1, lane 4 and lane 5) And both bands (c 39 and 465 kDa) were p

1, lane 4 and lane 5). And both bands (c. 39 and 46.5 kDa) were present in the supernatants of induced cultures of BL (Bi; Fig. 1, lane 3). It indicated that both Plu1961 and Plu1962 were expressed as soluble proteins in BL21 (DE3), no matter whether they were separately expressed or co-expressed. When Plu1961/Plu1962 was applied by mixing with diet, neither mortality nor growth inhibition of both H. armigera and S. exigua larvae was observed within the tested amounts

(15–150 μL) of BL (Bi) lysate. However, injection of 10 μL of supernatant of BL (Bi) lysate resulted in around 42% mortality of S. exigua fourth-instar larvae after 24 h. And the mortality rate rose Nintedanib with the increase in BL (Bi) lysate volume. When 100 μL of concentrated

supernatant of BL (Bi) lysate was injected into S. exigua fourth-instar larvae, 97% mortality rate was observed after 24 h (Fig. 2b). When compared with the control group (supernatant of BL21 (DE3) lysate and heat-inactivated supernatant of BL (Bi) lysate), the supernatant of BL (Bi) lysate caused extensive blackening of larvae (Fig. 2a). Blackening of S. exigua larvae suggested that injection of BL (Bi) lysate NVP-LDE225 resulted in the activation of phenoloxidase which was responsible for the synthesis of melanin, a key component in arthropod immunity and wound healing (Li et al., 2008). It demonstrated that Plu1961/Plu1962 had injectable toxicity against tested insect larvae, but no oral toxicity. MTT assay was performed

against insect midgut CF-203 cells to investigate the cytotoxicity of Plu1961/Plu1962. Neither component of binary toxin could affect the growth of CF-203 cells even after 4 days of incubation. In contrast, the mixture of Plu1961/Plu1962 caused a loss of cell viability after 24 h of incubation within the tested concentrations (0.2–1.6 μmol L−1). 0.2 μmol L−1 of binary toxin mixture resulted in 55% loss of cell viability. More than 90% of cells lost viability after treatment with 1.6 μmol L−1 of binary toxin (Fig. 2c). When compared with control cells (Fig. 3a), CF-203 cells treated with the mixture of Plu1961/Plu1962 showed Unoprostone marked swelling, formation of surface blisters, followed by membrane lysis and dispersal of the cytoplasmic organelles and swollen nuclear contents into the surrounding medium (Fig. 3d). In contrast, individual application of Plu1961 or Plu1962 alone had no morphological effect on CF-203 cells (Fig. 3b and c). Morphological changes in CF-203 cells exposed to Plu1961/Plu1962 mixture were further investigated by confocal microscope. The control cells and cells treated by Plu1961 alone displayed strong green fluorescence (microtubules) around the nuclei (strong blue fluorescence), the mitochondria (red fluorescence) appeared to be almost evenly distributed in the cytoplasm (Fig. 4a and b). In contrast, cells treated with the mixture of Plu1961/Plu1962 lost virtually all green and red fluorescence and exhibited only blue fluorescence (Fig. 4d).

Polyketides can

Polyketides can see more also be extracted from different algae, dinoflagellates and plants (Hopwood & Sherman, 1990; Austin & Noel, 2003), for which those compounds apparently serve as defensive substances against natural enemies (Manojlovic et al., 2000; Choi et al., 2004).

The probably most diverse group of polyketide producers are marine organisms like sponges, tunicates, and bryozoans. Such animals are a source of natural compounds with strong cytotoxic properties that are extremely interesting from a medical point of view (Piel, 2004, 2006; Moore, 2005, 2006; Piel et al., 2005). These substances belong to the pederin family, which currently comprises 36 members from eight different invertebrate animal genera (Narquizian & Kocienski, 2000; Simpson et al., 2000; Vuong et al., 2001; Paul et al., 2002). PFT�� concentration Polyketides are produced by hitherto uncultured, highly adapted bacterial endosymbionts. Cultivation of the pederin-producing bacterial endosymbionts of female Paederus rove beetles is not yet possible, and although chemical synthesis of pederin has been successfully reported by some groups

(Matsuda et al., 1988; Kocienski et al., 2000; Takemura et al., 2002; Jewett & Rawal, 2007), its low availability represents a serious impediment to drug development (Munro et al., 1999; Piel, 2002, 2004, 2006). Thus, tools are required for custom tailoring growth media for the enrichment and isolation of Paederus endosymbionts. Kellner (1999, 2001a, b, 2002a) demonstrated that a Pseudomonas-like endosymbiont is associated with the transfer of pederin production capabilities to the female progeny of Paederus beetles via endosymbiont-harbouring eggs. Analysis of metagenomic DNA from Paederus fuscipes beetles revealed the existence of a mixed modular polyketide synthase (pks)-gene cluster that is responsible for pederin biosynthesis (Piel, 2002). Specific PCR primers were designed from conserved regions of single cluster modules and utilized for the amplification of pks-gene fragments from endosymbionts in beetle or egg specimens (Piel, 2002).

However, direct evidence for the localization of Pseudomonas-like endosymbionts on eggs is lacking, and it is still unresolved, where such endosymbionts are located within Paederus beetles. FISH is an appropriate tool Clomifene for the in situ localization of specific phylogenetically defined groups of bacteria (Amann et al., 2001; Amann & Fuchs, 2008). Thus, the objectives were to (1) design and evaluate a specific 16S rRNA gene-targeted oligonucleotide probe for Pseudomonas-like Paederus riparius endosymbiont detection; (2) localize endosymbionts within serial egg thin-sections by FISH; and (3) determine where within the host symbionts are transferred to eggs by surface comparison of different egg stadiums using electron microscopy and pks-targeted PCR.

36, P < 001, ηρ2 = 064), with participants responding more quic

36, P < 0.01, ηρ2 = 0.64), with participants responding more quickly to the primary than to the secondary modality (Fig. 3A). This modality prevalence effect was present in both vision (t14 = −2.59, P = 0.02) and touch (t13 = −7.45, P< 0.01) individually but was stronger

if touch was the primary modality, as revealed by the interaction between modality prevalence and primary modality (F1,27 = 9,21, P < 0.01, ηρ2 = 0.25). The main effect of onset time was significant as well (F1,27 = 4,79, P = 0.037, ηρ2 = 0.15), showing that responses to targets appearing at the late time point were more efficient than responses to targets presented at the early time point. As in the RT analysis, for IE scores the critical interaction between modality prevalence and expected time was significant (F1,27 = 16,27, P < 0.01, ηρ2 = 0.38), attesting to Epigenetics inhibitor the decoupling

between temporal expectations across modalities (Fig. 3B). For the primary modality, participants responded more efficiently if the target appeared at the expected time point (t28 = −3.948, P< 0.01) whereas for the secondary modality a nearly significant trend in the opposite direction, that is, more efficient responses at the unexpected onset times, was observed (t28 = 1.87, P = 0.07). Investigating this pattern for each modality separately we found that, for touch as primary modality, BIBW2992 participants responded more efficiently if the stimulus was presented at the expected (vs. unexpected) time point (t13 = −3.125, P < 0.01). For secondary tactile targets a marginal trend towards more efficient responses at the unexpected time point was observable (t13 = 1.833, P = 0.090). For vision, participants responded more efficiently towards primary visual targets at expected vs. unexpected time points check details (t14 = −3.17, P < 0.01), whilst no response differences

between expected an unexpected onset times for visual secondary targets were observed (t14 = 0.71, P = 0.49). Neither the three-way interaction between modality prevalence, expected time and onset, nor any other effect or interaction, reached significance. In conclusion, the IE results were consistent with the RT results, confirming that there was no trade-off between accuracy and RTs in participants’ performance. For the upcoming discussion, we will therefore mainly focus on the RT results. According to the present results, temporal attention is not subject to a strong cross-modal synergy and, instead, it can be deployed in a relatively independent manner for separate sensory modalities. Considering only the primary modality, our results conform to the well-known effect of attention to time in single modality studies (Coull & Nobre, 1998; Miniussi et al., 1999; Griffin et al., 2001; Correa et al., 2004), meaning that performance improves for targets at expected, compared to unexpected, time points. It is noteworthy that, unlike Correa & Nobre (2008), we did not find a clear benefit in terms of accuracy.

1 mm, and a few beads of 2 mm diameter) three times for 45 s at 6

1 mm, and a few beads of 2 mm diameter) three times for 45 s at 6.5 m s−1. Samples were centrifuged, filtered (0.22 μm), diluted 1 : 20 with 70% MeOH, and infused at 120 μL h−1. ICR-FT/MS was externally calibrated on clusters of arginine (10 ppm in 70% MeOH). A time domain transient of 2 megawords was used, and 300 scans were accumulated for one spectrum. Spectra were internally calibrated

with an error of ≤ 0.1 ppm, exported with a signal-to-noise ratio of 3, and aligned within a 1 ppm window. Putative metabolites were annotated using MassTRIX (Wägele et al., 2012). Only masses found in all replicates were considered and analyzed in Genedata Expressionist for MS 7.6 (Genedata, Martinsried). Promoters were searched by bprom (Softberry Inc., New York) and terminators by webgester db (Mitra et al., 2011). Microarray PCI-32765 supplier data were accessed from the Gene Expression Database (genexpdb, http://genexpdb.ou.edu/index.php, see Table 1). Sequences were searched with Veliparib solubility dmso blastp or tblastn (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi, default parameters) using YaaW (Z0011) as query (Table S2). The evolutionary history of all species was inferred using the software package mega5 with a concatemer of 16s rRNA gene, atpD, adk, gyrB, purA, and recA by Minimum Evolution using p-distance. The bootstrap consensus was inferred from 1000 replicates

(Tamura et al., 2011). For some strains, not all sequences were available, and thus close relatives were used as surrogate, for example, some genes of Comamonas testosteroni CNB-2 were used for the yaaW-bearing strain ATCC 11996. The presence of htgA was detected using pairwise blastp

alignments with htgA (Z0012) as query (starting from the first GTG). htgA/yaaW sequences were examined for their nonsynonymous over synonymous rate ratio ω as described (Sabath et al., 2008; Sabath & Graur, 2010) including correction for multiple testing according to Benjamini & Hochberg (1995), after omitting alignment gaps (Tamura et al., 2011). 5′-RACE determined the major 5′-end of the + 1 transcription start of htgA to be 135 bp upstream. However, minor sites might be present, since Missiakas Ergoloid et al. (1993) found a site 82 bp upstream; others were predicted 98 (BProm) or 114 bp (Tutukina et al., 2007) upstream of the CTG-start codon of htgA. The upstream region of htgA was successfully tested for promoter activity using a promoterless gfp reporter. No terminator could be detected directly downstream of htgA but was detected downstream of dnaK (Fig. 1). Recently, strand-specific transcriptome sequencing showed that htgA is transcribed, albeit weakly, at some nonlaboratory growth conditions only (R. Landstorfer, S. Simon, S. Schober, D. Keim, S. Scherer & K. Neuhaus, unpublished data). The 5′-RACE major transcription start site of yaaW is 32 bp upstream of yaaI, but a minor site, 107 bp upstream of yaaW, was also detected.

Nevertheless, deeper into the gingival connective tissue, gingipa

Nevertheless, deeper into the gingival connective tissue, gingipain concentrations become gradually lower and stimulate, rather than inhibit, inflammation. This may in

turn induce connective tissue and bone destruction, which are the hallmarks of periodontitis. It is evident that P. gingivalis has developed mechanisms to invade and persist into the host, by astutely Ku-0059436 purchase adapting to its local niche. Its paradoxically opposing (stimulatory vs. inhibiting) effects on innate immune and inflammatory responses aim to subvert host defence mechanisms, in order to facilitate its survival in the tissues (Hajishengallis, 2009; Hajishengallis & Lambris, 2011). The net effect of this deregulated equilibrium is likely to determine if site-specific disease progresses beyond or remains at stationary phase. Whether inflammation is beneficial for P. gingivalis may depend on the stage of its establishment in the host (Hajishengallis, 2009; Pathirana et al., 2010). At early stages, suppression of inflammation and evasion of host recognition would aid P. gingivalis in colonizing, invading and establishing at the targeted site. At later stages, once P. gingivalis is well established, inducing inflammation may facilitate its increased demands in nutrients. Alternatively, P. gingivalis may

induce a ‘nonproductive inflammation’, one that fails to eliminate it, yet is sufficient to induce mediators of tissue destruction (Hajishengallis, 2009). Finally, as periodontitis is of polymicrobial

nature, it is reasonable to consider the role of different bacterial species within the context of (subgingival) Resveratrol biofilm http://www.selleckchem.com/products/lee011.html communities. Hence, P. gingivalis is likely to function in concerted action with other species, to their mutual benefit. For instance, complement manipulation by P. gingivalis may denote a coevolution strategy to support other species present in the biofilm, which may reciprocally provide further colonization opportunities and nutrient availability to P. gingivalis. Subsequent changes in the local microenvironment can differentially regulate expression of its virulence factors, and hence the proinflammatory or anti-inflammatory potentials of P. gingivalis. This is strongly indicated by recent evidence demonstrating that even at low abundance, this species qualitatively and quantitatively affects the composition of the oral commensal microbiota, which are in turn required for P. gingivalis-induced inflammatory bone loss (Hajishengallis et al., 2011). For these reasons, P. gingivalis is now considered a ‘keystone’ species in subgingival biofilms (Honda, 2011). This work was supported by the Institute of Oral Biology, Center of Dental Medicine, University of Zürich. “
“d-Xylulokinase catalyzes the phosphorylation of d-xylulose in the final step of the pentose catabolic pathway to form d-xylulose-5-phosphate.

This is a normal tendency of biofilm-forming bacteria such as myc

This is a normal tendency of biofilm-forming bacteria such as mycobacteria. On treatment with alcohol, most of the bacteria lose their cell shape and morphology and as a consequence remain unattached and occur mostly as single cells. Thus, the growth inhibitory activity of decanol can be attributed partly, if not exclusively, to its ability to damage the cellular envelope. Perhaps VX-809 datasheet this damage is a result of the well-known event of accumulation of alkanols in the membrane thus affecting the general membrane functions. Biofilm formation in many cases is important for bacterial virulence and survival (Parsek & Singh, 2003). So a successful attenuation of biofilm formation can be of wide interest for

the management of disease progression and elimination of the pathogen. An intact cellular envelope and its hydrophobicity helps in cell to cell adhesion and thus promotes biofilm formation in microorganisms such as mycobacteria. Thus, any damage to the cell envelope may hinder its ability to adhere to each other and subsequently inhibits biofilm formation. In this context we have assessed the Selleckchem DAPT ability of long-chain fatty alcohols in biofilm formation

by performing CV assay and acridine orange staining of the biofilm. Interestingly, our result showed that decanol concentrations of 0.1 and 0.2 mM, far lower than its MIC (0.4 mM), were able to attenuate biofilm formation (Fig. 3a). Furthermore, the quantitative CV assay also revealed that 9-decene-1-ol concentrations of 0.05 and 0.1 mM, again lower than its MIC (0.2 mM), were able to attenuate biofilm formation considerably (Fig. 3b). The same concentration of the alcohols tested had

no effect on planktonic growth as measured by OD600 nm. These results clearly suggest that a sublethal dose of both 1-decanol and 9-decene-1-ol is able to attenuate biofilm formation in vitro. This inhibition may result from the ability of these agents to damage the cellular envelope and thus in turn perturb the cell to cell adhesion, which Mirabegron is a key factor in biofilm formation. Exploring new agents that can attenuate biofilm formation and insight into the mechanism involved may shed light into therapeutic strategies for infections with microbes such as mycobacteria whose pathogenic potential strongly depends on successful biofilm formation within the host. Surface active agents such as surfactants and other membrane-damaging compounds are drawing significant attention in the field of antimicrobial chemotherapy. Drugs such as daptomycin clofazimine derivatives that are known to disrupt membrane integrity are already being used either clinically or are at the final stage of drug development (Adams et al., 1999; Pogliano et al., 2012). Membrane active agents generally have multiple target sites and diverse modes of action against the organism, reducing the chance of mutation at the target site (Andries et al., 2005; Koul et al., 2008).

In a symbiotic host system, collagen degradation could benefit th

In a symbiotic host system, collagen degradation could benefit the bacteria, but would be harmful for the eukaryotic host. Using a polyphasic approach, we investigated the presence of

collagenolytic activity in the bacterial community hosted by the marine sponge Cymbastela concentrica. Functional screening for collagenase activity using metagenomic library clones (227 Mbp) and cultured isolates of sponge’s bacterial community, as well as bioinformatic analysis of metagenomic shotgun-sequencing data (106 679 predicted genes) were used. The results show that the abundant members of the bacterial community contain very few genes encoding for collagenolytic enzymes, while some low-abundance see more sponge isolates possess collagenolytic activities. These findings indicate that collagen is not a preferred nutrient source for the majority of the members of the bacterial community associated with healthy C. concentrica, and that some low-abundance bacteria have collagenase activities that have the potential to harm the sponge through tissue degradation. Collagen is the major component of extracellular matrices of all metazoan life and represents an important protein conferring integrity and the physical form of eukaryotic organisms

(Harrington, 1996; Exposito et al., 2008). Sponges are among the oldest Metazoa and often contain collagen, which is either dispersed as this website thin fibrils or organized as bundles, termed spongin, in the intercellular matrix (Simpson, 1984; Brusca & Brusca, 1990). The expression of collagen is known to be essential for the development and structural integrity of sponges (Garrone et al., 1975; Shimizu & Yochizato, 1993; Krasko et al., 2000). Sponges harbour specific bacterial communities in different

cellular compartments, often for an extended period of time, and hence close associations between the microorganisms and the sponge host have been established (Taylor et al., 2007). Collagen is an essential and abundant part of the internal mesohyl structure of most sponges Oxaprozin (and in particular the Demospongia), where many microorganisms reside. As a rich source of nitrogen and carbon, collagen could provide nutrients for the sponge-associated microorganisms, and this may potentially have implications for the structural integrity of the host. A few cases of sponge diseases have been attributed to the presence of bacterial pathogens (Gaino & Pronzato, 1989; Webster et al., 2002; Mukherjee et al., 2009) and collagenolytic enzymes have been speculated to lead to tissue necrosis in sponges. Generally, bacterial collagenases, including the well-characterized enzymes from Clostridium sp. (Matsushita et al., 1994) and Vibrio sp. (Yu & Lee, 1999; Vaitkevicius et al., 2008), have been linked to pathogenicity and are regarded as virulence factors in human disease.

, 2011c) All these species have been involved in clinical infect

, 2011c). All these species have been involved in clinical infections and may bear several virulence genes, like those encoding Shiga toxins (stx1 and stx2), the type III secretion system

(TTSS) (ascF-G, Forskolin order ascV), flagella (fla) as well as several toxins (ast, act, alt, aexT) among others (Chacón et al., 2004; Aguilera-Arreola et al., 2005; Fehr et al., 2006; Chopra et al., 2009; Alperi & Figueras, 2010; Senderovich et al., 2012). Two new clinical species, Aeromonas taiwanensis and Aeromonas sanarellii, recovered from wound infections of hospitalized patients in Taiwan (although phenotypically misidentified as A. hydrophila and A. caviae, respectively) were recently discovered by sequencing the rpoD gene (Alperi et al., 2010a). Both species were described on the basis of a single strain (their type), and these were the only known Z-VAD-FMK purchase strains until two recent publications reported four isolates of A. sanarellii and one of A. taiwanensis in waste water in Portugal (Figueira et al., 2011), and a strain of A. taiwanensis recovered from the faeces of a female patient with diarrhoea in Israel (Senderovich et al., 2012). Isolates of the species A. sanarellii and A. taiwanensis were recorded in the course of a new study

that investigated the prevalence of Aeromonas populations in chironomid egg masses by culture and by real-time PCR methods (unpublished data). Considering the clinical relevance of these species, the Thalidomide present study describes for the first time the virulence genotypes and antibiotic susceptibility of these new species recovered from this new habitat and provides key phenotypic traits for their identification. Sampling for Aeromonas spp. populations was carried out in chironomid egg masses found in a waste stabilization pond in northern Israel between April and September 2009 using previously described procedures (Senderovich et al., 2008). Crushed egg masses were spread on M-Aeromonas agar (Biolife, Italy) for 24 h at 30 °C. Yellow, smooth, rounded colonies that were suspected Aeromonas species were then subcultured on Luria broth (LB) agar (Himedia, India). For each sample, about 15 Aeromonas isolates were identified to the species level using rpoD gene sequencing,

according to Soler et al. (2004). To observe the existence or not of clonally related isolates, DNA typing was carried out with the enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) technique using the primers and conditions described by Versalovic et al. (1991). Patterns with one or more different bands were considered different genotypes. In all A. sanarellii and A. taiwanensis strains, 24 phenotypic tests (Supporting information, Tables S1 and S2) were evaluated using conventional methods at 30 °C for 24 h up to 7 days as previously described (Abbott et al., 2003; Alperi et al., 2010b) with the exception of utilization of citrate, which was determined using the Simmons’s method (Cowan & Steel, 1993), and nitrate reduction (MacFaddin, 1976).

Positive for oxidase, catalase, nitrate reduction, and hydrolysis

Positive for oxidase, catalase, nitrate reduction, and hydrolysis of esculin, gelatin, Tween 40, and Tween 80. Negative for indole production, acid production from glucose (fermentation), arginine dihydrolase, urease, β-galactosidase, and hydrolysis of starch. In API ZYM system, cells are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, and naphthol-AS-BI-phosphohydrolase, but negative for all other enzymes. In API 50 CH tests, acid is produced oxidatively from d-glucose, esculin, and 5-ketogluconate (weakly positive). None of the other

carbon sources were oxidized in the API 50 CH tests. The cells utilize the following compounds as a carbon and energy source by conventional methods: d-glucose, trehalose, acetate, caprate, caproate, propionate, pyruvate, and l-alanine, but not the following compounds: N-acetyl-glucosamine, Selleck GSK126 l-arabinose, d-arabitol, d-fructose, d-galactose, d-mannitol, d-mannose, l-rhamnose, d-sorbitol, d-xylose, lactose, cellobiose, maltose, sucrose, glycerol, myo-inositol, adipate, citrate, formate, gluconate, lactate, dl-malate, succinate, l-asparagine, l-asparate, l-glutamate, l-histidine, l-leucine, l-serine,

DAPT cost l-threonine, l-phenylalanine, l-proline, benzoate, and 4-hydroxybenzoate. The DNA G + C content is 48.6 mol%. The type strain, KU41ET (=JCM 17778T), was isolated from seawater obtained from the coastal region of Ishigaki Island, Japan. We are grateful to Professor Hans Fluorouracil solubility dmso G. Trüper for his help with the genus and species name. This work was supported by ‘Strategic Project to Support

the Formation of Research Bases at Private Universities’: Matching Fund Subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2008–2012. “
“Bioinformatic and electron microscopy analyses indicate that the composition of the B. megaterium QM B1551 spore coat is likely to differ substantially from other Bacillus species. We report here on the identification and characterisation of novel B. megaterium proteins that appear to be abundant in the spore coat. All three proteins, encoded by loci BMQ_0737, BMQ_3035 and BMQ_4051, were identified by proteomic analysis of alkaline detergent extracts from mature spores. Putative spore coat proteins were characterised by transcriptional, reporter-fusion and mutagenesis analyses supported by fluorescence and transmission electron microscopy. These analyses revealed that BMQ_0737 is a novel morphogenetic protein that is required for the correct assembly of the B. megaterium outer spore coat and exosporium, both of which are structurally compromised or missing in BMQ_0737 null mutant spores. “
“Upon infection of the gastric epithelial cells, the Helicobacter pylori cytotoxin-associated gene A (CagA) virulence protein is injected into the epithelial cells via the type IV secretion system (TFSS), which is dependent on cholesterol.

coli’ pathway-based proteolytic system in E coli was performed u

coli’ pathway-based proteolytic system in E. coli was performed using homologous recombination technology. Using the strain constructed in ‘Replacement of the tnaA gene with the trpR gene’, the DNA fragment consisting of Ptrp, 5-Fluoracil purchase the kan gene, and ORF of the ubi4 gene was inserted in-frame upstream of the target gene (dnaB, fab, or pyrG) originally present in the chromosome

by homologous recombination (Product No. 33, 34, 60 in Table S2 and Fig. 1a). For the construction of the GlmU, DnaX, DnaG, IspA, Era, PyrH, or Der, we used a construction strategy that can be applied in cases where direct recombination of the essential target gene (the method described in ‘Construction of the bacterial strain targeting DnaB,FabB, or PyrG’) is unsuccessful or if the target gene has an operon structure. Using the strain constructed in ‘Replacement of the tnaA gene with the trpR gene’, the DNA fragment of ORF of the ubi4 gene fused

in-frame to ORF of the target gene (glmU, dnaX, dnaG, der, pyrH, era, or ispA) was inserted downstream of the tryptophan promoter present in the chromosome by homologous recombination, and then each original gene in the chromosome was deleted by homologous recombination with DNA fragment encoding the kan gene (Fig. 1b). Colony formation this website assay was performed using the strains

constructed in ‘Construction of tryptophan promoter-dependent expression system in E. coli’ (Table 2). No colony was detected in the LB agar plate containing Trp (1 mg mL−1) alone or containing Trp (1 mg mL−1) plus IPTG (10 mM), under the condition that colony Org 27569 formation (about 1000 CFU per plate) was observed in the LB agar plate containing IAA (25 μg mL−1). This result suggested that the Trp-mediated inhibition of the target gene expression was sufficient for evaluation of the colony-forming capacity as a phenotype. When the strain targeting DnaB was examined, colonies were detected in the LB agar without any supplement, suggesting that these strains do not require DnaB at the higher expression level induced by IAA for colony formation. By contrast, the number of colonies was not altered by inducing the proteolytic system alone in the strains targeting DnaB, although the size of colonies was very small (data not shown). In strains in which the tnaA gene was not replaced with the trpR gene, the inhibition of colony formation was not observed in the LB agar plates containing Trp, suggesting that the tryptophanase activity of endogenous TnaA interferes with the Trp-mediated inhibition of Ptrp in this system (data not shown).