Vibrio Cholerae Cells Research Articles

Modification of The Bacterial Abundance Properties of Water by Immersed Non-Activated Charcoal

The present studies aim at reducing the planktonic bacterial load in drinking water using non-activated charcoal. The evaluation is dependent on the incubation time and the different physiological states of the bacterial cells. Three bacteria (Vibrio cholerae, Escherichia coli and Staphylococcus aureus) and two charcoal substrates (Okan and Tali) were used for the experimentation. The Bacteria adhered to charcoal fragments in varying degrees. Overall, the concentrations of fixed Vibrio cholerae reached 53.4x107 CFU/cm2 and 39.6x107 CFU/cm2 after 9 hours of contact in exponential phase on the Okan and Tali respectively. Those of fixed Staphylococcus aureus reached 5.8x106 CFU/cm2 after 3 hours of contact in exponential phase and 3.4x106 CFU/cm2 after 3 and 6 hours of contact in the same phase, on the Okan and the Tali, respectively. The highest abundances of Escherichia coli adhered to the charcoal fragments were 50.4x107 CFU/cm² on Okan and 53.2x107 CFU/cm² on Tali after 9 hours of contact in the exponential phase. The highest adsorption coefficient (639.06 adhered cells/cm2) was noted on Okan in the exponential phase with Vibrio cholerae cells; and the lowest (1.02 adhered cells/cm2) on Tali in stationary phase with Staphylococcus aureus. The incubation time significantly (P<0.01) influenced the adhesion of bacterial cells to charcoal substrates. Although the adsorption capacity and intensity of Okan were relatively higher, the comparison of the adsorption potential of the two substrates considered did not reveal any significant difference (P>0.05), reflecting the absence of the influence of the physical properties of these substrates on cell retention.

Cell-lysis sensing drives biofilm formation in Vibrio cholerae

Matrix-encapsulated communities of bacteria, called biofilms, are ubiquitous in the environment and are notoriously difficult to eliminate in clinical and industrial settings. Biofilm formation likely evolved as a mechanism to protect resident cells from environmental challenges, yet how bacteria undergo threat assessment to inform biofilm development remains unclear. Here we find that population-level cell lysis events induce the formation of biofilms by surviving Vibrio cholerae cells. Survivors detect threats by sensing a cellular component released through cell lysis, which we identify as norspermidine. Lysis sensing occurs via the MbaA receptor with genus-level specificity, and responsive biofilm cells are shielded from phage infection and attacks from other bacteria. Thus, our work uncovers a connection between bacterial lysis and biofilm formation that may be broadly conserved among microorganisms.

Electrochemical Detection of Vibrio cholerae by Amine Functionalized Biocompatible Gadolinium Oxide Nanoparticles.

Herein, we report the biocompatible amine-functionalized gadolinium oxide nanoparticles (Gd2O3 NPs) for the possibility of electrochemical detection of Vibrio cholerae (Vc) cells. The microwave irradiation process is applied to synthesize Gd2O3 NPs. The amine (NH2) functionalization is carried out via overnight stirring with 3(Aminopropyl)triethoxysilane (APTES) at 55 °C. The size of NPs amine functionalized APETS@Gd2O3 NPs are determined by transmission electron microscopy (TEM). APETS@Gd2O3 NPs are further electrophoretically deposited onto indium tin oxide (ITO) coated glass substrate to obtain working electrode surface. The monoclonal antibodies (anti-CT) specific to cholera toxin associated to Vc cells are covalently immobilized onto the above electrodes using EDC-NHS chemistry and further BSA is added to obtain the BSA/anti-CT/APETS@Gd2O3/ITO immunoelectrode. Further, this immunoelectrode shows the response for cells in CFU range from 3.125 × 106 to 30 × 106 and is very selective with sensitivity and LOD 5.07 mA CFUs mL cm-2 and 0.9375 × 106 CFU respectively. To establish a future potential for APTES@Gd2O3 NPs in field of biomedical applications and cytosensing, the effect of APTES@Gd2O3 NPs on mammalian cells is also observed using in vitro cytotoxicity assay and cell cycle analysis.

Single-cell segmentation in bacterial biofilms with an optimized deep learning method enables tracking of cell lineages and measurements of growth rates.

Bacteria often grow into matrix-encased three-dimensional (3D) biofilm communities, which can be imaged at cellular resolution using confocal microscopy. From these 3D images, measurements of single-cell properties with high spatiotemporal resolution are required to investigate cellular heterogeneity and dynamical processes inside biofilms. However, the required measurements rely on the automated segmentation of bacterial cells in 3D images, which is a technical challenge. To improve the accuracy of single-cell segmentation in 3D biofilms, we first evaluated recent classical and deep learning segmentation algorithms. We then extended StarDist, a state-of-the-art deep learning algorithm, by optimizing the post-processing for bacteria, which resulted in the most accurate segmentation results for biofilms among all investigated algorithms. To generate the large 3D training dataset required for deep learning, we developed an iterative process of automated segmentation followed by semi-manual correction, resulting in >18,000 annotated Vibrio cholerae cells in 3D images. We demonstrate that this large training dataset and the neural network with optimized post-processing yield accurate segmentation results for biofilms of different species and on biofilm images from different microscopes. Finally, we used the accurate single-cell segmentation results to track cell lineages in biofilms and to perform spatiotemporal measurements of single-cell growth rates during biofilm development.

A Dueling-Competent Signal-Sensing Module Guides Precise Delivery of Cargo Proteins into Target Cells by Engineered Pseudomonas aeruginosa.

To recognize and manipulate a specific microbe of a crowded community is a highly challenging task in synthetic biology. Here we introduce a highly selective protein delivery platform, termed DUEC, which responds to direct contact of attacking cells by engineering the tit-for-tat/dueling response of H1-T6SS (type VI secretion system) in Pseudomonas aeruginosa. Using a Cre-recombinase-dependent reporter, we screened H1-T6SS-secreted substrates and developed Tse6N as the most effective secretion tag for Cre delivery. DUEC cells can discriminately deliver the Tse6N-Cre cargo into the cytosol of T6SS+ but not T6SS- Vibrio cholerae cells. DUEC could also deliver a nuclease cargo, Tse6N-NucSe1, to selectively kill provoking cells in a mixed community. These data demonstrate that the DUEC cell not only is a prototypical physical-contact sensor and delivery platform but also may be coupled with recombination-based circuits with the potential for complex tasks in mixed microbial communities.

Vibrio cholerae secretion system of the type VI

The review summarizes literature data on the Vibrio cholerae secretion system of the 6th type. This system is a contact-dependent macromolecular mechanism through which bacteria translocate toxic effector proteins into target cells. It is found in many Gram-negative bacteria, including Vibrio cholerae. V. cholerae infects phagocytic amoebae, nematodes, ciliates, bacteria belonging to different species, as well as unrelated strains of V. cholerae using this system. DNA released after lysis of competing bacteria can be taken up by Vibrio cholerae cells, which leads to the acquisition of new genetic material. The type VI secretion system is involved in the infectious process. The destruction of macrophages and microbiota contributes to the active reproduction of the pathogen and colonization of host epitheliocytes, and the production of effector proteins causes the development of diarrhea and intestinal inflammation. Cholera vibrio secretion system of the 6th type has a structure similar to other gram-negative bacteria. The genes encoding the proteins of this system are located in one large region of the second chromosome and in several additional clusters. It has been shown that toxigenic strains of V. cholerae contain an identical set of secretion system genes, while their composition is variable in non-toxigenic isolates. The regulation of secretion system protein expression differs in V. cholerae strains of different toxigenicity, depends on a number of environmental signals, and is associated with other cell regulatory networks. The paper provides experimental data on the analysis of the structure of the global regulatory gene, vasH, of the type VI secretion system in toxigenic and non-toxigenic V. cholerae O1, biovar El Tor strains isolated in the Russian Federation. Thus, the type VI secretion system is an important mechanism that facilitates the survival of V. cholerae in complex communities in vitro, protects against damaging factors of the macroorganism and increases virulence in vivo, and also provides evolutionary transformations of cholera vibrio. Further study of this system will allow a better understanding of the pathogen-host interaction processes, as well as the adaptation mechanisms of V. cholerae in the external environment.

Effects of chitosan nanoparticles loaded with mesenchymal stem cell conditioned media on gene expression in Vibrio cholerae and Caco-2 cells

Vibrio (V.) cholerae forms a pellicle for self-defense in the pathological conditions in the intestine, which protects it against antibiotics and adverse conditions. Targeting biofilm genes and Toll-like receptors (TLRs) is one of the new strategies to combat multidrug-resistant bacteria. The objective of this study was to evaluate the effect of mesenchymal stem cell conditioned media (MSC CM; 1000 µg), chitosan nanoparticles incorporated with mesenchymal stem cell conditioned media (MSC CM-CS NPs; 1000 µg + 0.05%), and chitosan nanoparticles (CS NPs; 0.05%) on the expression of bap1 and rbmC biofilm genes in V. cholerae and TLR2 and TLR4 genes in Caco-2 cells. The bacteria were inoculated in the presence or absence of MSC CM, MSC CM-CS NPs, and CS NPs for 24 h at 37 °C to evaluate the expression of biofilm genes. The Caco-2 cells were also exposed to V. cholerae for 1 h and then MSC CM, MSC CM-CS NPs, and CS NPs for 18 h at 37 °C. After these times, RNA was extracted from Caco-2 cells and bacteria exposed to the compounds, and the expression of target genes was evaluated using real-time PCR. Caco-2 cell viability was also assessed by MTT assay. After adding MSC CM, MSC CM-CS NPs, and CS NPs to V. cholerae medium, the percentage reduction in gene expression of bap1 was 96, 91, and 39%, and rbmC was 93, 92, and 32%, respectively. After adding MSC CM, MSC CM-CS NPs, and CS NPs to the Caco-2 cell medium, the percentage reduction in the gene expression of TLR4 was 89, 90, and 82%, and TLR2 was 41, 43, and 32%, respectively. MTT showed that Caco-2 cell viability was high and the compounds had little toxicity on these cells. Finally, it suggests that MSC CM-CS NPs designed may be a therapeutic agent to combat inflammation and biofilm formation in multidrug-resistant V. cholerae. However, further studies in vivo are also recommended.

Outer Membrane Vesicles Facilitate Trafficking of the Hydrophobic Signaling Molecule CAI-1 between Vibrio harveyi Cells.

Many bacteria use extracellular signaling molecules to coordinate group behavior, a process referred to as quorum sensing (QS). However, some QS molecules are hydrophobic in character and are probably unable to diffuse across the bacterial cell envelope. How these molecules are disseminated between bacterial cells within a population is not yet fully understood. Here, we show that the marine pathogen Vibrio harveyi packages the hydrophobic QS molecule CAI-1, a long-chain amino ketone, into outer membrane vesicles. Electron micrographs indicate that outer membrane vesicles of variable size are predominantly produced and released into the surroundings during the stationary phase of V. harveyi, which correlates with the timing of CAI-1-dependent signaling. The large vesicles (diameter, <55 nm) can trigger a QS phenotype in CAI-1-nonproducing V. harveyi and Vibrio cholerae cells. Packaging of CAI-1 into outer membrane vesicles might stabilize the molecule in aqueous environments and facilitate its distribution over distances.IMPORTANCE Formation of membrane vesicles is ubiquitous among bacteria. These vesicles are involved in protein and DNA transfer and offer new approaches for vaccination. Gram-negative bacteria use hydrophobic signaling molecules, among others, for cell-cell communication; however, due to their hydrophobic character, it is unclear how these molecules are disseminated between bacterial cells. Here, we show that the marine pathogen Vibrio harveyi packages one of its QS molecules, the long-chain ketone CAI-1, into outer membrane vesicles (OMVs). Isolated CAI-1-containing vesicles trigger a QS phenotype in CAI-1 nonproducing V. harveyi and also in Vibrio cholerae cells. Packaging of CAI-1 into OMVs not only solubilizes, stabilizes, and concentrates this class of molecules, but facilitate their distribution between bacteria that live in aqueous environments.

Interplay of Nanoparticle Resonance Frequency and Array Surface Coverage in Live-Cell Plasmon-Enhanced Single-Molecule Imaging

Super-resolution imaging has provided new insights into nanoscale optics. Plasmonic gold nanotriangle arrays created by nanosphere lithography can enhance single-molecule fluorescence intensity to further improve imaging. Here, gold nanotriangle arrays on glass coverslips were used as inexpensive, facile, and broadly applicable imaging substrates for living Vibrio cholerae cells expressing photoactivatable fluorescent proteins—the red PAmCherry or the green PAGFP—and resulted in fluorescence enhancements upon coupling living cells to nanotriangle arrays. Within the requirements for this wide-field coupling geometry, we analyze and optimize the coupling as a function of local surface plasmon resonance frequency and particle coverage.

Cryo-EM structure of the extended type VI secretion system sheath-tube complex.

The bacterial type VI secretion system (T6SS) uses contraction of a long sheath to quickly thrust a tube with associated effectors across membranes of eukaryotic and bacterial cells 1-5 . Only limited structural information is available about the inherently unstable precontraction state of the T6SS. Here, we obtain a 3.7 Å resolution structure of a non-contractile sheath-tube complex using cryo-electron microscopy and show that it resembles the extended T6SS inside Vibrio cholerae cells. We build a pseudo-atomic model of the complete sheath-tube assembly, which provides a mechanistic understanding of coupling sheath contraction with pushing and rotating the inner tube for efficient target membrane penetration. Our data further show that sheath contraction exposes a buried recognition domain to specifically trigger the disassembly and recycling of the T6SS sheath by the cognate ATP-dependent unfoldase ClpV.

Detection of Viable Vibrio cholerae Cells in Seafood Using a Real-Time Visual Loop-Mediated Isothermal Amplification Combined with Propidium Monoazide

Vibrio cholerae is an important foodborne pathogen causing severe intestinal infectious diseases that have high incidence and mortality. Almost all of rapid testing methods including immunological and molecular assays for V. cholerae are incapable of distinguishing live cells from dead ones, which may overestimate the number of bacteria and result in many false positive results. To address the problems, live cell-specific dye such as propidium monoazide (PMA) is employed. The loop-mediated isothermal amplification (LAMP) assay is a nucleic acid amplification method that is fast, specific, and sensitive. In this study, we developed a real-time visual LAMP assay using PMA dye to detect thyA gene, thereby identifying viable V. cholerae cells. The results showed that only V. cholarae strains could be detected, and there was no cross-reaction with non-V. cholarae strains. Besides, the sensitivity of the PMA-LAMP assay was 1.1 × 102 CFU/mL and the entire reaction could be accomplished within 1 h. The sensitivity was on par with that of the PMA-qPCR assay. The detection limit in different artificially inoculated samples was 5 CFU/25 g materials for the tested pathogens. In the practical test, the PMA-LAMP assay performed well in comparison with PMA-qPCR and the culture method. Hence, PMA-LAMP assay can provide a highly effective and rapid approach for detecting viable V. cholerae.

Stepwise changes in viable but nonculturable Vibrio cholerae cells.

Many bacterial species are known to become viable but nonculturable (VBNC) under conditions that are unsuitable for growth. In this study, the requirements for resuscitation of VBNC-state Vibrio cholerae cells were found to change over time. Although VBNC cells could initially be converted to culturable by treatment with catalase or HT-29 cell extract, they subsequently entered a state that was not convertible to culturable by these factors. However, fluorescence microscopy revealed the presence of live cells in this state, from which VBNC cells were resuscitated by co-cultivation with HT-29 human colon adenocarcinoma cells. Ultimately, all cells entered a state from which they could not be resuscitated, even by co-cultivation with HT-29. These characteristic changes in VBNC-state cells were a common feature of strains in both V. cholerae O1 and O139 serogroups. Thus, the VBNC state of V. cholerae is not a single property but continues to change over time.

Enhanced Resistance to Several Abiotic Stresses in Vibrio cholerae during Starvation.

In this study, we investigated whether Vibrio cholerae cells exposed to nutrient-limited conditions developed resistance to abiotic stresses because of which most, if not all, starved cells turn coccoid in shape and exhibited marked resistance to freezing, low pH, and chlorine, but not against bile.

ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNA-binding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and site-directed modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria.

Understanding the Pathogenicity of Vibrio Cholerae via Two-Color Live-Cell Super-Resolution Microscopy

Cholera afflicts more than 5 million people annually. Here, we investigate the virulence pathway of this epidemic disease with live-cell single-molecule fluorescence imaging in Vibrio cholerae cells, the bacterium responsible for producing the cholera toxin. In the Gram-negative pathogen V. cholerae, virulence gene expression is under control of an unusual set of membrane proteins. Here, a membrane complex including two activators, ToxR and TcpP, binds the toxT promoter, recruits RNA polymerase, and activates toxT gene expression leading to activation of ToxT-controlled virulence genes. To circumvent the diffraction limit of light, which bounds the resolution of optical microscopy to ∼250 nm, we use single-molecule tracking and super-resolution techniques like Photoactivated Localization Microscopy (PALM) to achieve resolutions more than an order of magnitude better than the diffraction limit. We have created fusions of the membrane-bound transcription activators TcpP and ToxR with orthogonal photo-(re)activatable fluorescent proteins, and in this study, we examine the dynamics and co-localization patterns of single PAmCherry-TcpP and mCitrine-ToxR molecules in the virulence pathway. We also image V. cholerae cells that have knockouts of ToxR and/or TcpP to determine if the protein dynamics change in the absence of the binding partner. This work aims to identify characteristics of TcpP and ToxR motion to understand their regulatory behavior in the transcriptional activation of the gene toxT and subsequent activation of downstream virulence genes, and to establish a model for the formation of the ToxR/TcpP/toxT protein-DNA complex important in early pathogenesis. In addition to elucidating the regulatory pathway of V. cholerae, the impact of this work will be to further provide a general model for outer-membrane-bound transcription control in bacteria and nuclear-membrane-bound transcription in eukaryotic cells.

Two Case Studies where Electron Cryotomography has Revealed Mechanism: ESCRT and the Type VI Secretion System

Sometimes all that is really needed to distinguish between competing models is a few really good pictures. Electron cryotomography has increasingly met that need by delivering unprecedented 3-D images of intact cells in a near-native (frozen-hydrated) state to “macromolecular” resolution. I will present two case studies. In the first, cryotomograms of dividing Sulfolobus acidocaldarius cells revealed a thin layer of material at the leading edge of ingressing membranes whose structure and development point uniquely to an “hourglass” model for how the ESCRT machinery drives membrane scission. In the second, cryotomograms of Vibrio cholerae cells revealed extended cytoplasmic tubes in two different conformational states. Identification of the tubes as Type VI Secretion Systems led immediately to a “spring-loaded dagger” model for their ability to deposit toxins into neighboring cells.

Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

A new protocol for rapid, specific, and sensitive cell-based quantification of Vibrio cholerae/Vibrio mimicus in water samples was developed. The protocol is based on catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6 V. cholerae cells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific for V. cholerae and V. mimicus) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantify V. cholerae/V. mimicus via solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 × 10(1) cells ml(-1) in the large saline lake Neusiedler See to 5.6 × 10(4) cells ml(-1) in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantify V. cholerae/V. mimicus in ecological studies as well as for risk assessment and monitoring programs.

Construction of novel vaccine strains of Vibrio cholerae co-expressing the Inaba and Ogawa serotype antigens

The approach of inducing protective immunity against cholera by oral vaccination with killed whole Vibrio cholerae cells is effective, but the complexity of current cholera vaccines makes them difficult and relatively expensive to manufacture, especially if recombinant cholera toxin B subunit is included in the formulation. In an effort to simplify the composition of a new generation of oral cholera vaccines we have generated a novel non-toxigenic candidate vaccine strain of V. cholerae O1 that stably expresses both the Ogawa and Inaba serotype antigens on its surface. This was done by introducing a functional wbeT gene without a functional promoter into the chromosome of an O1 Inaba strain. The resulting low levels of expression of the wbeT gene product allowed for the desired partial serotype switching. This strain (MS1342) can potentially replace the three virulent strains used in currently manufactured cholera vaccines. Oral immunization of mice with formalin-killed MS1342 bacteria gave rise to Ogawa-specific, Inaba-specific and cross-reactive serum antibodies that were detectable both by lipopolysaccharide (LPS)-specific ELISAs and as vibriocidal antibodies that are considered to predict protective efficacy. These responses as well as intestinal mucosal IgA anti-LPS antibody responses were fully comparable with those obtained by immunization with the internationally licensed oral cholera vaccine Dukoral®. We propose that such a strain may form the basis of a single strain killed whole cell cholera vaccine protecting against cholera caused by either the Inaba or Ogawa serotype of V. cholerae O1.

Synthesis of nanosilver particles by reverse micelle method and study of their bactericidal properties

Nanosilver particles have been synthesized by the reverse micelle method, where AgNO3 was used as a silver ions source, NaBH4 and quercetin – as reducing agents, CTAB, SDOSS and AOT- as surfactants, while the stabilizer was Vietnamese chitosan. Studying the factors influencing the process of nanosilver particle formation, it was shown that the particle size of the nanosilver products depends on the concentration of the reaction components and their stoichiometric ratio. It was also shown that the reaction system using AOT surfactant is capable of producing nanosilver particles with smallest nanoparticles (ϕav ∼ 5 nm) and good particle-size distribution. The study on bactericidal activity of the nanosilver products indicated that the disinfecting solution with a nanosilver concentration of 3 ppm was able to inhibit all E.coli and Coliforms, TPC and fungi at 15 ppm, while Vibrio cholerae cells were inactivated completely with 0.5 ppm of nanosilver after 30 minutes exposition.

An avirulent Vibrio cholerae strain—A producer of the cholera toxin B subunit: Obtainment and molecular genetic analysis

The conjugative recombinant plasmid pIEM3 (KmR TcR) was constructed in order to introduce the cloned ctxB gene encoding the cholera toxin B subunit into the Vibrio cholerae cells. The plasmid was obtained as a result of co-integration of two plasmids: a conjugative plasmid, pIEM1(KmR), carrying mini-kan transposon and IS1 element, as well as the pCTdelta27(TcR) plasmid that is a derivative of the pBR322 which carries the cloned ctxB gene. The avirulent Vibrio cholerae strain eltor biovar deprived (according to the PCR analysis) of the key structural and regulatory pathogenicity genes and carrying a mutation in a single gene of the O1 antigen was chosen as the pIEM3 plasmid carrier strain. The cointegrate uncoupling was shown to take place in 5% the cholera vibrio cells followed by retention of only the multi-copy pCTdelta7 plasmid. This event leads to the formation of the TcRKmS clones characterized by high levels of the cholera toxin secreted B subunit production (10 to 14 microg/ml), one of these (KM93) being selected as a strain-producer of the protein. Molecular-genetic and biochemical assays were used to elucidate peculiar features of inheritance and expression of the cloned ctxAB gene within the KM93 cells. The expression of the cloned ctxB gene was shown to be independent of the presence of the toxR, tcpP, tcpH, toxT regulatory genes suggesting the existence of some other mechanisms that might exert their control over the transcriptional activity of the cholera toxin B subunit gene. Effective production of the cholera toxin B subunit would be also observed if the constructed producer strain was cultured under the conditions of industrial process. This indicates a possibility of its employment as a source of this protein involved in manufacturing cholera immunodiagnostic and prophylactic preparations.