Kil Gene Research Articles

Simple and efficient genome recombineering using kil counter-selection in Escherichia coli

Seamless modification of the Escherichia coli genome using positive selection / negative selection is widely used in metabolic engineering and functional genome analysis. Some excellent negative selection systems have been reported, of which tetA-sacB and inducible toxins system are prominent. To expand the existing negative selection toolkit, we constructed a new negative selection marker system based on kil gene of lambda prophage. The selection stringency of kil was measured and compared with the most widely used counter-selection gene, sacB, at the lacI, ack, and dbpa loci using different E. coli strains. At all these loci of tested strains, the selection stringency of kil significantly exceeds that of sacB by 2- to 28-fold. When dsDNA fragments were employed for recombination, the efficiency for isolating the correct recombinant of kil was significantly higher than that of sacB. This new negative selection system does not require special media or extended incubation time. However, our system cannot be used in host strains containing temperature-sensitive kil gene. A Red system providing plasmid without kil gene is recommended for use together with our system. Our counter-selection system is expected to be an addition to the engineering arsenal of E. coli.

Enhanced Biofilm Formation and Membrane Vesicle Release by Escherichia coli Expressing a Commonly Occurring Plasmid Gene, kil.

Escherichia coli is one of the most prevalent microorganisms forming biofilms on indwelling medical devices, as well as a representative model to study the biology and ecology of biofilms. Here, we report that a small plasmid gene, kil, enhances biofilm formation of E. coli. The kil gene is widely conserved among naturally occurring colicinogenic plasmids such as ColE1 plasmid, and is also present in some plasmid derivatives used as cloning vectors. First, we found that overexpression of the kil gene product dramatically increased biofilm mass enriched with extracellular DNA in the outer membrane-compromised strain RN102, a deep rough LPS mutant E. coli K-12 derivative. We also found that the kil-enhanced biofilm formation was further promoted by addition of physiologically relevant concentrations of Mg2+, not only in the case of RN102, but also with the parental strain BW25113, which retains intact core-oligosaccharide LPS. Biofilm formation by kil-expressing BW25113 strain (BW25113 kil+) was significantly inhibited by protease but not DNase I. In addition, a large amount of proteinous materials were released from the BW25113 kil+ cells. These materials contained soluble cytoplasmic and periplasmic proteins, and insoluble membrane vesicles (MVs). The kil-induced MVs were composed of not only outer membrane/periplasmic proteins, but also inner membrane/cytoplasmic proteins, indicating that MVs from both of the outer and inner membranes could be released into the extracellular milieu. Subcellular fractionation analysis revealed that the Kil proteins translocated to both the outer and inner membranes in whole cells of BW25113 kil+. Furthermore, the BW25113 kil+ showed not only reduced viability in the stationary growth phase, but also increased susceptibility to killing by predator bacteria, Vibrio cholerae expressing the type VI secretion system, despite no obvious change in morphology and physiology of the bacterial membrane under regular culture conditions. Taken together, our findings suggest that there is risk of increasing biofilm formation and spreading of numerous MVs releasing various cellular components due to kil gene expression. From another point of view, our findings could also offer efficient MV production strategies using a conditional kil vector in biotechnological applications.

Screening for conditions of enhanced production of a recombinant β-glucanase secreted into the medium by Escherichia coli

The extracellular production of a hybrid bacterial beta-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a beta-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength. By far the highest extracellular glucanase activity (>200 U ml(-1)) was achieved when a strain harbouring the kil gene under control of a strong synthetic stationary-phase promoter and the glucanase gene under control of a strong synthetic constitutive promoter was cultivated on a complex medium mainly composed of casein peptone, yeast extract, and glycerol.

Factors that influence the extracellular expression of streptavidin in Escherichia coli using a bacteriocin release protein

Aiming to increase production of recombinant streptavidin in Escherichia coli, the effect of different leader sequences, different promoter strengths of the bacteriocin release protein (kil), host strain and medium composition on the expression and secretion into the medium was investigated. Expression vectors containing an expression or secretion unit were constructed with different combinations of leader sequence for the streptavidin gene and promoters for the kil gene and streptavidin gene. Results showed that a high-level extracellular production of streptavidin could be accomplished with E. coli BL21(DE3) by using the leader sequence of the phoA gene, a strong stationary-phase promoter for the kil gene and supplementation of the medium by glycine. Using a stationary-phase promoter for the expression of streptavidin had a negative effect.

Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter

By using a beta-glucanase from Bacillus as a model protein, we investigated whether the secretion competence based on the action of the kil gene can be improved using stronger promoters for the expression of the kil gene. Since the production of extracellular target proteins also depends on the promoter strengths of the target gene, we constructed four expression vectors with all possible combinations of a weak and a strong stationary-phase promoter for the kil gene, and a weak and a strong constitutive promoter, respectively, for the beta-glucanase gene. The results of batch fermentations showed that the use of stronger promoters generally decreased the cell density. However, a drastic increase of productivity of the cells to produce and secrete beta-glucanase resulted in a significantly higher activity of extracellular beta-glucanase. The yield of extracellular beta-glucanase can be increased (to 168 %) by using a strong promoter for the beta-glucanase alone. However, the increase was much higher when the weak promoter of the kil gene was replaced by a strong stationary-phase promoter (to 221 %). An even higher yield of extracellular beta-glucanase was reached when beta-glucanase was expressed by a strong promoter in addition indicating a combinatorial effect. This shows that the extracellular production of a recombinant target gene can be optimized by tuning the promoter strengths of components, the kil gene and the target gene.

Induction of the SOS response by bacteriophage lytic development in Salmonella enterica

Infection of Salmonella enterica with lytic mutants of either P22 or SE1 bacteriophages triggers the expression of its DNA damage-inducible SOS response through a lexA-dependent pathway. This induction of the SOS system strictly requires the presence of the bacteriophage kil gene. Accordingly, plasmid overexpression of the kil gene also promotes the S. enterica SOS network induction. Furthermore, S. enterica Gifsy prophages are induced following the infection with SE1 and P22 lytic derivatives. The observed data reveal a hitherto unknown SOS system-mediated fail-safe mechanism of resident prophages against infection with heteroimmune lytic bacteriophages and suggest a novel role for the kil family of proteins.

Inhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: Experiments with a model system

We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context.

E.coli cell-cycle regulation by bacteriophage lambda.

We re-examined the old but surprising claim of Kourilsky and Knapp that transient expression of genes located downstream of the p(L) promoter of bacteriophage lambda can induce cell-cycle synchrony in a population of Escherichia coli cells. Although we were unable to reproduce a lasting synchrony, a cessation of division, followed by one or two fairly synchronous cell divisions was observed. This line up of the cell cycle was found to be due to two genetically separable events: a temporary block of cell division and, at the same time, a block to the initiation of new rounds of DNA replication. These blocks then release after about one mass doubling so that chromosome replication and cell division occur during a short time interval in all the cells in the population. The cell division block is a result of the transient expression of the lambda kil gene. The block to initiation of DNA replication requires a region that we term bin (blocks initiation) immediately upstream of the xis gene. The region consists of ea22 and ea8.5 and two small open reading frames (ORFs) that flank them. Deletion-substitution mutagenesis suggests that all four ORFs may be required for the initiation block. The ability of the phage to modify two aspects of the host cell cycle presumably reflects a stratagem that provides the phage with an advantage for lysogeny or lytic growth.

Secretory production of recombinant human C-reactive protein in Escherichia coli, capable of binding with phosphorylcholine, and its characterization

Recombinant human CRP (rhCRP) was secreted into culture supernatant of Escherichia coli by co-expressing kil gene that has a function to secrete colicin E1 outside the cell. Highly purified 5 g rhCRP was produced from 180 L culture supernatant by affinity chromatography. The purified rhCRP was indistinguishable from the native one with respect to Ca 2+-dependent binding ability to phosphorylcholine, electrophoretic behavior, N-terminal amino acid analysis, and immunochemical properties. The molecular weight of rhCRP monomer was determined to be 23059.7 Da by TOF/MS analysis. These results indicate that rhCRP has the same protein structure as native one and that rhCRP has the potential as a reference material and/or calibrator of high-sensitivity CRP assay to predict the risk of cardiovascular disease.

Cell toxicity caused by products of the pL operon of bacteriophage lambda

Induction of a λ prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the λpL operon. We genetically modified the λ prophage to determine which λpL operon functions were involved in cell killing. Viability assays and flow cytometry were used to monitor cell death and filamentation. The kil gene was shown to cause cell death and filamentation as described previously. Another killing activity was mapped within the pL operon to the gam gene. Inspection of the DNA sequence showed that there are two possible translation start points for both kil and gam. In both cases, the shorter of the two possible products could cause cell killing. The shorter products were also sufficient for the known filamentation and recombination activities of the respective Kil and Gam functions. The expression level of the pL operon is down-regulated by Cro repressor. In the absence of Cro, higher pL expression levels allow either Kil or Gam to be lethal or growth inhibitory, whereas at lowered expression in Cro-repressed conditions, only Kil is lethal. The filamentation function of Kil and recombination activity of Gam are unaffected at Cro-repressed levels of expression.

High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium.

The Tn5-based transposon Tn5-KIL3 (Miksch et al. 1997c) bearing the kil gene of the ColE1 plasmid of Escherichia coli, which mediates controlled export of periplasmic proteins into the culture medium, was stably integrated into the chromosome of Klebsiella planticola with high transposition frequency. A Bacillus hybrid beta-glucanase located on an RSF1010-derived plasmid was mobilized from E. coli to K. planticola and used as a reporter protein to select strains with high expression and secretion competence. During fermentation experiments it was shown that the production of beta-glucanase in K. planticola was improved to an unexpectedly high level when the enzyme was secreted into the medium. Due to the stationary-phase promoter used for the expression of the kil gene the secretion of beta-glucanase into the medium started at the transition from the exponential to the stationary phase, as in E. coli, and the fraction of secreted protein reached 90%. The results showed that K. planticola may represent an interesting organism for the production of heterologous proteins.

Controlled secretion into the culture medium of a hybrid beta-glucanase by Acetobacter methanolicus mediated by the kil gene of Escherichia coli located on a Tn5-derived transposon.

A Tn5-based transposon bearing the kil gene (killing protein), mediating controlled export of periplasmic proteins into the culture medium, was constructed (Tn5-KIL3). This transposon contained the kil gene of the ColEl plasmid under the growth-phase-dependent promoter of the fic gene (filamentation induced by cAMP) of Escherichia coli, an interposon located upstream of kil, a kanamycin/neomycin-resistance gene, a multiple cloning site and the mob site. The transposition of Tn5-KIL3 to Acetobacter methanolicus showed a moderate transposition frequency (10(-5) -10(-6). By insertion of a Bacillus hybrid beta-glucanase (bgl) as a model protein into the transposon (Tn5-LF3) it was shown that the secretion function as well as the gene of the target protein had been transferred to and stably integrated into the chromosome of A. methanolicus, and that the transposition of Tn5-LF3 was non-specific. beta-Glucanase was highly overexpressed and secreted into the medium during stationary phase. Total and extra-cellular production of beta-glucanase varied depending on the integration site of the transposon. The viability of the bacterial cells was not affected, and cell lysis did not occur.

The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase

Heterologous gene products produced by Escherichia coli cells can be exported into the culture medium by the action of the kil gene of the ColE1 plasmid, which encodes a bacterial release protein. The kil gene was fused with the stationary-phase promoter of the fic gene of E. coli, and a secretion cassette (Kil-Km cassette) containing the regulated kil gene, the Km-resistance gene, and multiple cloning sites for the integration of target genes was constructed. Using the gene for beta-glucanase (bgl) as a target gene, it was shown that the protein produced was only secreted into the medium during the stationary phase. Quasi-lysis and lethality were not observed. The primary effect of the induction of the kil gene was the overproduction of beta-glucanase. The total amount produced per milliliter of bacterial culture was almost threefold higher than that of the corresponding Kil- control. The protein pattern of periplasm and culture medium was analyzed before and after induction of the kil gene expression, indicating that the release of periplasmic proteins is semiselective. This secretion system is the first to use a growth-phase-regulated promoter for the expression of the kil gene.

Extracellular production of a hybrid beta-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors.

Cultivation conditions for the extracellular production of a hybrid beta-glucanase from Bacillus were established by using Escherichia coli JM 109 carrying the plasmid pLF3. This plasmid contained a novel secretion system consisting of the kil gene (killing protein) of plasmid ColE1 under the stationary-phase promoter of either the fic or the bolA gene, an omega interposon (Prentki and Krisch 1984) located upstream of the promoters and a hybrid beta-glucanase gene of Bacillus. When controlled by the fic promoter, the kil gene led to a higher total production of beta-glucanase and a higher protein secretion than when it was under control of the bolA promoter. When the effect of different distances between the stationary-phase promoters and the kil gene was investigated, a shorter distance was generally found to result in a higher secretion. With a complex growth medium, the kinetics of extracellular production of the enzyme depended on several operating variables, such as the salt concentration (NaCl) and the oxygen supply, which were varied by changing the culture volume and the shaking speed. In defined media the secretion of beta-glucanase into the medium was increased significantly by the addition of glycerol as a carbon source and by prolonged cultivation. The strain with the highest production and secretion yield of beta-glucanase [E. coli JM 109(pLF3)] was tested on the fermenter scale.

Identification of a new inhibitor of essential division gene ftsZ as the kil gene of defective prophage Rac.

A gene function carried by a plasmid, causing arrest of cell division in Escherichia coli, has been identified as the product of a short open reading frame of the prophage Rac, previously designated orfE, expressed only under conditions of prophage induction. Because Rac carries a killing function expressed under conditions of zygotic induction, an orfE-defective Rac+ strain was constructed. This strain had lost the killing function, indicating that orfE is kil. Division inhibition by kil was specifically relieved by overexpression of essential division gene ftsZ. The kil gene product acts independently of the min operon, and its effects are increased in conditions of high cyclic AMP (cAMP) receptor protein-cAMP complex levels in the cell. Furthermore, at high levels of expression, kil product distorts the rod shape of the cells. These features distinguish kil-encoded protein from the inhibitory product of gene dicB, which occupies a similar genetic location in Kim (Qin), another defective prophage of Escherichia coli.

Production of Soluble and Active Recombinant Murine Interleukin-2 in Escherichia coli: High Level Expression, Kil-Induced Release, and Purification

We describe the production of soluble murine interleukin-2 (mIL2) and its purification following regulated release in the growth medium of Escherichia coli. The system is based on the ability of the Kil protein of pMB9 to release periplasmic proteins into the growth medium. As the kil gene is under control of the strong, but well regulatable pL promoter, the kil bearing plasmid is stably maintained in the cell. mIL2, fused to the outer membrane protein A (OmpA) signal peptide, was secreted into the periplasm and subsequently released into the growth medium after induction of the kil gene. This strategy allows a quick and easy purification of the heterologous protein without using strong denaturants or detergents, yielding a native protein with a specific biological activity equal to the natural mIL2, The system permits the production of mIL2 at levels up to 16 mg/liter. From a 12-liter fermentation, a final yield of about 30 mg of pure mIL2 was obtained.

Efficient specific release of periplasmic proteins from Escherichia coli using temperature induction of cloned kil gene of pMB9

We have cloned the kil gene of pMB9 under control of the tightly regulated leftward promoter (pL) of coliphage lambda. Three types of plasmids were constructed. In all cases the activity of the lambda promoter is controlled by a thermosensitive cl repressor (product of the c/857 gene) supplied form a resident defective prophage or cloned onto a compatible p 15A-derived plasmid. Induction of the kil protein is brought about by a temperature shift of the culture from 28 degrees C to 42 degrees C. Plasmid pPLc28K1 contains the kil gene including its natural ribosome-binding site and preceded by a transcription termination site. Using a bacterial strain with antitermination properties (e.g., M5219), periplasmic proteins can upon induction be gradually the growth of the host strain. The second plasmid pPLc321K1, contains the kil-coding sequence preceded by an engineered ribosome binding site derived from the attenuator of the Escherichia coli tryptophan operon. With this plasmid induction of the Kil protein is very rapid and specific release of the periplasmic proteins in essentially complete within 30 min after induction. In a third construct, pcl857K1, the pL-kil cassette together with c/857 allele are present on the same replicon, which is compatible with ColE1-derived expression vectors. This configuration allows accumulation in the periplasm of cloned gene products, induced by, e.g., tac or trp promoters at low temperature and subsequent release into the medium following increase of the temperature of the culture. Under repressed conditions (growth at low temperature) all plasmids are perfectly stable in a large number of E. coli strains tested, also when cultivated on a 20-L fermentor scale. Controlled, heat-induced release of periplasmic proteins is highly specific and applicable at relatively high cell densities. The method therefore is an attractive alternative to cumbersome osmotic shock procedures for large-scale cultures.

A new DNA cloning/sequencing vector with a built-in mechanism for generation of nested deletions using transposon Tn3

We have constructed a new cloning/sequencing vector suitable for sequencing of DNA fragments too long to be sequenced in a single step. This vector plasmid contains inverted repeats (IR) of transposon Tn3, the kil and cI857 genes of phage λ, and multiple cloning sites (MCS). Escherichia coli cells harboring plasmids containing nested deletions from one of the Tn3 IR ends, across kil, to variable end points, can be positively selected by plating at 42°C. The deletion products, fractionated according to their size by agarose-gel electrophoresis, can be sequenced by using a synthetic primer whose 3′-end is located within the Tn3 IR, and the total sequence of the insert can be constructed from the partial sequences.

Cold-sensitive phenotype of Escherichia coli cells harboring a plasmid carrying the kil gene of phage lambda brought under control of cI857 gene promoters

A plasmid (pI-14), containing part of the phage λ control region ( EcoRI-D fragment of λ cJ857) with a large deletion between genes rex and kil, confers a cold-sensitive (cs) phenotype on the host bacteria, whereas the parent plasmid (pMM200) without the deletion made the host bacteria ( Escherichia coli strain DOO) high-temperature sensitive. This phenomenon could be explained on the basis of the sequence analysis of the deletion. Upon this deletion, the λ kil gene, which was originally under the control of the p L promoter, was brought under the control of the λ cI promoters, resulting in the reversal of the host cell response to temperature. This example shows that gene circuits showing diametrically opposite responses to environmental factors (in this case, temperature) can be constructed from the same elements when the effector gene (here, kil) is connected in different ways to the sensor gene (here, cI857). This cold-dependent killing activity was also dependent on the crp + state of the host.

Lambda Kil-Mediated Lysis Requires the Phage Context

The λ kil gene has been shown to be responsible for premature lysis effected by addition of chloramphenicol between 15 and 20 min after thermal induction of a λ prophage. Here, we localized the kil reading frame. The kil gene, represented by λ orf47, overlaps genes cIII and gam. Expression of the plasmid-borne kil gene resulted in growth arrest, a reduction of colony-forming units and filament formation. However, kil-mediated cell lysis could not be triggered by chloramphenicol when the plasmid borne kil gene was expressed, suggesting that kil-induced cell lysis requires the phage context.