Regulation of <Emphasis Type="Italic">lac</Emphasis> Transcription in Antibiotic-treated <Emphasis Type="Italic">E. coli</Emphasis>

Chloramphenicol stabilizes pre-existing lac mRNA, but inhibits further accumulation by allowing rapid degradation of nascent message. Puromycin accelerates decay of pre-existing and new lac message by discharging protective ribo-somes. Both effects are partially reversed by the suA mutation.     

Regulation of the <Emphasis Type="Italic">htpX</Emphasis> Gene of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> and Its Expression in <Emphasis Type="Italic">E. coli</Emphasis>

Xylella fastidiosa was the first phytopathogen to be completely sequenced, and its genome revealed several interesting features to be used in functional studies. In the present work, the htpX gene, which encodes a protein involved in the heat shock response in other bacteria, was analyzed by RT-PCR by using cells derived from different cultural conditions. This gene was induced after a temperature upshift to 37°C after growth in minimal medium, XDM, but showed constitutive expression in rich medium or in XDM plus plant extracts. Sequences upstream to the htpX gene, containing a putative regulatory region, were also transferred to E. coli, and the thermoregulation was maintained in the new host, since it was constitutively transcribed at 37°C or 45°C in all culture media tested, but not at 28°C in minimal culture medium. The gene was also cloned into the expression vector pET32Xa/LIC, and the expression of the corresponding protein was confirmed by Western blotting.     

Epidemiological investigation of <Emphasis Type="Italic">eaeA</Emphasis>-positive <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Escherichia albertii</Emphasis> strains isolated from healthy wild birds

Escherichia coli has commonly been associated with diarrheal illness in humans and animals. Recently, E. albertii has been reported to be a potential pathogen of humans and animals and to be carried by wild birds. In the present study, the prevalence and genetic characteristics of intimin-producing E. coli and E. albertii strains were evaluated in wild birds in Korea. Thirty one of 790 Enterobacteriaceae strains from healthy wild birds were positive for the intimin gene (eaeA) and twenty two of the 31 strains were identified as atypical enteropathogenic E. coli (aEPEC) that did not possess both EAF and bfpA genes. A total of nine lactose non-fermenting coliform bacterial strains were identified as E. albertii by PCR and sequence analysis of housekeeping genes. A total of 28 (90.3%) eaeA-positive strains were isolated from waterfowl. Fifteen aEPEC (68.2%) and two E. albertii (22.2%) strains had a β-intimin subtype and 14 aEPEC strains harboring β-intimin belonged to phylogenetic group B2. AU eaeA-positive E. albertii and 3 aEPEC strains possessed the cytolethal distending toxin gene (cdtB). The eaeA-positive E. coli and E. albertii strains isolated from healthy wild birds need to be recognized as a potential pathogroup that may pose a potential threat to human and animal health. These findings indicate that eaeA-positive E. coli as well as E. albertii can be carried by wild birds, posing a potential threat to human and animal health.     

<Emphasis Type="SmallCaps">L</Emphasis>-Tyrosine production by deregulated strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The excretion of the aromatic amino acid l-tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this l-tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing l-tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that l-tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final l-tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of l-tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

Viability and survival capability of quinolone-resistant uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Uropathogenic strains of E. coli isolated from urine of patients with urinary tract infections were tested for antibiotic sensitivity using bio-Merieux kits and ATB-UR 5 expression system. The virulence of strains was evaluated by serum bactericidal assay, macrophage “killing” and bacterial adhesive tests. Survival capability of strains was assessed under starvation in saline. The results showed that quinolone-resistant uropathogenic strains of E. coli exhibit significantly reduced adhesive potential but relatively high resistance to serum and macrophage bactericidity. In contrast to laboratory strains, the quinolone-resistant uropathogenic clinical isolate demonstrated increased viability during starvation in saline. Our study suggests that quinolone-resistant uropathogenic strains are highly adaptable clones of E. coli, which can exhibit compensatory viability potential under unfavorable conditions. The clinical occurrence of such phenotypes is likely to contribute to the survival, persistence and spread strategy of resistant bacteria.     

Cloning of <Emphasis Type="Italic">srfA</Emphasis> operon from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C9 and its expression in <Emphasis Type="Italic">E. coli</Emphasis>

The srfA operon is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin. The srfA operon is composed of the four genes, srfAA, srfAB, srfAC, and srfAD, encoding the surfactin synthetase subunits, plus the sfp gene that encodes phosphopantetheinyl transferase. In the present study, 32 kb of the srfA operon was amplified from Bacillus subtilis C9 using a long and accurate PCR (LA-PCR), and ligated into a pIndigoBAC536 vector. The ligated plasmid was then transformed into Escherichia coli DH10B. The transformant ET2 showed positive signals to all the probes for each open reading frame (ORF) region of the srfA operon in southern hybridization, and a reduced surface tension in a culture broth. Even though the surface-active compound extracted from the E. coli transformant exhibited a different R _f value of 0.52 from B. subtilis C9 or authentic surfactin (R _f = 0.63) in a thin layer chromatography (TLC) analysis, the transformant exhibited a much higher surface-tension-reducing activity than the wild-type strain E. coli DH10B. Thus, it would appear that an intermediate metabolite of surfactin was expressed in the E. coli transformant harboring the srfA operon.     

A recombinant <Emphasis Type="Italic">E. coli</Emphasis> bioprocess for hyaluronan synthesis

An Escherichia coli strain, JM109, was successfully engineered into an efficient hyaluronic acid (HA) producer by co-expressing the only known class-II HA synthase from a Gram-negative bacterium (Pasteurella multocida) and uridine diphosphate-glucose dehydrogenase from E. coli K5 strain. The engineered strain produced about 0.5 g/L HA in shake flask culture and about 2.0–3.8 g/L in a fed-batch fermentation process in a 1-L bioreactor. The sharp increase in viscosity associated with HA accumulation necessitated pure oxygen supplement to maintain fermentation in aerobic regime. Precursor supply during HA synthesis was probed by glucosamine supplement, which shortens biosynthesis pathway and eliminates one step requiring ATP. HA synthesis was increased with glucosamine supplement from 2.7 to 3.7 g/L (37%), which was mirrored with a concomitant 42% decrease in pure oxygen input, suggesting a close connection between energy metabolism and precursor supply. Decoupling HA synthesis from cell growth by using fosfomycin (an inhibitor for cell wall synthesis) led to a 70% increase in HA synthesis, suggesting detrimental effects on HA synthesis from cell growth via precursor competition. This study demonstrates a potentially viable process for HA based on a recombinant E. coli strain. In addition, the precursor supply limitation identified in this study suggests new engineering targets in subsequent metabolic engineering efforts.     

A new methanol assimilating yeast, <Emphasis Type="Italic">Ogataea</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>, the ascosporic state of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>

Ogataea parapolymorpha sp. n. (NRRL YB-1982, CBS 12304, type strain), the ascosporic state of Candida parapolymorpha, is described. The species appears homothallic, assimilates methanol as is typical of most Ogataea species and forms hat-shaped ascospores in asci that become deliquescent. O. parapolymorpha is closely related to Ogataea angusta and Ogataea polymorpha. The three species can be resolved from gene sequence analyses but are unresolved from fermentation and growth reactions that are typically used for yeast identification. On the basis of multiple isolates, O. angusta is known only from California, USA, in association with Drosophila and Aulacigaster flies, O. parapolymorpha is predominantly associated with insect frass from trees in the eastern USA but O. polymorpha has been isolated from various substrates in the USA, Brazil, Spain and Costa Rica.     

Genome-wide analysis of <Emphasis Type="Italic">E. coli</Emphasis> cell-gene interactions

Background

The pursuit of standardization and reliability in synthetic biology has achieved, in recent years, a number of advances in the design of more predictable genetic parts for biological circuits. However, even with the development of high-throughput screening methods and whole-cell models, it is still not possible to predict reliably how a synthetic genetic construct interacts with all cellular endogenous systems. This study presents a genome-wide analysis of how the expression of synthetic genes is affected by systematic perturbations of cellular functions. We found that most perturbations modulate expression indirectly through an effect on cell size, putting forward the existence of a generic Size-Expression interaction in the model prokaryote Escherichia coli.

Results

The Size-Expression interaction was quantified by inserting a dual fluorescent reporter gene construct into each of the 3822 single-gene deletion strains comprised in the KEIO collection. Cellular size was measured for single cells via flow cytometry. Regression analyses were used to discriminate between expression-specific and gene-specific effects. Functions of the deleted genes broadly mapped onto three systems with distinct primary influence on the Size-Expression map. Perturbations in the Division and Biosynthesis (DB) system led to a large-cell and high-expression phenotype. In contrast, disruptions of the Membrane and Motility (MM) system caused small-cell and low-expression phenotypes. The Energy, Protein synthesis and Ribosome (EPR) system was predominantly associated with smaller cells and positive feedback on ribosome function.

Conclusions

Feedback between cell growth and gene expression is widespread across cell systems. Even though most gene disruptions proximally affect one component of the Size-Expression interaction, the effect therefore ultimately propagates to both. More specifically, we describe the dual impact of growth on cell size and gene expression through cell division and ribosomal content. Finally, we elucidate aspects of the tight control between swarming, gene expression and cell growth. This work provides foundations for a systematic understanding of feedbacks between genetic and physiological systems.

     

Function of the <Emphasis Type="Italic">rel</Emphasis> Gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sokawa et al. suggest that rel^- strains of Escherichia coli possess abnormal protein synthesizing machinery, which cannot carry out normal protein synthesis when the supply of amino-acids is limited.     

Enhanced production of recombinant galactose oxidase from <Emphasis Type="Italic">Fusarium graminearum</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

The gene gaoA encoding the copper-dependent enzyme galactose oxidase (GAO) from Fusarium graminearum PH-1 was cloned and successfully overexpressed in E. coli. Culture conditions for cultivations in shaken flasks were optimized, and optimal conditions were found to be double-strength LB medium, 0.5% lactose as inducer, and induction at the reduced temperature of 25°C. When using these cultivation conditions ~24 mg of active GAO could be produced in shaken flasks per litre medium. Addition of copper to the fermentation medium decreased the enzyme production significantly. The His-tagged recombinant enzyme could be purified conveniently with a single affinity chromatography step. The purified enzyme showed a single band on SDS–PAGE with an apparent molecular mass of 66 kDa and had kinetic properties similar to those of the fungal wild-type enzyme.     

Extracellular expression of keratinase Ker P from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

Keratinase from Pseudomonas aeruginosa KS-1 was expressed constitutively as an extracellular protein in Escherichia coli with high specific activity of 3.7 kU/mg. It was purified fourfold as a 33 kDa monomeric protein by Q-Sepharose ion exchange chromatography with a recovery of 95%. It is a serine protease with optimal activity at pH 9 and 50°C. It was stable from pH 4 to 12 for 1 h with a t_1/2 of 12 min at 70°C. It hydrolyzed haemoglobin > fibrin > feather keratin > azo-casein > casein > meat protein > gelatin. Among synthetic substrates, it efficiently hydrolyzed N-Suc-ala-ala-pro-phe-pNA, N-Suc-ala-ala-ala-pNA, N-Suc-ala-ala-pro-leu-pNA and also plasmin substrate, d-Val-Leu-Lys-pNA     

Common genomic features of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">doylei</Emphasis> strains distinguish them from <Emphasis Type="Italic">C. jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis>

Background  

Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

Minimizing acetate formation in <Emphasis Type="Italic">E. coli</Emphasis> fermentations

Escherichia coli remains the best-established production organism in industrial biotechnology. However, when aerobic fermentation runs at high growth rates, considerable amounts of acetate are accumulated as by-product. This by-product has negative effects on growth and protein production. Over the last 20 years, substantial research efforts have been expended on reducing acetate accumulation during aerobic growth of E. coli on glucose. From the onset it was clear that this quest would not be a simple or uncomplicated one. Simple deletion of the acetate pathway reduced the acetate accumulation, but other by-products were formed. This mini review gives a clear outline of these research efforts and their outcome, including bioprocess level approaches and genetic approaches. Recently, the latter seems to have some promising results.     

Characterization of the effects of <Emphasis Type="Italic">n</Emphasis>-butanol on the cell envelope of <Emphasis Type="Italic">E. coli</Emphasis>

Biofuel alcohols have severe consequences on the microbial hosts used in their biosynthesis, which limits the productivity of the bioconversion. The cell envelope is one of the most strongly affected structures, in particular, as the external concentration of biofuels rises during biosynthesis. Damage to the cell envelope can have severe consequences, such as impairment of transport into and out of the cell; however, the nature of butanol-induced envelope damage has not been well characterized. In the present study, the effects of n-butanol on the cell envelope of Escherichia coli were investigated. Using enzyme and fluorescence-based assays, we observed that 1 % v/v n-butanol resulted in the release of lipopolysaccharides from the outer membrane of E. coli and caused ‘leakiness’ in both outer and inner membranes. Higher concentrations of n-butanol, within the range of 2–10 % (v/v), resulted in inner membrane protrusion through the peptidoglycan observed by characteristic blebs. The findings suggest that strategies for rational engineering of butanol-tolerant bacterial strains should take into account all components of the cell envelope.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Cloning and Expression of <Emphasis Type="Italic">H. influenzae</Emphasis> 49247 IgA Protease in <Emphasis Type="Italic">E. coli</Emphasis>

IgA protease is secreted by various mucosal pathogenic bacteria which can cleave human immunoglobulin A1 (IgA1) in its hinge region. In addition to be considered as a virulence factor, it's reported that IgA protease can also be used for IgA nephropathy (IgAN) treatment. Our previous study identified bacteria H. influenzae 49247 expressed high activity of IgA protease with promised application in IgAN therapy. In this study, we cloned the IgA protease gene of H. influenzae 49247 with degenerate primers. Alignment analysis indicated that H. influenzae 49247 IgA protease showed unique DNA and amino acid sequence but with typical endopeptidase domain and beta transporter domain compared with known IgA proteases from the same species. To facilitate expression and purification, the H. influenzae 49247 IgA protease gene was sub-cloned into the pET28-A(+) vector with insertion of a 6xHis tag downstream of the endopeptidase domain and upstream of the potential autocleavage site. The recombined IgA protease can be constitutively expressed in E. coli and secreted into the culture medium. With a simple nickel affinity binding, the secreted IgA protease can be purified with high purity (95%) and a molecular weight of about 130 kDa. The identity of the IgA protease was validated by the presence of 6xHis tag in the purified protein by western blotting and its ability to cleave human IgA1 molecule. Collectively, the successful cloning, expression and purification of H. influenzae 49247 IgA protease will augment its therapeutic study in IgAN treatment.     

Antimicrobial effects of zero-valent iron nanoparticles on gram-positive <Emphasis Type="Italic">Bacillus</Emphasis> strains and gram-negative <Emphasis Type="Italic">Escherichia coli</Emphasis> strains

Background

Zero-valent iron nanoparticles (ZVI NPs) have been used extensively for the remediation of contaminated soil and groundwater. Owing to their large active surface area, they serve as strong and effective reductants. However, the ecotoxicity and bioavailability of ZVI NPs in diverse ecological media have not been evaluated in detail and most studies have focused on non-nano ZVI or Fe⁰. In addition, the antimicrobial properties of ZVI NPs have rarely been investigated, and the underlying mechanism of their toxicity remains unknown.

Results

In the present study, we demonstrate that ZVI NPs exhibited significant toxicity at 1000 ppm against two distinct gram-positive bacterial strains (Bacillus subtilis 3610 and Bacillus thuringiensis 407) but not against two gram-negative strains (Escherichia coli K12 and ATCC11634). Specifically, ZVI NPs caused at least a 4-log and 1-log reductions in cell numbers, respectively, in the two Bacillus strains, whereas no change was detected in the two E. coli strains. X-ray photoelectron spectroscopy, X-ray absorption near-edge, and extended X-ray absorption fine structure spectra confirmed that Bacillus cells exposed to ZVI NPs contained mostly Fe₂O₃ with some detectable FeS. This finding indicated that Fe⁰ nanoparticles penetrated the bacterial cells, where they were subsequently oxidized to Fe₂O₃ and FeS. RedoxSensor analysis and propidium iodide (PI) staining showed decreased reductase activity and increased PI in both Bacillus strains treated with a high (1000 ppm) concentration of ZVI NPs.

Conclusion

Taken together, these data show that the toxicity of ZVI NPs was derived from their oxidative properties, which may increase the levels of reactive oxygen species and lead to cell death.