap), via AHL mediated SmaR derepression (indicated using dashed arrows). Rap then further activates carA and pigA expression (indicated using solid arrows). This results in upregulation of Pig and Car production. Multiple studies have linked Pi limitation to enhanced secondary metabolite production [17]. However,

the complex molecular mechanisms underlying phosphate-mediated regulation have proven difficult to elucidate. Extensive studies in Streptomyces species have shown that PhoPR (PhoBR) activates secondary metabolism in response to Pi limitation, including biosynthesis of undecylprodigiosin, a tripyrrole closely related to Pig [40, 41]. However, in Streptomyces, inactivation of PhoP or deletion of phoPR also activates secondary metabolism [41]. In contrast, deletion of phoB and/or phoR in Serratia 39006 had no impact on secondary metabolism, demonstrating clear differences between the regulatory Fosbretabulin cell line mechanisms employed by these distantly related bacteria. Although

the requirement for increased secondary metabolism under conditions of phosphate limitation is unclear, it has been proposed that enhanced secondary metabolism allows the production of compounds which may, for example, directly antagonise other microorganisms or act as signalling molecules, thereby providing producing organisms with a competitive advantage under nutrient deprived conditions [40, 42, 43]. Conclusion In conclusion, we have established that via the global transcriptional regulators PhoB, SmaR Protein kinase N1 and Rap, multiple inter-linked pathways are acting to upregulate secondary metabolism in Serratia 39006 under conditions of Pi limitation, highlighting the importance of Pig and Car production under these conditions. Methods Bacterial strains, plasmids, phage and culture conditions Bacterial strains and plasmids are listed in Additional File 1[44–49]. Serratia sp. ATCC 39006 derivative strains were grown at 30°C and E. coli strains were grown at 37°C in Luria broth (LB; 5 g l-1 yeast extract, 10 g l-1 bacto tryptone and 5 g l-1 NaCl), minimal media (0.1% w/v (NH4)2SO4, 0.41 mM MgSO4, 0.2% w/v glucose, 40 mM K2HPO4, 14.7 mM KH2PO4, pH 6.9–7.1) or in phosphate limiting (PL) media (0.1% w/v (NH4)2SO4, 0.41 mM MgSO4, 0.2% w/v glucose, 0.1 M HEPES, pH 6.9–7.