An immune-induced reeler protein is involved in the Bombyx mori melanization cascade

In this study, we isolated two reeler cDNAs from bacteria-challenged larval fat bodies of the silkworm, Bombyx mori. A reeler domain spanned most of the coding regions of these two cDNAs, and their expression patterns were different in B. mori larval tissues. The reeler1 gene was strongly induced by Escherichia coli K12 and Bacillus subtilis in B. mori larval hemocytes, fat bodies and midguts, but reeler2 was expressed at extremely low levels in these tissues. We focused on the reeler1 gene for functional analysis. Interference by double-stranded reeler1 RNA in vivo led to reduced nodule formation in bacteria-injected larvae, while the injection of recombinant Reeler1 promoted nodule formation in reeler1 gene-silenced larvae, indicating that Reeler1 is involved in the nodulation response. Knockdown of the reeler1 gene significantly decreased phenoloxidase activity in bacteria-challenged larval hemolymph, while injection of recombinant Reeler1 enhanced phenoloxidase activity, suggesting that Reeler1 is involved in the prophenoloxidase activation cascade. Our results provide new mechanistic evidence about the melanization cascade in the insect immunity.     

The effect of antibiotics and photodynamic therapy on extended-spectrum beta-lactamase (ESBL) positive of Escherichia coli and Klebsiella pneumoniae in urothelial cells

Background/aimUrinary tract infections are commonly caused by the bacteria Escherichia coli and Klebsiella pneumoniae (UTI). The emergence of extended-spectrum -lactamase (ESBL)-producing bacteria strains has made UTI treatment more difficult.Materials and methodsThe aim of this study was to characterize E. coli and K. pneumoniae strains' cytotoxic effects, antibiotic sensitivity, interaction with urothelial cells, and reaction to photodynamic therapy.ResultsAs demonstrated by the higher number of colonies formed, the ESBL + E. coli and K. Pneumonia showed a higher degree of binding with human urothelial cells. With the urothelial cells, K. Pneumonia had the highest binding ability. The cytotoxicity of non-ESBL generating E. coli and K. Pneumonia, on the other hand, was higher. With longer incubation, the discrepancy between the cytotoxic effects of non-ESBL producer and ESBL + E. coli decreased. K. Pneumonia was the opposite. The concentration of ESBL-negative E. coli was easily decreased by photodynamic therapy; however, after a two-hour incubation period, the number of E. coli ESBL + colonies increased from 124 percent to 294 percent.ConclusionWith the duration of the incubation period, the number of non-ESBL-producing K. Pneumonia increased. Even with longer incubation times, the number of K. Pneumonia ESBL + colonies decreased, contrary to expectations. The findings show that the two bacterial species differed in terms of cytotoxicity, interaction with urothelial cells, and photodynamic therapy response.     

Intervention study of the association of antibiotic utilization measures with control of extended-spectrum β-lactamase (ESBL)-producing bacteria

We investigated the effects of replacing third-/fourth-generation cephalosporins with piperacillin–tazobactam on the rate of acquisition of extended spectrum β-lactamase-producing Klebsiella pneumoniae and Escherichia coli by patients hospitalized in a Department of Respiratory Medicine.This 9-month, prospective, non-controlled, intervention study comprised two phases: a 3-month pre-intervention phase (Phase I) and a 6-month intervention phase (Phase II), during which the use of third-/forth-generation cephalosporins was restricted and replaced by piperacillin–tazobactam. Rectal swabs were obtained within 24 h after admission (baseline screening), weekly, and 48 h before discharge during Phase I and the last 3 months of Phase II (Phase IIb). Swabs were tested for E. coli and K. pneumoniae, and extended spectrum β-lactamase production was detected with the double disc test.Use of third/fourth-generation cephalosporins decreased by 63.0% and 100%, respectively; while the use of piperacillin–tazobactam increased by 28-fold. The rate of acquisition of extended spectrum β-lactamase-producing E. coli and K. pneumoniae together in rectal swab specimens decreased in Phase IIb as compared with Phase I (19.5% vs 29.5%). Few rectal swab specimens were positive for extended spectrum β-lactamases-producing K. pneumoniae, and no substantial decrease in the rate of its acquisition was observed.     

Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sp. strains

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bongori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperature phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.     

Genome Sequences of Escherichia coli B strains REL606 and BL21(DE3)

Escherichia coli K-12 and B have been the subjects of classical experiments from which much of our understanding of molecular genetics has emerged. We present here complete genome sequences of two E. coli B strains, REL606, used in a long-term evolution experiment, and BL21(DE3), widely used to express recombinant proteins. The two genomes differ in length by 72,304 bp and have 426 single base pair differences, a seemingly large difference for laboratory strains having a common ancestor within the last 67 years. Transpositions by IS1 and IS150 have occurred in both lineages. Integration of the DE3 prophage in BL21(DE3) apparently displaced a defective prophage in the λ attachment site of B. As might have been anticipated from the many genetic and biochemical experiments comparing B and K-12 over the years, the B genomes are similar in size and organization to the genome of E. coli K-12 MG1655 and have > 99% sequence identity over ∼ 92% of their genomes. E. coli B and K-12 differ considerably in distribution of IS elements and in location and composition of larger mobile elements. An unexpected difference is the absence of a large cluster of flagella genes in B, due to a 41 kbp IS1-mediated deletion. Gene clusters that specify the LPS core, O antigen, and restriction enzymes differ substantially, presumably because of horizontal transfer. Comparative analysis of 32 independently isolated E. coli and Shigella genomes, both commensals and pathogenic strains, identifies a minimal set of genes in common plus many strain-specific genes that constitute a large E. coli pan-genome.     

Identification and Mutational Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> Sorbitol-Enhanced Glucose-Repressed <Emphasis Type="Italic">srlA</Emphasis> Promoter Expressed in LB Medium by Using Homologous Recombination and One-Round PCR Products

Escherichia coli has been used for recombinant protein production for many years. However, no native E. coli promoters have been found for constitutive expression in LB medium. To obtain high-expression E. coli promoters active in LB medium, we inserted various promoter regions upstream of eEmRFP that encodes a red fluorescent protein. Among the selected promoters, only colonies of srlA promoter transformants turned red on LB plate. srlA is a gene that regulates sorbitol utilization. The addition of sorbitol enhanced eEmRFP expression but glucose and other sugars repressed, indicating that srlAp is a sorbitol-enhanced glucose-repressed promoter. To analyze the srlAp sequence, a novel site-directed mutagenesis method was developed. Since we demonstrated that homologous recombination in E. coli could occur between 12-bp sequences, 12-bp overlapping sequences were attached to the set of primers that were designed to produce a full-length plasmid, denoted “one-round PCR product.” Using this method, we identified that the srlA promoter region was 100 bp. Further, the sequence adjacent to the start codon was found to be essential for high expression, suggesting that the traditionally used restriction enzyme sites for cloning in the promoter region have hindered expression. The srlA-driven expression system and DNA manipulation with one-round PCR products are useful tools in E. coli genetic engineering.     

Escherichia coli K1 induces pterin production for enhanced expression of Fcγ receptor I to invade RAW 264.7 macrophages

Macrophages serve as permissive niches for Escherichia coli (E. coli) K1 to attain high grade bacteremia in the pathogenesis of meningitis in neonates. Although pterin levels are a diagnostic marker for immune activation, the role of macrophages in pterin production and in the establishment of meningitis is unknown. Here, we demonstrate that macrophages infected with E. coli K1 produce both neopterin and biopterin through increased expression of GTP-cyclohydrolase 1 (GCH1). Of note, increased production of biopterin enhances the expression of Fc-gamma receptor I (CD64), which in turn, aided the entry of E. coli K1 in macrophages while increased neopterin suppresses reactive oxygen species (ROS), thereby aiding bacterial survival. Inhibition of GCH1 by 2, 4-Diamino-6-hydroxypyrimidine (DAHP) prevented the E. coli K1 induced expression of CD64 in macrophages in vitro and the development of bacteremia in a newborn mouse model of meningitis. These studies suggest that targeting GCH1 could be therapeutic strategy for preventing neonatal meningitis by E. coli K1.     

Effect of Simulated Microgravity on E. coli K12 MG1655 Growth and Gene Expression

This study demonstrates the effects of simulated microgravity on E. coli K 12 MG1655 grown on LB medium supplemented with glycerol. Global gene expression analysis indicated that the expressions of hundred genes were significantly altered in simulated microgravity conditions compared to that of normal gravity conditions. Under these conditions genes coding for adaptation to stress are up regulated (sufE and ssrA) and simultaneously genes coding for membrane transporters (ompC, exbB, actP, mgtA, cysW and nikB) and carbohydrate catabolic processes (ldcC, ptsA, rhaD and rhaS) are down regulated. The enhanced growth in simulated gravity conditions may be because of the adequate supply of energy/reducing equivalents and up regulation of genes involved in DNA replication (srmB) and repression of the genes encoding for nucleoside metabolism (dfp, pyrD and spoT). In addition, E. coli cultured in LB medium supplemented with glycerol (so as to protect the cells from freezing temperatures) do not exhibit multiple stress responses that are normally observed when cells are exposed to microgravity in LB medium without glycerol.     

99mTc-MORF oligomers specific for bacterial ribosomal RNA as potential specific infection imaging agents

PurposeRadiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents.Methods and resultsIdentical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were ^99mTc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from Escherichia coli strains SM101 and K12 was 4- and 150-fold higher compared to PNA and PS-DNA, respectively. Subsequently MORF oligomer in fluorescence in situ hybridization showed a stronger signal with study MORF compared to control in fixed preparations of two E. coli strains and Klebsiella pneumoniae. Flow cytometry analysis showed study MORF accumulation to be 8- and 80-fold higher compared to the control in live K. pneumoniae and Staphylococcus aureus, respectively. Further, fluorescence microscopy showed increased accumulation of study MORF over control in live E. coli and K. pneumonia. Binding of ^99mTc-study MORF to RNA from E. coli SM101 and K12 was 30.4 and 117.8 pmol, respectively, per 10¹⁰ cells. Mice with K. pneumoniae live or heat-killed (sterile inflammation) in one thigh at 90 min for both ^99mTc-study MORF and control showed higher accumulation in target thighs than in blood and all other organs expect for kidneys and small intestine. Accumulation of ^99mTc-study MORF was significantly higher (p = 0.009) than that of the control in the thigh with sterile inflammation.ConclusionA ^99mTc-MORF oligomer complimentary to the bacterial 16S rRNA demonstrated binding to bacterial RNA in vitro with specific accumulation into live bacteria. Radiolabeled MORF oligomers antisense to the bacterial rRNA may be useful to image bacterial infection.     

Construction and performance of heterologous polyketide‐producing K‐12‐ and B‐derived Escherichia coli

Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.     

Effect of Clove (Syzygium aromaticum) spice as microbial inhibitor of resistant bacteria and Organoleptic Quality of meat

For centuries, spices have been utilized as flavourants, colourants and as preservatives in food. Moreover, spices possess various antimicrobial properties with massive health benefits for the treatment and management of ailments and diseases. The present study was focused on three (3) aspects; (1) isolation and molecular identification of bacteria from the meat; (2) to determine the antimicrobial activity of the spices against the pathogens; (3) to assess the organoleptic properties of the spiced meat. A total of twelve (n = 12) spices evaluated against forty (n = 40) spoilage food-borne pathogenic bacteria (Escherichia coli and Enterococci spp.). The spice extracts were tested using disk diffusion method to determine the inhibition abilities. The results show that clove and black seed cumin extract exhibited excellent antibacterial activity against most pathogenic bacteria. Clove displayed the highest inhibition zone of 18 mm against E. coli (EcFwS1). Clove extract was the most inhibitor followed by black cumin, whereas extracts of thyme and cinnamon showed weak antibacterial activities against the tested strains. The most sensitive strain to spice extracts was Enterococcus spp. (EnFmL1) and the most resistant strain being E. coli. (EcFmS1 and EcFpL1). Untreated meat showed that E. coli and Enterococcus spp. count was 4.4 * 10⁵ ± 3.4 * 10⁵ and 2.2 * 10⁵ ± 3.6 * 10⁴ cfu/mL respectively after 7 days while the single dose of clove showed 5.4 * 10⁴ ± 4.4 * 10² cfu/mL of E. coli and 1.7 * 10⁵ ± 4.1 * 10⁴ cfu/mL of Enterococcus spp. The organoleptic characteristics such as colour, texture, odour, pH, shape of the single dose of clove on the meat was overall acceptable.     

Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157

We have sequenced the genome of Shigella flexneri serotype 2a, the most prevalent species and serotype that causes bacillary dysentery or shigellosis in man. The whole genome is composed of a 4 607 203 bp chromosome and a 221 618 bp virulence plasmid, designated pCP301. While the plasmid shows minor divergence from that sequenced in serotype 5a, striking characteristics of the chromosome have been revealed. The S.flexneri chromosome has, astonishingly, 314 IS elements, more than 7-fold over those possessed by its close relatives, the non-pathogenic K12 strain and enterohemorrhagic O157:H7 strain of Escherichia coli. There are 13 translocations and inversions compared with the E.coli sequences, all involve a segment larger than 5 kb, and most are associated with deletions or acquired DNA sequences, of which several are likely to be bacteriophage-transmitted pathogenicity islands. Furthermore, S.flexneri, resembling another human-restricted enteric pathogen, Salmonella typhi, also has hundreds of pseudogenes compared with the E.coli strains. All of these could be subjected to investigations towards novel preventative and treatment strategies against shigellosis.     

Expression of orexin receptors 1 (OX1R) and 2 (OX2R) in the porcine pituitary during the oestrous cycle

Orexin A and B, also termed hypocretin 1 and 2, are associated with the stimulation of food intake and arousal. The biological actions of the hormones are mediated via two distinct G protein-coupled receptors, termed orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). OX1R is selective for orexin A and OX2R binds orexin A and orexin B with similar affinity. The present study analyzed mRNA and protein expressions of OX1R and OX2R in adenohypophysis (AP) and neurohypophysis (NP) of cycling pigs. The tissue samples were harvested on days 2–3, 10–12, 14–16, and 17–19 of the oestrous cycle. Using quantitative real-time PCR higher OX1R gene expression was detected in AP on days 2–3 relative to days 10–12, 14–16 and 17–19 (p < 0.05). In NP the OX1R mRNA level was elevated on days 10–12 compared to the remaining stages (p < 0.05). OX2R gene expression in AP was the lowest on days 10–12 (p < 0.05 compared to days 2–3 and 17–19) and the expression peak occurred on days 17–19 (p < 0.05 vs. the all studied stages). In NP the highest (p < 0.05) expression of OX2R mRNA was noted on days 17–19 in relation to the remaining periods. OX1R protein content in AP was greatest on days 10–12 (p < 0.05), whereas in NP it was greatest on days 2–3 and 14–16 (p < 0.05 vs. days 10–12 and 17–19). In both cases the lowest OX1R protein expression was observed during follicular phase (p < 0.05 in relation to three remaining studied stages). OX2R protein in AP was lower (p < 0.05) on days 2–3 and 14–16 compared to days 10–12 and 17–19. In NP the lowest (p < 0.05) expression of this protein was on days 17–19 and the highest on days 10–12 (p < 0.05 compared to days 2–3 and 17–19). In summary, the present findings provide the first evidence that OX1R and OX2R mRNAs and proteins occur in the pituitary of the pig and indicate the dependence of orexin receptor expression on the endocrine reproductive state.     

Molecular properties and immune defense of two ferritin subunits from freshwater pearl mussel,Hyriopsis schlegelii

Ferritin is a conserved iron-binding protein involved in cellular iron metabolism and host defense. In the present study, two distinct cDNAs for ferritins in the freshwater pearl mussel Hyriopsis schlegelii were identified (designated as HsFer-1 and HsFer-2) by SMART RACE approach and expressed sequence tag (EST) analysis. The full-length cDNAs of HsFer-1 and HsFer-2 were of 760 and 877 bp, respectively. Both of the two cDNAs contained an open reading frame (ORF) of 522 bp encoding for 174 amino acid residues. Sequence characterization and homology alignment indicated that HsFer-1 and HsFer-2 had higher similarity to H-type subunit of vertebrate ferritins than L-type subunit. Analysis of the HsFer-1 and HsFer-2 untranslated regions (UTR) showed that both of them had an iron response element (IRE) in the 5′-UTR, which was considered to be the binding site for iron regulatory protein (IRP). Quantitative real-time PCR (qPCR) assays were employed to examine the mRNA expression profiles. Under normal physiological conditions, the expression level of both HsFer-1 and HsFer-2 mRNA were the highest in hepatopancreas, moderate in gonad, axe foot, intestine, kidney, heart, gill, adductor muscle and mantle, the lowest in hemocytes. After stimulation with bacteria Aeromonas hydrophila, HsFer-1 mRNA experienced a different degree of increase in the tissues of hepatopancreas, gonad and hemocytes, the peak level was 2.47-fold, 9.59-fold and 1.37-fold, respectively. Comparatively, HsFer-2 showed up-regulation in gonad but down-regulation in hepatopancreas and hemocytes. Varying expression patterns indicate that two types of ferritins in H. schlegelii might play different roles in response to bacterial challenge. Further bacteriostatic analysis showed that both the purified recombinant ferritins inhibited the growth of A. hydrophila to a certain degree. Collectively, our results suggest that HsFer-1 and HsFer-2 are likely to be functional proteins involved in immune defense against bacterial infection.     

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP⁺ for acetyl-CoA production. After 24 h of cultivation, a 3.7-fold increase in NADPH/NADP⁺ ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48 h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2–3-fold over the base strain (up to 0.8 g/L), and in combination to 1.4 g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6 g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16 g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.