Gene <Emphasis Type="Italic">yddG</Emphasis> of <Emphasis Type="Italic">Escherichia coli</Emphasis> encoding the putative exporter of aromatic amino acids: Constitutive transcription and dependence of the expression on the cell growth rate

Gene yddG of Escherichia coli encodes a protein of the inner membrane. Data obtained earlier demonstrated that under conditions of aromatic amino acids overproduction YddG promotes their export from E. coli cells. In this work, a method of primer extension was used to localize the P _yddG promoter, which corresponds to E. coli promoters recognized by RNA polymerase in complex with σ⁷⁰ or σ^S subunits. By constructing a gene of the hybrid protein YddG’-LacZ at the intrinsic site of gene yddG location in the E. coli chromosome and analyzing the activity of β-galactosidase in cells growing on laboratory media LB and M9, the constitutive type of yddG expression at a low level was demonstrated (the activity was about 3 to 4% of the LacZ level under induction of the lac operon in E. coli wild-type cells). The expression of yddG had a twofold increase under conditions of retarded cell growth upon the stress caused by the high NaCl content (0.6 M) or by the presence of phenylalanine excess quantities (>1 mM) in the culture medium.     

Comparative expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> of the native and hybrid genes for acyl-lipid Δ<Superscript>9</Superscript> desaturase

Expression of the desC gene coding for acyl-lipid Δ⁹ desaturase of thermophilic cyanobacterium Synechocystis sp. PCC6803 was studied in Escherichia coli cells. In a hybrid gene constructed (desC-licBM3), a sequence of the native acyl-lipid Δ⁹ desaturase was fused in frame with the reporter gene coding for thermostable lichenase. Lichenase contained in the hybrid protein simplified selection and analysis of the expression of membrane desaturase in the heterologous host. Comparisons of the expression for the native and hybrid genes in bacterial cells showed that lichenase remained active and thermostable in the hybrid protein, while desaturase retains the capability of introducing a double bound in the corresponding position of fatty acid residues.     

Cloning of <Emphasis Type="Italic">rec</Emphasis>A gene of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and phenotypic complementation of <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant deficient strain

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM^®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm², while non-transformed cells tolerated only up to dose 0.08 J/cm². It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.     

Fusion expression of the PGLa-AM1 with native structure and evaluation of its anti-<Emphasis Type="Italic">Helicobacter pylori</Emphasis> activity

Helicobacter pylori (H. pylori) shows increasingly enhanced resistance to various antibiotics, and its eradication has become a major problem in medicine. The antimicrobial peptide PGLa-AM1 is a short peptide with 22 amino acids and exhibits strong antibacterial activity. In this study, we investigated whether it has anti-H. pylori activity for the further development of anti-H. pylori drugs to replace existing antibiotics. However, the natural antimicrobial peptide PGLa-AM1 shows a low yield and is difficult to separate, limiting its application. A good strategy to solve this problem is to express the antimicrobial peptide PGLa-AM1 using gene engineering at a high level and low cost. For getting PGLa-AM1 with native structure, in this study, a specific protease cleavage site of tobacco etch virus (TEV) was designed before the PGLa-AM1 peptide. For convenience to purify and identify high-efficiency expression PGLa-AM1, the PGLa-AM1 gene was fused with the polyhedrin gene of Bombyx mori (B. mori), and a 6 × His tag was designed to insert before the amino terminus of the fusion protein. The fusion antibacterial peptide PGLa-AM1 (FAMP) gene codon was optimized, and the gene was synthesized and cloned into the Escherichia coli (E. coli) pET-30a (+) expression vector. The results showed that the FAMP was successfully expressed in E. coli. Its molecular weight was approximately 34 kDa, and its expression level was approximately 30 mg/L. After the FAMP was purified, it was further digested with TEV protease. The acquired recombinant antimicrobial peptide PGLa-AM1 exerted strong anti-H. pylori activity and therapeutic effect in vitro and in vivo.     

Conjugative chromosomal gene transfer in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Conjugative transfer of 20-kb chromosomal fragment carrying genes encoding tetracycline (tet ^r ) and lincomycin (lin ^r ) resistance in the soil strain Bacillus subtilis 19 is described. Transfer was preceded by this fragment insertion into the large conjugative p19cat plasmid producing a hybrid plasmid. Insertion frequency was 10^?4?10^?5. Then genes tet ^r and lin ^r were transferred to the recipient strains. The transfer of chromosomal genes inserted into the plasmid and plasmid gene cat occurred sequentially and resembled sexduction, which represents chromosomal gene transfer by F′ and R′ plasmids during conjugation in Escherichia coli and other gram negative bacteria.     

Comparative Analysis of Mono- and Bifunctional Chorismate Synthases in <Emphasis Type="Italic">Escherichia coli</Emphasis> Cells Capable and Incapable of Phenylalanine Production

The activity of chorismate synthase, the terminal enzyme of the common aromatic pathway, is absolutely dependent on reduced flavin mononucleotide. The bifunctional chorismate synthase of Saccharomyces cerevisiae (product of the ARO2 gene) can reduce flavin in a reaction that involves NADPH, in contrast to the monofunctional chorismate synthase of Escherichia coli (product of the _aro C gene). The latter enzyme does not have the capacity for flavin reduction, and its activity therefore depends on the flavin reductase function of the cell. Chemical synthesis of the structural part of the ARO2 gene that involved the substitution of rare E. coli codons was performed for an in vivo comparison of the two types of chorismate synthase. ARO2 expression was tested in the T7 system, and isogenic E. coli strains TG1Δ _aro CP_tac-ARO2 and TG1Δ _aro CP_tac- _aro C were obtained. Comparative analysis of proteins from the cell extracts of these strains and in silico assessment of hybrid RBS efficiency showed that the level of AroC protein synthesis in TG1Δ _aro CP_tac- _aro C was higher than the level of ARO2 synthesis in the TG1Δ _aro CP_tac-ARO2 cells. The introduction of Ptac-ARO2 and Ptac- _aro C modifications led to complete recovery of the growth of the aromatic auxotroph TG1Δ _aro C on minimal mineral medium supplemented with glucose and restored phenylalanine production in the E. coli strain DV1017Δ _aro C, which lacked chorismate synthase activity. The similar positive effects of P_tac- _aro C and P_tac-ARO2 on phenylalanine biosynthesis in the DV1017ΔtyrR strain, in which chorismate synthase played a “bottleneck” role, indicated the absence of a limiting effect of reduced flavin on monofunctional chorismate synthase overexpressed in E. coli cells.     

Engineering the growth pattern and cell morphology for enhanced PHB production by <Emphasis Type="Italic">Escherichia coli</Emphasis>

E. coli JM109?envC?nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109?minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109?envC?nlpD and E. coli JM109?minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109?minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109?envC?nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.     

Enhancement of crystallinity of cellulose produced by <Emphasis Type="Italic">Escherichia coli</Emphasis> through heterologous expression of <Emphasis Type="Italic">bcsD</Emphasis> gene from <Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>

Objectives

To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001.

Results

The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD.

Conclusion

The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

     

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.     

Construction of an alternative glycerol-utilization pathway for improved β-carotene production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Glycerol, which is an inevitable by-product of biodiesel production, is an ideal carbon source for the production of carotenoids due to its low price, good availability and chemically reduced status, which results in a low requirement for additional reducing equivalents. In this study, an alternative carbon-utilization pathway was constructed in Escherichia coli to enable more efficient β-carotene production from glycerol. An aldehyde reductase gene (alrd) and an aldehyde dehydrogenase gene (aldH) from Ralstonia eutropha H16 were integrated into the E. coli chromosome to form a novel glycerol-utilization pathway. The β-carotene specific production value was increased by 50% after the introduction of alrd and aldH. It was found that the glycerol kinase gene (garK), alrd and aldH were the bottleneck of the alternative glycerol metabolic pathway, and modulation of garK gene with an mRS library further increased the β-carotene specific production value by 13%. Finally, co-modulation of genes in the introduced aldH–alrd operon led to 86% more of β-carotene specific production value than that of the strain without the alternative glycerol-utilization pathway and the glycerol-utilization rate was also increased. In this work, β-carotene production of E. coli was significantly improved by constructing and optimizing an alternative glycerol-utilization pathway. This strategy can potentially be used to improve the production of other isoprenoids using glycerol as a cheap and abundant substrate, and therefore has industrial relevance.     

The <Emphasis Type="Italic">yajC</Emphasis> gene from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and its role in ethanol tolerance

The yajC gene (Lbuc_0921) from Lactobacillus buchneri NRRL B-30929 was identified from previous proteomics analyses in response to ethanol treatment. The YajC protein expression was increased by 15-fold in response to 10 % ethanol vs 0 % ethanol. The yajC gene encodes the smaller subunit of the preprotein translocase complex, which interacts with membrane protein SecD and SecF to coordinate protein transport and secretion across cytoplasmic membrane in Escherichia coli. The YajC protein was linked to sensitivity to growth temperatures in E. coli, involved in translocation of virulence factors during Listeria infection, and stimulating a T cell-mediated response of Brucella abortus. In this study, the L. buchneri yajC gene was over-expressed in E. coli. The strain carrying pET28byajC that produces YajC after isopropyl β-d-1-thiogalactopyranoside induction showed tolerance to 4 % ethanol in growth media, compared to the control carrying pET28b. This is the first report linking YajC to ethanol stress and tolerance.     

Molecular cloning and functional characterization of a Cu/Zn superoxide dismutase gene (<Emphasis Type="Italic">CsCSD1</Emphasis>) from <Emphasis Type="Italic">Cucumis sativus</Emphasis>

Superoxide dismutase (SOD) proteins, which are widely present in the plant kingdom, play vital roles in response to abiotic stress. However, the functions of cucumber SOD genes in response to environmental stresses remain poorly understood. In this study, a SOD gene CsCSD1 was identified and functionally characterized from cucumber (Cucumis sativus). The CsCSD1 protein was successfully expressed in E. coli, and its overexpression significantly improved the tolerance of host E. coli cells to salinity stress. Besides, overexpression of CsCSD1 enhanced salinity tolerance during germination and seedling development in transgenic Arabidopsis plants. Further analyses showed that the SOD and CAT (catalase) activities of transgenic plants were significantly higher than those of wild-type (WT) plants under normal growth conditions as well as under NaCl treatment. In addition, the expression of stress-response genes RD22, RD29B and LEA4-5 was significantly elevated in transgenic plants. Our results demonstrate that the CsCSD1 gene functions in defense against salinity stress and may be important for molecular breeding of salt-tolerant plants.     

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.     

Antimicrobial properties of ternary eutectic aluminum alloys

Several Escherichia coli deletion mutants of the Keio collection were selected for analysis to better understand which genes may play a key role in copper or silver homeostasis. Each of the selected E. coli mutants had a deletion of a single gene predicted to encode proteins for homologous recombination or contained functions directly linked to copper or silver transport or transformation. The survival of these strains on pure copper surfaces, stainless steel, and alloys of aluminum, copper and/or silver was investigated. When exposed to pure copper surfaces, E. coli ΔcueO was the most sensitive, whereas E. coli ΔcopA was the most resistant amongst the different strains tested. However, we observed a different trend in sensitivities in E. coli strains upon exposure to alloys of the system Al–Ag–Cu. While minor antimicrobial effects were detected after exposure of E. coli ΔcopA and E. coli ΔrecA to Al–Ag alloys, no effect was detected after exposure to Al–Cu alloys. The release of copper ions and cell-associated copper ion concentrations were determined for E. coli ΔcopA and the wild-type E. coli after exposure to pure copper surfaces. Altogether, compared to binary alloys, ternary eutectic alloys (Al–Ag–Cu) had the highest antimicrobial effect and thus, warrant further investigation.     

<Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> RecX and <Emphasis Type="Italic">Escherichia coli</Emphasis> RecX proteins are capable to replace each other in vivo and in vitro

A plasmid carrying the Deinococcus radiodurans recX gene under the control of a lactose promoter decreases the Escherichia coli cell resistance to UV irradiation and γ irradiation and also influences the conjugational recombination process. The D. radiodurans RecX protein functions in the Escherichia coli cells similarly to the E. coli RecX protein. Isolated and purified D. radiodurans RecX and E. coli RecX proteins are able to replace each other interacting with the E. coli RecA and D. radiodurans RecA proteins in vitro. Data obtained demonstrated that regulatory interaction of RecA and RecX proteins preserves a high degree of conservatism despite all the differences in the recombination reparation system between E. coli and D. radiodurans.     

Probiotic Evaluation of Antimicrobial <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> VJC38 Isolated from the Crop of Broiler Chicken

The aim of this study was to evaluate probiotic properties of antimicrobial Lactobacillus plantarum VJC38 in vitro. L. plantarum VJC38 was isolated from the crop of broiler chicken and characterized using dnaK gene sequence. The inhibitory activities of L. plantarum VJC38 against bacterial and fungal pathogens were evaluated. Antifungal compounds secreted by the strain VJC38 were identified using Gas Chromatography and Mass Spectrometry (GC-MS). The strain was evaluated for its tolerance to low pH, resistance to bile salts, auto-aggregation, co-aggregation with pathogenic Escherichia coli, cell surface hydrophobicity, cholesterol lowering activity, β-galactosidase production, adhesion ability to Caco-2 cells, mucin degradation, hemolytic activity and biogenic amine production. Phylogenetic analysis of dnaK gene of bacterial strain VJC38 showed 99% sequence similarity to Lactobacillus plantarum var. plantarum. It showed effective inhibition against food spoiling and pathogenic organisms like Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Aspergillus niger, Penicillium expansum and Eurotium species. The antifungal compound phenol- 2,4-bis(1,1-dimethylethyl) (PD) was identified in the culture filtrate of L. plantarum VJC38 and reported to have inhibition against Aspergillus species. L. plantarum VJC38 exhibited tolerance to low pH, resistance to bile salts, bile salt hydrolase activity, auto-aggregation (87.5%), co-aggregation with Escherichia coli (55.7%), cholesterol lowering activity (64%), β-galactosidase production (1206 MU), adherence to Caco-2 cells (11%), negative for mucin degradation, hemolytic activity and biogenic amine production. L. plantarum VJC38 could be a good candidate for further investigation in vivo to elucidate its health benefits and to evaluate its technological properties as a bio-protective strain.     

A homomeric geranyl diphosphate synthase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> and its combinatorial optimization for production of geraniol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Geranyl diphosphate (GPP), the unique precursor for all monoterpenoids, is biosynthesized from isopentenyl diphosphate and dimethylallyl diphosphate via the head-to-tail condensation reaction catalyzed by GPP synthase (GPPS). Herein a homomeric GPPS from Camptotheca acuminata, a camptothecin-producing plant, was obtained from 5′- and 3′-rapid amplification of cDNA ends and subsequent overlap extension and convenient PCR amplifications. The truncate CaGPPS was introduced to replace ispA of pBbA5c-MevT(CO)-MBIS(CO, ispA), a de novo biosynthetic construct for farnesyl diphosphate generation, and overexpressed in Escherichia coli, together with the truncate geraniol synthase-encoding gene from C. acuminata (tCaGES), to confirm CaGPPS-catalyzed reaction in vivo. A 24.0 ± 1.3 mg L^?1 of geraniol was produced in the recombinant E. coli. The production of GPP was also validated by the direct UPLC-HRMS^E analyses. The tCaGPPS and tCaGES genes with different copy numbers were introduced into E. coli to balance their catalytic potential for high-yield geraniol production. A 1.6-fold increase of geraniol production was obtained when four copies of tCaGPPS and one copy of tCaGES were introduced into E. coli. The following fermentation conditions optimization, including removal of organic layers and addition of new n-decane, led to a 74.6 ± 6.5 mg L^?1 of geraniol production. The present study suggested that the gene copy number optimization, i.e., the ratio of tCaGPPS and tCaGES, plays an important role in geraniol production in the recombinant E. coli. The removal and addition of organic solvent are very useful for sustainable high-yield production of geraniol in the recombinant E. coli in view of that the solubility of geraniol is limited in the fermentation broth and/or n-decane.