Engineering chimeric two-component system into <Emphasis Type="Italic">Escherichia coli</Emphasis> from <Emphasis Type="Italic">Paracoccus denitrificans</Emphasis> to sense methanol

Escherichia coli does not have the methanol sensing apparatus, was engineered to sense methanol by employing chimeric two-component system (TCS) strategy. A chimeric FlhS/EnvZ (FlhSZ) chimeric histidine kinase (HK) was constructed by fusing the sensing domain of Paracoccus denitrificans FlhS with the catalytic domain of E. coli EnvZ. The constructed chimeric TCS FlhSZ/OmpR could sense methanol by the expression of ompC and gfp gene regulated by ompC promoter. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed the dynamic response of the chimeric TCS to methanol. The expression of ompC and the gfp fluorescence was maximum at 0.01 and 0.5% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric HK FlhSZ. This strategy can be employed for the construction of several chimeric TCS based bacterial biosensors for the development of biochemical producing recombinant microorganisms.     

<Emphasis Type="BoldItalic">In situ</Emphasis> real-time evaluation of radiation-responsive promoters in the extremely radioresistant microbe <Emphasis Type="BoldItalic">Deinococcus radiodurans</Emphasis>

A third generation promoter probe shuttle vector pKG was constructed, using the green fluorescent protein as a reporter, for in situ evaluation of Deinococcal promoter activity in Escherichia coli or Deinococcus radiodurans. The construct yielded zero background fluorescence in both the organisms, in the absence of promoter sequences. Fifteen Deinococcal promoters, either harbouring Radiation and Desiccation Response Motif (RDRM) or not, were cloned in vector pKG. Only the RDRM-promoter constructs displayed (i) gamma radiation inducible GFP expression in D. radiodurans, following gamma irradiation, (ii) DdrO-mediated repression of GFP expression in heterologous E. coli, or (iii) abolition in GFP induction following gamma irradiation, in pprI mutant of D. radiodurans. Utility of pKG vector for real-time in situ assessment of Deinococcal promoter function was, thus, successfully demonstrated.     

Engineered fumarate sensing Escherichia coli based on novel chimeric two-component system

DcuS/DcuR two component system (TCS) was firstly employed for the expression of the gfp gene under the dcuB gene promoter in aerobic condition to develop high throughput screening system able to screen microorganisms producing high amount of fumarate. However, the DcuS/DcuR TCS could not produce a signal strong enough to mediate the expression of the gfp gene responding fumarate concentration. Thus, DcuS/DucR TCS was engineered by recruiting the EnvZ/OmpR system, the most-studied TCS in E. coli. A chimeric DcuS/EnvZ (DcuSZ) TCS was constructed by fusing the sensor histidine kinase of DcuS with the cytoplasmic catalytic domain of EnvZ, in which the expression of the gfp gene or the ompC gene was mediated by the ompC gene promoter through the cognate response regulator, OmpR. The output signals produced by the chimeric DcuSZ TCS were enough to detect fumarate concentration quantatively, in which the expressions of the gfp gene and the ompC gene were proportional to the fumarate concentration in the medium. Moreover, principal component analysis of C₄-dicarboxylates showed that DcuSZ chimera was highly specific to fumarate but could also respond to other C₄-dicarboxylates, which strongly suggests that TCS-based high throughput screening system able to screen microorganisms producing target chemicals can be developed.     

Screening of promoters from <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. CGMCC 3584 using a green fluorescent protein reporter system

Available molecular and genetic tools for the genetic manipulation of Arthrobacter species are limited until now. In gene engineering, a continuous set of promoters with various strengths are of importance for fine-tuning gene expression in metabolic optimization and control analysis. Here, for the first time, we constructed a promoter trap system using green fluorescence protein (GFP) as a reporter, for screening and characterizing functional Arthrobacter promoters. Twenty-three Arthrobacter transformants of various GFP fluorescence strengths were isolated and characterized through the analysis of DNA sequences. Among the 23 putative promoters, 2 were selected for deletion analysis of promoter elements. As a result, the deletion of the upstream of the putative promoter P8 and P13 caused a 43.8% decrease and a 29.1% increase in the fluorescence signals, respectively. Finally, we obtained the strongest promoter P13-3 which was 4.4 times more potent than the promoter of 6–hydroxyl–d–nicotine oxidase gene which was previously reported in Arthrobacter nicotinovorans, and the obtained promoter was used to improve the production of cyclic adenosine monophosphate in Arthrobacter sp. CGMCC 3584. The screening strategy together with obtained promoters in this study would contribute to the future engineering of Arthrobacter species.     

High yield production of four-carbon dicarboxylic acids by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Several metabolic engineered Escherichia coli strains were constructed and evaluated for four-carbon dicarboxylic acid production. Fumarase A, fumarase B and fumarase C single, double and triple mutants were constructed in a ldhA adhE mutant background overexpressing the pyruvate carboxylase from Lactococcus lactis. All the mutants produced succinate as the main four-carbon (C4) dicarboxylic acid product when glucose was used as carbon source with the exception of the fumAC and the triple fumB fumAC deletion strains, where malate was the main C4-product with a yield of 0.61–0.67 mol (mole glucose)^?1. Additionally, a mdh mutant strain and a previously engineered high-succinate-producing strain (SBS550MG-Cms pHL413-Km) were investigated for aerobic malate production from succinate. These strains produced 40.38 mM (5.41 g/L) and 50.34 mM (6.75 g/L) malate with a molar yield of 0.53 and 0.55 mol (mole succinate)^?1, respectively. Finally, by exploiting the high-succinate production capability, the strain SBS550MG-Cms243 pHL413-Km showed significant malate production in a two-stage process from glucose. This strain produced 133 mM (17.83 g/L) malate in 47 h, with a high yield of 1.3 mol (mole glucose)^?1 and productivity of 0.38 g L^?1 h^?1.     

Secreted xylanase XynA mediates utilization of xylan as sole carbon source in <Emphasis Type="Italic">Candida utilis</Emphasis>

The fodder yeast Candida utilis is able to use xylose mono- and oligomers as sources of carbon but not the abundant polymer xylan. C. utilis transformants producing the Penicillium simplicissimum xylanase XynA were constructed using expression vectors encoding fusions of the Saccharomyces cerevisiae Mfα1 pre-pro secretion leader to XynA. The Mfα1-XynA fusion was efficiently processed in transformants and XynA was secreted almost quantitatively into the culture medium. Secreted XynA was enzymatically active and allowed transformants to grow on xylan as the sole carbon source. Addition of a second expression unit for the heterologous green fluorescent protein (GFP) generated C. utilis transformants, which showed intracellular GFP fluorescence during growth on xylan. The results suggest that xylanase-producing C. utilis is suited as a cost-effective host organism for heterologous protein production and for other biotechnical applications.     

Simultaneous release of recombinant cellulases introduced by coexpressing colicin E7 lysis in <Emphasis Type="Italic">Escherichia coli</Emphasis>

With the increasingly serious global environment problem caused by slather use of fossil fuels, numerous efforts have been made in developing renewable alternatives. Converting lignocellulose to bioenergy has accomplished by cellulases but the production is limited, therefore the recombinant expression platforms in E. coli have been established. Since E. coli is partly restricted by its inability to secrete enzymes to extracellular membrane, we constructed a synthetic circuit to coexpress cellulases and colicin E7 lysis to solve this problem. The pBAD-RBS-lysis-TT (BBa_K117010) sequence from iGEM was added to form a chimeric plasmid based on pET system, which harboring cellulases from Bacillus subtilis or Thermobifida fusca. The presence of arabinose for the promoter pBAD, leading to induce lysis and further breakdown of the host membrane to release cellulase to extracellular membrane. The extracellular activities increase to 48.1 and 55.5% under lysis gene functioned on Cel5 and CD1, respectively. This is the first attempt to show that cellulases have successfully expressed and released to extracellular membrane without cumbersome steps. Finally, this synthetic circuit simplifies and achieves the simultaneous release and heterologous expression of the cellulases as well as obtain a long-term stable enzyme in E. coli system.     

Evaluation of microbial globin promoters for oxygen-limited processes using <Emphasis Type="Italic">Escherichia coli</Emphasis>

Oxygen-responsive promoters can be useful for synthetic biology applications, however, information on their characteristics is still limited. Here, we characterized a group of heterologous microaerobic globin promoters in Escherichia coli. Globin promoters from Bacillus subtilis, Campylobacter jejuni, Deinococcus radiodurans, Streptomyces coelicolor, Salmonella typhi and Vitreoscilla stercoraria were used to express the FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker. FbFP fluorescence was monitored online in cultures at maximum oxygen transfer capacities (OTR_max) of 7 and 11 mmol L^?1 h^?1. Different FbFP fluorescence intensities were observed and the OTR_max affected the induction level and specific fluorescence emission rate (the product of the specific fluorescence intensity multiplied by the specific growth rate) of all promoters. The promoter from S. typhi displayed the highest fluorescence emission yields (the quotient of the fluorescence intensity divided by the scattered light intensity at every time-point) and rate, and together with the promoters from D. radiodurans and S. coelicolor, the highest induction ratios. These results show the potential of diverse heterologous globin promoters for oxygen-limited processes using E. coli.     

Genomic characterisation,detection of genes encoding virulence factors and evaluation of antibiotic resistance of <Emphasis Type="Italic">Trueperella pyogenes</Emphasis> isolated from cattle with clinical metritis

Trueperella pyogenes is one of the most important microorganisms causing metritis in post-partum cattle. Co-infection with other bacterial species such as Escherichia coli or Fusobacterium necrofurom increases the severity of the disease and the persistence of bacteria in utero. The aim of this study was to investigate the frequency of T. pyogenes strains, and their virulence and antimicrobial resistant profiles in metritis cases. The study was carried out on 200 samples obtained from metritis discharges of postpartum cattle on 18 farms around Tehran, Iran. Sixty-five T. pyogenes isolates (32.5%) were identified, of which 16 isolates were detected as pure cultures and the other 49 isolates from cultures most commonly mixed with E. coli or F. necrofurom. In terms of diversity in biochemical characteristic of T. pyogenes strains, 8 different biotypes were identified among the isolates. Single or multi antimicrobial resistance was observed in 48 isolates (73.9%), which was mostly against trimethoprim sulfamethoxazole, azithromycin, erythromycin and streptomycin. The tetracycline resistance gene tetW and macrolide resistance genes ermB and ermX were detected in 30, 18 and 25 isolates, respectively. In the screening of genes encoding virulence factors, fimA and plo genes were identified in all tested isolates. Genes encoding nanP, nanH, fimC, fimG, fimE and cbpA were detected in 50, 54, 45, 40, 50 and 37 of isolates, respectively. Thirteen different genotypes were observed in these T. pyogenes isolates. A significant association between clonal types and virulence factor genes, biochemical profile, CAMP test result, severity of the disease and sampling time was detected.     

Autodisplay of an avidin with biotin-binding activity on the surface of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli.

Results

Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and β domains, whereas the second consisted of a 314 amino acid from α and truncated β domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA).

Conclusions

Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

     

Inducing flocculation of non-floc-forming <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

The present article reviews several approaches for inducing flocculation of Escherichia coli cells. The common industrially used bacterium E. coli does not naturally have floc-forming ability. However, there are several approaches to induce flocculation of E. coli cells. One is induction by flocculants—polyvalent inorganic salts, synthetic polymeric flocculants, or bio-based polymeric materials, including polysaccharide derivatives. Another method is the induction of spontaneous flocculation by changing the phenotypes of E. coli cells; several studies have shown that physical treatment or gene modification can endow E. coli cells with floc-forming ability. Coculturing E. coli with other microbes is another approach to induce E. coli flocculation. These approaches have particular advantages and disadvantages, and remain open to clarification of the flocculation mechanisms and improvement of the induction processes. In this review, several approaches to the induction of E. coli flocculation are summarized and discussed. This review will be a useful guide for the future development of methods for the flocculation of non-floc-forming microorganisms.     

Co-transformation of canola by chimeric chitinase and <Emphasis Type="Italic">tlp</Emphasis> genes towards improving resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T₂ generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29 mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant.     

Study of ectoparasitism of ultramicrobacteria of the genus <Emphasis Type="Italic">Kaistia</Emphasis>, strains NF1 and NF3 by electron and fluorescence microscopy

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gramnegative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria (Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated [1], but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes.     

Histone-antihistone interactions in the <Emphasis Type="Italic">Escherichia coli-Tetrahymena pyriformis</Emphasis> association

Using the Escherichia coli-Tetrahymena pyriformis model system, we revealed the involvement of bacterial antihistone activity and protozoan histones in interactions between pro-and eukaryotic microorganisms. Antihistone activity enhanced the viability of E. coli in association with T. pyriformis, according to our data on the dynamics of E. coli cell numbers. The strain with antihistone activity induced incomplete phagocytosis in the infusorians, resulting in cytological changes and ultrastructural alterations that indicated the retention of bacterial cells in phagosomes. Bacteria with antihistone activity located in the T. pyriformis cytoplasm influenced the eukaryotic nucleus. This was accompanied by in macronucleus decompactization and a decrease in the average histone content in the population of infusorians. The data obtained suggest that protozoan histone inactivation by bacteria is one of the mechanisms involved in prokaryote persistence in associations with eukaryotic microorganisms.     

Phosphoenolpyruvate:glucose phosphotransferase system modification increases the conversion rate during <Emphasis Type="SmallCaps">l</Emphasis>-tryptophan production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli FB-04(pta1), a recombinant l-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (l-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key l-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher l-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, l-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g^?1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.     

Production of caffeoylmalic acid from glucose in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli.

Results

We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli–E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L.

Conclusions

Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.

     

The role of thiol redox systems in the response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to far-UV irradiation

Escherichia coli mutants deficient in glutathione (gshA), glutaredoxin (grxA), thioredoxin (trxA), and thioredoxin reductase (trxB) synthesis were studied with respect to their resistance to far-UV (UV₂₅₄) exposure. The trxA, trxB, and grxA mutants subjected to a short-term UV exposure were found to be more resistant to UV irradiation than the parent cells. Under the same conditions, the trxA and trxB mutants demonstrated a high level of induction of the sulA gene, a component of the SOS regulon. The mutagenic effect of long-term UV exposure of all the mutants with redox deficiencies was more pronounced than in the case of the parent strain, and the trxA and trxB mutants were found to be the least viable microorganisms. Pretreatment of the cells with low concentrations of the thiol-oxidizing agent diamide enhanced the sulA gene expression; however, high concentrations of diamide inhibited sulA expression. The data obtained indicate that the thiol redox systems of E. coli are involved in its response to far-UV irradiation.     

Conjugative plasmid transfer from <Emphasis Type="Italic">Escherichia coli</Emphasis> is a versatile approach for genetic transformation of thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Geobacillus</Emphasis> species

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli–Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1 × 10^?7 and 3.8 × 10^?2/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.