OmpC Porin Research Articles

Rapid detection of porins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

The rapid and cost-efficient determination of carbapenem resistance is an important prerequisite for the choice of an adequate antibiotic therapy. A MALDI-TOF MS-based assay was set up to detect porins in the current study. A loss of the components of porin alone such as OmpK35/OmpK36 or together with the production of carbapenemases will augment the carbapenem resistance. Ten strains of Escherichia coli and eight strains of Klebsiella pneumoniae were conducted for both sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF MS analysis. MALDI-TOF/TOF MS analysis was then performed to verify the correspondence of proteins between SDS-PAGE and MALDI-TOF MS. The results indicated that the mass spectrum of ca. 35,000, 37,000, and 38,000-m/z peaks of E. coli ATCC 25922 corresponded to OmpA, OmpC, and OmpF with molecular weight of approximately ca. 38, 40, and 41 kDa in SDS-PAGE gel, respectively. The band of OmpC and OmpF porins were unable to be distinguished by SDS-PAGE, whereas it was easy to be differentiated by MALDI-TOF MS. As for K. pneumoniae isolates, the mass spectrum of ca. 36,000 and 38,600-m/z peaks was observed corresponding to OmpA and OmpK36 with molecular weight of approximately ca. 40 and 42 kDa in SDS-PAGE gel, respectively. Porin OmpK35 was not observed in the current SDS-PAGE, while a 37,000-m/z peak was found in K. pneumoniae ATCC 13883 and carbapenem-susceptible strains by MALDI-TOF MS which was presumed to be the characteristic peak of the OmpK35 porin. Compared with SDS-PAGE, MALDI-TOF MS is able to rapidly identify the porin-deficient strains within half an hour with better sensitivity, less cost, and is easier to operate and has less interference.

Single cell super-resolution imaging of E. coli OmpR during environmental stress.

Two-component signaling systems are a major strategy employed by bacteria, and to some extent, yeast and plants, to respond to environmental stress. The EnvZ/OmpR system in E. coli responds to osmotic and acid stress and is responsible for regulating the protein composition of the outer membrane. EnvZ is a histidine kinase located in the inner membrane. Upon activation, it is autophosphorylated by ATP and subsequently, it activates OmpR. Phosphorylated OmpR binds with high affinity to the regulatory regions of the ompF and ompC porin genes to regulate their transcription. We set out to visualize these two-components in single bacterial cells during different environmental stress conditions and to examine the subsequent modifications to the bacterial nucleoid as a result. We created a chromosomally-encoded, active, fluorescent OmpR-PAmCherry fusion protein and compared its expression levels with RNA polymerase. Quantitative western blotting had indicated that these two proteins were expressed at similar levels. From our images, it is evident that OmpR is significantly less abundant compared to RNA polymerase. In cross-sectional axial images, we observed OmpR molecules closely juxtaposed near the inner membrane during acidic and hyposomotic growth. In acidic conditions, the chromosome was compacted. Surprisingly, under acidic conditions, we also observed evidence of a spatial correlation between the DNA and the inner membrane, suggesting a mechanical link through an active DNA-OmpR-EnvZ complex. This work represents the first direct visualization of a response regulator with respect to the bacterial chromosome.

Immunization of main and minority Salmonella Typhi porins induce protective antibodies against bacteria.

Immunization of main and minority Salmonella Typhi porins induce protective antibodies against bacteria.

Investigation of the EnvZ/OmpR Bacterial Signaling System using Single Particle Tracking and Single Molecule Force Spectroscopy

The EnvZ/OmpR two-component regulatory system in bacteria regulates the porins OmpF and OmpC in response to changes in osmolality of the environment. The cytoplasmic portion of the sensor kinase EnvZ (EnvZc) has been shown to be the osmo-sensor (Wang et al., EMBO J, 2012). At high osmolality, the four-helix bundle domain of the EnvZc dimer is relaxed, increasing the autophosphorylation of EnvZ. On the other hand, at low osmolality, the four-helix bundle domain is stretched, leading to lower autophosphorylation. After EnvZ is autophosphorylated, the phosphoryl group is transferred to the DNA binding protein OmpR. OmpR∼P binds to the ompF and ompC promoters to regulate their expression. Since the osmo-sensing capability of EnvZ is associated with stretch-relaxation dynamics, we wanted to understand and characterize the mechanical stability of EnvZ in the absence and presence of ATP- or OmpR/OmpR∼P-binding. In our initial experiments, we used magnetic tweezers to stretch a single EnvZc molecule. This gives two unfolding events from the dimerization and ATP binding domains. Although there is a wealth of in vitro biochemical studies of EnvZ and OmpR, an understanding of the signal transduction from EnvZ to OmpR in vivo is poorly understood. We used single-particle tracking of an OmpR-PAmcherry fusion to examine the relative abundance of DNA-bound/free OmpR obtained from the mobility of the OmpR-PAmCherry tracks. This was then used to determine how the fraction of bound/free OmpR changed in response to environmental stress. Our preliminary results indicate that the bound fraction of OmpR is localized at certain cellular positions, which could be the location of the genes regulated by OmpR. Supported by Mechanobiology RCE from the Ministry of Education, Singapore and VA-IO1BX000372 to LJK.

How do the protonation states of E296 and D312 in OmpF and D299 and D315 in homologous OmpC affect protein structure and dynamics? Simulation studies

In this study, the structural and dynamic properties of two major porins (OmpF and OmpC) in Escherichia coli are investigated using molecular dynamics (MD) simulations. Both porins have the extracellular loop L3 folded halfway through the pore to form a constriction area. The solute influx and efflux are controlled by the L3 movement. E296 and D312 in OmpF and homologous D299 and D315 in OmpC located on the barrel wall are found to play a key role in L3 gating activity. All possible charged states of both E296(D299) and D312(315) are applied in this study to observe changes in overall structure and especially L3 movement. The results show that different protonation states of both residues cause the large-scale deviations in structure and pore cavity especially in OmpF. Fully charged E296(D299) and D312(315) increase the protein flexibility significantly. Deprotonating at least one of E296(D299) and D312(315) helps to fasten L3 to the barrel wall and maintain pore size. Lacking of interactions with D312(315) can lead to the pore closure in OmpF. Comparing with OmpC, not only is OmpF less stable, but it is also more sensitive to the charge states of both E296(D299) and D312(315).

High Salt Concentrations Increase Permeability through OmpC Channels of Escherichia coli

OmpF and OmpC porin channels are responsible for the passage of small hydrophilic solutes across the outer membrane of Escherichia coli. Although these channels are two of the most extensively studied porin channels, what had yet remained elusive was the reason why OmpC shows markedly lower permeability than OmpF, despite having little difference in its channel size. The OmpC channel, however, is known to contain a larger number of ionizable residues than the OmpF channel. In this study, we examined the channel property of OmpF and OmpC using the intact cell of E. coli, and we found that the permeability of several β-lactams and lactose through OmpC became increased to the level comparable with OmpF with up to 0.3 m salt that may increase the Debye-Hückel shielding or with 2% ethanol or 0.3 m urea that may perturb the short range ordering of water molecules. Replacing 10 pore-lining residues that show different ionization behavior between OmpC and OmpF led to substantial conversion of channel property with respect to their permeability and response to external salt concentration. We thus propose that the overall configuration of ionizable residues in the channel that may orient water molecules and the electrostatic profile of the channel play a decisive role in defining the channel property of the OmpC porin rather than its channel size.

Scale-down effect on the extracellular proteome of Escherichia coli: correlation with membrane permeability and modulation according to substrate heterogeneities

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane permeability and the synthesis of several outer-membrane components allowing to cope with substrate limitation commonly found in high cell density culture. A comparative analysis of protein leakage has thus been performed in well-mixed bioreactors and in scale-down devices. The extracellular proteome of E.coli has been investigated by 2D-gel electrophoresis and identified by subsequent MALDI-TOF analysis. On 110 picked spots, 67 proteins have been identified and the sub-localisation and the molecular function of these proteins have been determined. A majority of the extracellular proteome was composed of outer-membrane and periplasmic proteins (64 %) confirming the fact that leakage is involved in high cell density cultures. About 50 % of this extracellular proteome was composed of transport and binding proteins. Furthermore, the more abundant spots on the gel corresponded to porin proteins and periplasmic transporters. In particular, the OmpC porin was found to be very abundant. Moreover, the scale-down effect on this extracellular proteome has been investigated by two-dimensional differential in-gel electrophoresis analysis (2D-DIGE), and significant differences have been observed by comparison with culture carried out in well-mixed systems. Indeed, since substrate limitation signal is alleviated in this kind of apparatus, cell permeability was lowered as shown by flow cytometry. In scale-down conditions, protein leakage was thus less abundant.

Chemical Structures and Transit Kinetics of Carbapenems Translocating Through E. Coli OmpC

Previously, we proposed a novel mechanism of antibacterial translocation through the general diffusion porin OmpC in Gram-negative E. coli (Abstract 12-A-3857-BPS, 2668-Pos). Our proposed mechanism explains the observed rapid translocation of substrates capable of replacing the H-bonds of solvating water in the channel constriction zone upon evacuation of this water to bulk solvent. This hypothesis is based on previous work linking protein-ligand association and dissociation barriers in general to the costs of water transfer to/from binding sites. In the current study, we investigated a set of 8 carbapenems (including known drugs and antibacterials in development) for their structure-translocation kinetic relationships with OmpC using electrophysiology and microbiology methods. Taking into account the supporting electrophysiology and microbiology data, we demonstrated that these molecules likewise translocate through OmpC porin at rates that depend on their polar composition and H-bond replacement ability. The additional acidic group of ertapenem compared to other analogs promoted the highest entry rate into OmpC (kon ∼2x104 M-1s-1). Zwitterionic compounds with highly polar groups attached to the penem-2 ring (i.e. panipenem, imipenem and doripenem) exhibited faster kon (>104 M-1s-1), while those zwitterionic analogs with fewer exposed polar groups (i.e. meropenem and biapenem) exhibited slower kon (∼5x103 M-1s-1). Tebipenem pivoxil, a pro-dug developed for oral absorption, and razupenem with a change to thiazol-2-yl-thio moiety exhibited the slowest kon rates in to OmpC (∼1.5x103 M-1s-1) and also showed interaction with the phospholipid membrane. Our findings may help better understand the molecular mechanisms underlying antibiotic uptake through the outer membrane of Gram-negative bacteria, which is a key step in achieving antibiotic exposure and efficacy at intracellular targets.

Molecular Competition between Large and Small Polyethylene-Glycols (PEGs) Partitioning into OMPC Porin Channels

Molecular crowding by polyethylene glycols (PEGs) has been employed in a wide range of inquiries into molecular structure and dynamics. Investigating the partitioning of crowded polymers into biological nanopores reveals properties also seen in, e.g., DNA ejection and compaction into viral capsids, as well as in facilitated molecule transport. Capitalizing on our previous work on the partitioning of binary PEG mixtures into the benchmark Alpha-Hemolysin nanopore, we have now studied partitioning of mono- and polydisperse polymer solutions into comparably sized, yet differently structured, diffusion channels. We find that in the presence of large non-partitioning PEG 3400, the small, otherwise equipartitioning PEG 200 partitions disproportionately. Specifically, with 15% PEG 3400, an added 15% PEG 200 reduces channel conductance by 65% (compared with channel conductance in polymer-free solutions), while 15% PEG 200 alone reduces channel conductance by only 45%. We employed open-channel noise analysis to investigate channel diffusion properties of individual polymers, and to compare them with their properties in bulk water solution. In this way we demonstrate and quantify crowding-assisted transport through nanopores in mixed-polymer systems.

Emergence of Escherichia coli Sequence Type 131 Isolates Producing KPC-2 Carbapenemase in China

Twenty-two KPC-2-producing Escherichia coli isolates were obtained from three hospitals in Hangzhou, China, from 2007 to 2011. One isolate, with OmpC porin deficiency, exhibited high-level carbapenem resistance. Pulsed-field gel electrophoresis showed that few isolates were indistinguishable or closely related. Multilocus sequence typing indicated that sequence type 131 (ST131) was the predominant type (9 isolates, 40.9%), followed by ST648 (5 isolates), ST405 (2 isolates), ST38 (2 isolates), and 4 single STs, ST69, ST2003, ST2179, and ST744. Phylogenetic analysis indicated that 9 group B2 isolates belonged to ST131, and 5 of 11 group D isolates belonged to ST648. Only one group B1 isolate and one group A isolate were identified. A representative plasmid (pE1) was partially sequenced, and a 7,788-bp DNA fragment encoding Tn3 transposase, Tn3 resolvase, ISKpn8 transposase, KPC-2, and ISKpn6-like transposase was obtained. The blaKPC-2-surrounding sequence was amplified by a series of primers. The PCR results showed that 13 isolates were consistent with the genetic environment in pE1. It is the first report of rapid emergence of KPC-2-producing E. coli ST131 in China. The blaKPC-2 gene of most isolates was located on a similar genetic structure.

Role of porin proteins in acquisition of transferrin iron by enteropathogens

Acquisition of iron from key innate immune defence proteins such as transferrin (Tf) and lactoferrin is an important mechanism by which pathogenic bacteria obtain essential iron for growth within their host. Bacterial species that do not produce siderophores often use specific Tf-binding proteins, the best characterized being the Neisseriaceae-type Tf-binding proteins TbpA and TbpB. Previous work from our laboratory has shown that siderophore-producing enteric species such as Escherichia coli also readily bind Tf, although no genomic evidence exists for Tbp-like Tf-binding proteins. Application of proteomic analyses and molecular mutagenesis strategies to an enteropathogenic E. coli identified the OmpA and OmpC porins as Tf-binding proteins. Mutagenesis of the ompA or ompC genes affected E. coli Tf binding and, furthermore, compromised the ability of the ompA mutant to respond to growth promotion by certain catecholamine stress hormones. Evidence was also found to implicate the OmpA porin as an entry point for catecholamine stress hormones. Further proteomic analyses in other bacterial pathogens revealed wide-scale involvement of porins in Tf binding: Salmonella typhimurium (OmpC), and Shigella sonnei, Shigella flexneri and Shigella boydii (OmpC and/or OmpA). This study shows that in addition to their existing housekeeping functions, the Gram-negative porin family of proteins can also act as Tf-capture proteins for those bacteria that lack the classical Neisseriaceae-type Tf-binding proteins.

Discrimination of E. coli B E BL21(DE3) porin mutants by attenuated total reflectance Fourier-transformed infrared (ATR-FTIR) spectroscopy

Attenuated total reflectance Fourier-transformed infrared (ATR-FTIR) spectroscopy measures the changes that occur in a totally internally reflected infrared beam when the evanescent wave it produces penetrates only a few micrometers (0.5-5μm) into the sample, deposited on top of an optically dense crystal (Figure 1) [1]. Both conventional FTIR and ATR-FTIR have been successfully used for bacteria discrimination and classification, as each species presents an unique spectral “fingerprint”, making it a promising, fast and reliable screening method for pathogen identification in a clinic setting [2-3]. In this study, ATR-FTIR is used in an attempt to discriminate between Escherichia (E.) coli BE BL21(DE3) and a derived mutant lacking the major E. coli porins OmpF and OmpC (Table 1). Porins are transmembrane proteins located in the outer membrane of Gram-negative bacteria such as E. coli that allow the passage of various solutes, including antibiotics, playing therefore an important role in the development of antibiotic resistance. The ability to effectively distinguish between strains differing only in the porin profile would mean more than just information on structural differences, but also an initial indication on resistance mechanisms [4]. Strain Relevant genotype

Antibiotic Trapping by Plasmid-Encoded CMY-2 β-Lactamase Combined with Reduced Outer Membrane Permeability as a Mechanism of Carbapenem Resistance in Escherichia coli

A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CR E. coli lacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CR E. coli possessed bla(CTX-M-15) and bla(OXA-1). The CR E. coli strain additionally harbored bla(CMY-2) and demonstrated a >15-fold increase in β-lactamase activity against nitrocefin, but no hydrolysis of meropenem was detected. However, nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation either in vitro or in vivo with meropenem, indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into various E. coli strains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance. In conclusion, carbapenem resistance emerged by a combination of elevated β-lactamase production and lack of porin expression. Due to the reduced outer membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the large amount of the β-lactamase. This study, therefore, provides evidence that the mechanism of "trapping" by CMY-2 β-lactamase plays a role in carbapenem resistance.

OmpC-like porin from outer membrane of Yersinia enterocolitica: Molecular structure and functional activity

OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the "warm" variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the "cold" variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 β-strands connected by short "periplasmic" and longer "extracellular" loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the "extracellular" loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential.

Frequent emergence of porin-deficient subpopulations with reduced carbapenem susceptibility in ESBL-producing Escherichia coli during exposure to ertapenem in an in vitro pharmacokinetic model

Ertapenem resistance is increasing in Enterobacteriaceae. The production of extended-spectrum β-lactamases (ESBLs) and reduced expression of outer membrane porins are major mechanisms of resistance in ertapenem-resistant Klebsiella pneumoniae. Less is known of ertapenem resistance in Escherichia coli. The aim of this study was to explore the impact of ESBL production in E. coli on the antibacterial activity of ertapenem. Two E. coli strains, with and without ESBL production, were exposed to ertapenem in vitro for 48 h at concentrations simulating human pharmacokinetics with conventional and higher dosages. Isolates with non-susceptibility to ertapenem (MICs 0.75-1.5 mg/L) were detected after five of nine time-kill experiments with the ESBL-producing strain. All of these isolates had ompR mutations, which reduce the expression of outer membrane porins OmpF and OmpC. Higher dosage did not prevent selection of porin-deficient subpopulations. No mutants were detected after experiments with the non-ESBL-producing strain. Compared with other experiments, experiments with ompR mutants detected in endpoint samples showed significantly less bacterial killing after the second dose of ertapenem. Impaired antibacterial activity against E. coli with ESBL production and ompR mutation was also demonstrated in time-kill experiments with static antibiotic concentrations. The combination of ESBL production and porin loss in E. coli can result in reduced susceptibility to ertapenem. Porin-deficient subpopulations frequently emerged in ESBL-producing E. coli during exposure to ertapenem at concentrations simulating human pharmacokinetics. Inappropriate use of ertapenem should be avoided to minimize the risk of selection of ESBL-producing bacteria with reduced susceptibility to carbapenems.

Involvement of MarR and YedS in Carbapenem Resistance in a Clinical Isolate of Escherichia coli from China

A carbapenem-resistant clinical isolate of Escherichia coli, which lacked OmpF and OmpC porins, carried a marR mutation and expressed a functional yedS, a normally nontranslated gene. MarR and YedS are described here as having effects on the ability of this strain to resist carbapenems. Additionally, expression of YedS was regulated by the small RNA MicF in a MarA-dependent way. These findings illustrate how broadly bacteria can mutate within a selective clinical setting, in this case, resistance to carbapenems, by altering three porin genes and one regulatory gene.

Expression of OmpC and OmpF porin proteins and survival of Escherichia coli under photooxidative stress in Black Sea water

In the present study, the synthesis of OmpC and OmpF porins (outer membrane pro- teins C and F, respectively) of Escherichia coli under photooxidative stress in Black Sea water and the roles of RpoS (the starvation/stationary phase sigma factor, σ 38 ) and EnvZ (a transmembrane environmental sensor protein) in changing porin protein expression were investigated. The t99 (time required for a 2 log reduction) values for wild-type E. coli W3110 and MSZ31 (envZ − ) under photooxidative stress were observed to be 14.3 and 16.8 h, respectively, while for porin-deficient mutants MKC505 (ompC − ), MH621 (ompF − ), MKCF36 (ompF − ompC − ), and MSR31 (ompR � ) they were 10.6, 10.2, 9.8 and 9.1 h, respectively. Further, when the porins or ompR were deleted, the resistance to photooxidative stress decreased. OmpC and OmpF porin expression increased in all studied transcriptional fusion strains (wild type and mutants) in control microcosms, and almost completely decreased under photooxidative stress in the test microcosms, which consisted of light exposure with methylene blue added as a photosensitizer. Thus, it could be concluded that RpoS and EnvZ regulation of the synthesis of OmpC and OmpF is not photooxidation-dependent. This study determined for the first time that the expression of ompC in brackish water is influenced by RpoS.

Butanol as a regulatory factor of ompC gene expression in E. coli cells

The influence of butanol on the expression of ompC gene encoding synthesis of OmpC porin in the MG 1655 strain of E. coli and butanol-tolerant mutant ButR was studied. It was shown that in the case of wild bacteria, the addition of butanol to the growth medium results in an increased level of ompC transcription. However, OmpC porin is not detected in the membrane fraction of cells. ButR mutant exhibits a higher level of ompC gene expression. A direct correlation is observed between the level of OmpC porin expression and its content in the membrane fraction of ButR mutant cells. In the regulatory region of the ompC gene of the ButR mutant, three nucleotide substitutions located in the binding sites of OmpR and CpxR activator proteins were identified. It was shown that mutations in the regulatory region of the ompC gene in the ButR mutant are responsible for the decreased level of OmpC porin expression under normal growth conditions. However, these mutations lead to an increased level of OmpC porin synthesis in the presence of butanol. These data suggest an additional mechanism of ompC gene regulation with the participation of butanol as a positive transcription effector.

Antibacterial effects of silver nanoparticles on gram-negative bacteria: Influence on the growth and biofilms formation, mechanisms of action

Antibacterial action of silver nanoparticles (AgNP) on Gram-negative bacteria (planctonic cells and biofilms) is reported in this study. AgNP of 8.3nm in diameter stabilized by hydrolyzed casein peptides strongly inhibited biofilms formation of Escherichia coli AB1157, Pseudomonas aeruginosa PAO1 and Serratia proteamaculans 94 in concentrations of 4–5μg/ml, 10μg/ml and 10–20μg/ml, respectively. The viability of E. coli AB1157 cells in biofilms was considerably reduced by AgNP concentrations above 100 to −150μg/ml. E. coli strains with mutations in genes responsible for the repair of DNA containing oxidative lesions (mutY, mutS, mutM, mutT, nth) were less resistant to AgNP than wild type strains. This suggests that these genes may be involved in the repair of DNA damage caused by AgNP. E. coli mutants deficient in excision repair, SOS-response and in the synthesis of global regulators RpoS, CRP protein and Lon protease present similar resistance to AgNP as wild type cells. LuxI/LuxR Quorum Sensing systems did not participate in the control of sensitivity to AgNP of Pseudomonas and Serratia. E. coli mutant strains deficient in OmpF or OmpC porins were 4–8 times more resistant to AgNP as compared to the wild type strain. This suggests that porins have an important function related AgNP antibacterial effects.

Calcium Chloride Made E. coli Competent for Uptake of Extraneous DNA Through Overproduction of OmpC Protein

In the standard method of transformation of Escherichia coli with extraneous DNA, cells are made competent for DNA uptake by incubating in ice-cold 100 mM CaCl(2). Analysis of the whole protein profile of CaCl(2)-treated E. coli cells by the techniques of one- and two-dimensional gel electrophoresis, MALDI-MS and immunoprecipitation revealed overproduction of outer membrane proteins OmpC, OmpA and heat-shock protein GroEL. In parity, transformation efficiency of E. coli ompC mutant by plasmid pUC19 DNA was found to be about 40 % lower than that of the wild type strain. Moreover, in E. coli cells containing groEL-bearing plasmid, induction of GroEL caused simultaneous overproduction of OmpC. On the other hand, less OmpC was synthesized in E. coli groEL mutant compared to its wild type counterpart, by CaCl(2)-shock. From these results it can be suggested that in the process of CaCl(2)-mediated generation of competence, the heat-shock chaperone GroEL has specific role in DNA entry into the cell, possibly through the overproduced OmpC and OmpA porins.