Recombinant Bacterial Cells Research Articles

Live bacterial cells as analytical tools for speciation analysis: Hypothetical or practical?

This review tries to show the capability of live bacterial cells in speciation analysis, so it emphasizes speciation analysis of metalloid and organometalloid species by combining an uptake process by live bacterial cells with a specific detector. Especially covered in depth is the uptake process, because better understanding of equilibrium and retention mechanisms provides the basis for optimizing the use of bacterial cells. We outline the main parameters affecting the uptake process and the analytical characteristics. We also cover bacteria-based biosensors that represent a research field of growing interest in using live bacteria. We also highlight some future considerations about the role of recombinant bacterial cells in analytical chemistry.

Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

BackgroundNative as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099) of the S-layer protein and EGFP (enhanced green fluorescent protein), in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells.ResultsUpon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua)-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S.) cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase) promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM) studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE) promoter.ConclusionThe mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of otherwise instable proteins in their native conformation. In addition the self assembly properties of the fusion proteins allow their simple purification. Moreover the binding properties of the S-layer part can be used to immobilize the fusion proteins to various surfaces. Arrays of highly ordered and densely structured proteins either immobilized on surfaces or within living cells may be advantageous over the respective soluble variants with respect to stability and their potential interference with cellular metabolism.

Expression and one-step ion-exchange purification of (AAR)IL-8 (human IL-8 receptor antagonist)

Interleukin-8 (IL-8) is C-X-C chemokine, which is produced by a variety of cells. IL-8 plays an important role in the inflammatory response and may be a therapeutic target for some inflammatory diseases. To develop an IL-8 receptor antagonist, (AAR)IL-8 (IL-8 receptor antagonist) was constructed and successfully expressed in Escherichia coli. (AAR)IL-8 could be easily purified by one-step SP–Sepharose fast flow column after the lysate of recombinant bacterial cells was heated at 70 °C for 10 min. The purity of (AAR)IL-8 is more than 95%. This purification process resulted in final purified yields of 4.29 mg (AAR)IL-8/g cell paste. In addition, the purified (AAR)IL-8 can significantly inhibit the chemotaxis that was induced by human IL-8 in vitro and in vivo. These results showed that this purification process is very simple and effective. It could be easily amplified at a larger scale. (AAR)IL-8 might find use as a new therapeutic IL-8 receptor antagonist for some acute and chronic inflammatory diseases.

Asymmetric Dihydroxylation of Cinnamonitrile to trans‐3‐[(5S,6R)‐5,6‐Dihydroxycyclohexa‐1,3‐dienyl]‐acrylonitrile using Chlorobenzene Dioxygenase in Escherichia coli (pTEZ30)

AbstractAsymmetric cis‐dihydroxylations of aromatic compounds are catalyzed by bacterial dioxygenases. In order to prevent through conversion, either dihydrodiol dehydrogenase blocked mutant strains or recombinant bacterial cells are used as biocatalysts for synthetic purposes. We characterized the cis‐dihydroxylation of cinnamonitrile by chlorobenzene dioxygenase (CDO) in recombinant E. coli on different reaction scales. The absolute stereochemistry of the product was determined to be trans‐3‐[(5S,6R)‐5,6‐dihydroxycyclohexa‐1,3‐dienyl]‐acrylonitrile. The cells showed a maximum specific activity of 3.76 U/g cdw in shake‐flask experiments. Stable expression of the dioxygenase genes in E. coli JM101 (pTEZ30) resulted in increasing volumetric productivities. The maximum volumetric productivities of 80 and 92 mg product/L/h were achieved on 2‐L and 30‐L scales, respectively. The specific growth rate correlated with the volumetric productivity during the biotransformations. An average volumetric productivity of 40 mg product/L/h in reactors on 2‐L and 30‐L scales resulted in 0.96 and 16.4 g of isolated product at the end of the biotransformations. This points out the need for metabolically active cells and controllable expression systems for achieving high volumetric productivities for cofactor dependent biooxidations. We have now applied this concept for the asymmetric dihydroxylation of the non‐natural substrate cinnamonitrile using multicomponent CDO in tailored E. coli JM101 in long‐term reactions.

Construction of recombinant S-layer proteins (rSbsA) and their expression in bacterial ghosts – a delivery system for the nontypeable Haemophilus influenzae antigen Omp26

This study has investigated the feasibility of a combination of recombinant surface layer (S-layer) proteins and empty bacterial cell envelopes (ghosts) to deliver candidate antigens for a vaccine against nontypeable Haemophilus influenzae (NTHi) infections. The S-layer gene sbsA from Bacillus stearothermophilus PV72 was used for the construction of fusion proteins. Fusion of maltose binding protein (MBP) to the N-terminus of SbsA allowed expression of the S-layer in the periplasm of Escherichia coli. The outer membrane protein (Omp) 26 of NTHi was inserted into the N-terminal and C-terminal regions of SbsA. The presence of the fused antigen Omp26 was demonstrated by Western blot experiments using anti-Omp26 antisera. Electron microscopy showed that the recombinant SbsA maintained the ability to self-assemble into sheet-like and cylindrical structures. Recombinant E. coli cell envelopes (ghosts) were produced by the expression of SbsA/Omp26 fusion proteins prior to gene E-mediated lysis. Intraperitoneal immunization with these recombinant bacterial ghosts induced an Omp26-specific antibody response in BALB/c mice. These results demonstrate that the NTHi antigen, Omp26, was expressed in the S-layer self-assembly product and this construct was immunogenic for Omp26 when administered to mice in bacterial cell envelopes.

Immunogenicity of an HIV-1 gag DNA vaccine carried by attenuated Shigella

The use of live attenuated invasive bacteria as a carrier for DNA-based vaccines has been reported recently. In this study, we used a Shigella flexneri serotype 2a rfbF mutant for immunization of a DNA vaccine coding for HIV-1 SF2 Gag. The recombinant bacterial vector delivered gag DNA to mammalian cells in vitro resulting in Gag protein expression, and was found to have a low level of pathogenicity among a number of Shigella cell spread defective mutants tested. Intranasal immunization of mice with live recombinant bacterial cells induced a gag-specific cellular immune response similar to that seen with i.m. injection of naked DNA. Importantly, a strong boosting effect was observed in mice primed with DNA, suggesting utility of bacterial vectors in prime-boost vaccination regimens.

A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen.

The mature crystalline bacterial cell surface (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and assembles into an oblique lattice type. As the deletion of up to 179 C-terminal amino acids did not interfere with the self-assembly properties of SbsC, the sequence encoding the major birch pollen allergen (Bet v1) was fused to the sequence encoding the truncated form rSbsC(31-920). The S-layer fusion protein, termed rSbsC/Bet v1, maintained the ability to self-assemble into flat sheets and open-ended cylinders. The presence and the functionality of the fused Bet v1 sequence was proved by blot experiments using BIP1, a monoclonal antibody against Bet v1 and Bet v1-specific IgE-containing serum samples from birch pollen allergic patients. The location and accessibility of the allergen moiety on the outer surface of the S-layer lattice were demonstrated by immunogold labeling of the rSbsC/Bet v1 monolayer, which was obtained by oriented recrystallization of the S-layer fusion protein on native cell wall sacculi. Thereby, the specific interactions between the N-terminal part of SbsC and a distinct type of secondary cell wall polymer were exploited. This is the first S-layer fusion protein described that had retained the specific properties of the S-layer protein moiety in addition to those of the fused functional peptide sequence.

ChemInform Abstract: Recombinant Bacterial Cells as Efficient Biocatalysts for the Production of Nucleosides

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Oxygenation reactions of various tricyclic fused aromatic compounds using Escherichia coli and Streptomyces lividans transformants carrying several arene dioxygenase genes.

Bioconversion (biotransformation) experiments on arenes (aromatic compounds), including various tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the cells of Escherichia coli transformants expressing several arene dioxygenase genes. E. coli carrying the phenanthrene dioxygenase (phdABCD) genes derived from the marine bacterium Nocardioides sp. strain KP7 converted all of these tricyclic aromatic compounds, while E. coli carrying the Pseudomonas putida F1 toluene dioxygenase (todC1C2BA) genes or the P. pseudoalcaligenes KF707 biphenyl dioxygenase (bphA1A2A3A4) genes was not able to convert these substrates. Surprisingly, E. coli carrying hybrid dioxygenase (todC1::bphA2A3A4) genes with a subunit substitution between the toluene and biphenyl dioxygenases was able to convert fluorene, dibenzofuran, and dibenzothiophene. The cells of a Streptomyces lividans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconversion of various tricyclic fused aromatic compounds. The ability of this actinomycete in their conversion was similar to that of E. coli carrying the corresponding genes. Products converted from the aromatic compounds with these recombinant bacterial cells were purified by column chromatography on silica gel, and identified by their MS and 1H and 13C NMR analyses. Several products, e.g., 4-hydroxyfluorene converted from fluorene, and cis-1,2-dihydroxy-1,2-dihydrophenanthridine, cis-9,10-dihydroxy-9,10-dihydrophenanthridine, and 10-hydroxyphenanthridine, which were converted from phenanthridine, were novel compounds.

Optimizing initial plasmid copy number distribution for improved protein activity in a recombinant fermentation

Recombinant bacterial cells in a fermentation broth rarely contain the same number of plasmids, even though this simplification is often used. Recent work has however indicated limitations of the simplified approach. Based on these studies, the distribution of plasmid copy numbers per cell has been represented macroscopically here in a Gaussian form for the fraction of biomass as a function of the copy number. Applying this distribution and an experimentally validated kinetic model to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) synthesis by Escherichia coli containing the plasmid pBR Eco gap, it is seen that GAPDH production in a batch fermentation is maximized by a particular initial (non-zero) copy number variance and an optimal duration. To implement this distribution in a bioreactor, it is suggested that the profile may be discretized, inocula corresponding to the mean copy number of each fraction prepared, and then combined to obtain the seed culture.

Time Course of SDS–alkaline lysis of recombinant bacterial cells for plasmid release

SDS–alkaline lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of lysis reaction. The results of the experiments showed that cell lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number. The high molecular weight globular proteins and chromosomal DNA were denatured, and the resulting changes in rheometric measurements characterised the denaturation time. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 768–770, 1998.

Time Course of SDS–alkaline lysis of recombinant bacterial cells for plasmid release

SDS-alkaline lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of lysis reaction. The results of the experiments showed that cell lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number. The high molecular weight globular proteins and chromosomal DNA were denatured, and the resulting changes in rheometric measurements characterised the denaturation time.

Simultaneous topographic and fluorescence imagings of recombinant bacterial cells containing a green fluorescent protein gene detected by a scanning near-field optical/atomic force microscope.

A scanning near-field optical/atomic force microscope (SNOAM) system was applied for simultaneous topographic and fluorescence imaging of biological samples in air and liquid. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic force microscopy cantilever and as a scanning near-field optical microscopy probe. Optical resolution of this system was about 50-100 nm in fluorescence mode for fluorescent latex beads on a quartz glass plate. Green fluorescent protein (GFP) is a convenient indicator of transformation and should allow cells to be separated by fluorescence-activated cell sorting. The gene coding to GFP was cloned in recombinant Escherichia coli. The SNOAM system used 458- or 488-nm irradiation from a multiline Ar ion laser for excitation of GFP, since a native GFP has been known to give a maximum at 395 nm and a broad absorption spectrum until 500 nm. Topographic and fluorescence images of recombinant E. coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope. A nanoscopic GFP fluorescence spectrum was obtained by positioning the optical fiber probe above the bright area of the E. coli cells. Comparing topographic and fluorescence images, it can be seen that individual E. coli cells expressed different fluorescence intensities. Fluorescence obtained by SNOAM indicated that GFP oxidation possibly occurred near the cell surface. A SNOAM system also indicated the possibility of precise imaging of native cells in liquid.

Molecular cloning and characterization of rat sperm surface antigen 2B1, a glycoprotein implicated in sperm-zona binding.

The rat sperm surface antigen, 2B1, that has been proposed to play a key role in sperm adhesion to the zona pellucida, has been cloned and its entire cDNA sequenced. Northern blot analysis indicates that 2B1 is encoded by a 2.2-kb RNA transcript that is abundantly expressed in the testis. The deduced protein sequence contains 512 amino-acid residues with a strong candidate signal sequence and C-terminal transmembrane domain. Data base searches reveal a high degree of sequence similarity to guinea pig, rabbit, monkey, and human PH20 sperm surface antigens, and a lower degree of similarity to honey bee and whiteface hornet venom hyaluronidases. Rat 2B1 antigen also possesses hyaluronidase activity, suggesting that it is a bifunctional protein with putative roles in the dispersion of cumulus oophorus cells as well as zona adhesion. However, while it would appear that 2B1 is the rat homologue of the guinea pig PH20 antigen, they differ in a number of important biochemical respects (including their mode of attachment to the sperm membrane and distribution between soluble and membrane-bound fractions), as well as in their localization on the sperm membrane. Expression of regions of the 2B1 protein in recombinant bacterial cells has allowed a preliminary mapping of the 2B1 epitope, and has provided more definitive information on the endoproteolytic processing of 2B1 during epididymal transit.

Synthesis of mammalian prolylendopeptidase in Escherichia coli and analysis of the recombinant protein

Prolylendopeptidase is a cytoplasmic serine proteinase which hydrolyses peptide bonds at the C-terminal side of prolines. Here, the expression in Escherichia coli of the cDNA coding for the enzyme from porcine brain is reported. Furthermore, the purification of active prolylendopeptidase from the extract of recombinant bacterial cells is described. The large amounts of protein obtained were used to gain insight in the substrate specificity of recombinant prolylendopeptidase, by using internally quenched fluorescent substrates.

An adjustable expression system for controlling growth rate, plasmid maintenance, and culture dynamics

Recombinant bacterial cells express various levels of model product proteins if the genes of interest are regulated by controllable promoters. The level of gene expression influences the growth-rate differential between plasmid-bearing and plasmid-free cells, and thereby affects the culture dynamics of a plasmid-containing cell population. An expression system has been designed in which host Escherichia coli cells contain the pil operon controlled by a tac promoter; these cells are transformed with plasmids that contain the repressor gene, lacl, for the tac promoter, in combination with an expression system for a model protein, chloramphenicol acetyl transferase (CAT). Experimental and theoretical results show that plasmid-bearing cells can be maintained as dominant in continuous cultures without selective pressure when 12% or less of the cells' total protein is the model product protein, CAT. This is because the segment cells produce pili greatly in excess of normal wild-type levels, and thus have more of a metabolic burden than do the plasmid-bearing cells that overproduce CAT. However, when the level of the plasmid-directed CAT expression is increased above 12% of the cells' total protein, the growth rate of the plasmid-bearing cells decreases to a value lower than that of the segregant cells. Therefore, plasmid-containing cells lose their selective advantage at this expression level, and cannot be maintained as the dominant cell type in a continuous culture unless antibiotic or other positive selection methods are used. By controlling the growth rate differential of this bacterial host/plasmid system, a variety of interesting competitive culture dynamics is investigated. All experimental measurements for continuous cultures are in very good agreement with theory using kinetic parameters determined from independent batch experiments.

Analysis of two-stage recombinant bacterial fermentations using a structured kinetic model

A structured kinetic model has been employed to analyze the performance of a two-stage continuous fermentation of a recombinant Escherichia coli. Separating the cell growth phase from the gene expression phase in two fermentors minimizes the growth rate difference between the recombinant cells and the plasmid-free cells in the first fermentor, thereby increasing the plasmid stability. The plasmid-harboring cells from the first fermentor are continuously fed into the second fermentor, in which the foreign protein synthesis is turned on by the addition of the inducer. Consequently, the recombinant cells experience an immediate reduction in growth rates as soon as they enter the second stage and then recover to synthesize the foreign protein. To analyze the fermentation performance contributed by these cells with different intracellular foreign protein levels and growth rates, a novel method for determining the residence time distribution of the growing cells in the second stage has been formulated. Combined with this method, the structured kinetic model for recombinant bacterial cells is used to predict the plasmid stability and foreign productivity at various operation conditions, such as induction strength and dilution rates. This model can provide us with thorough understanding of the characteristics of the two-stage fermentations, and is useful for the development of large scale continuous cultures of recombinant bacteria.