Determination of ion permeability through the channels made of porins from the outer membrane ofSalmonella typhimurium in lipid bilayer membranes

Summary The three types of porin (matrix-proteins) fromSalmonella typhimurium with molecular weights of 38,000, 39,000 and 40,000 were reconstituted with lipid bilayer membranes either as a trimer or as an oligomer (complex I). The specific conductance of the membranes increased several orders of magnitude after the addition of the porins into the aqueous phase bathing the membranes. A linear relationship between protein concentration in the aqueous phase and membrane conductance was found. In the case of lower protein concentrations (10^–12 m), the conductance increased in a stepwise fashion with a single conductance increment of 2.3 nS in 1m KCl. For a given salt the conductance increment was found to be largely independent of the particular porin (38 K, 39K or 40 K) and on the state of aggregation, although porin oligomers showed an up to 10 times smaller conductance increase in macroscopic conductance measurements. The conductance pathway has an ohmic current voltage characteristic and a poor selectivity for different alkali ions. Further information on the structure of the pores formed by the different porins fromSalmonella was obtained from the selectivity for various ions. From the permeability of the pore for large ions (Tris⁺, glucosamine⁺, Hepes^–_ a minimum pore diameter of 0.8 nm is estimated. This value is in agreement with the size of the pore as calculated from the conductance data for 1m KCl (1.4 nm for a pore length of 7.5 nm). The pore diameter may well account for the sugar permeability which has been found in reconstituted vesicles. The findings reported here are consistent with the assumption that the different porins form large aqueous channels in the lipid bilayer membranes and that the single condutance unit is a trimer. In addition, it is suggested that one trimer contains only one pore rather than a bundle of pores.     

Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: Role of voltage and pH

The bifunctional adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis invades target cells via transport through the cytoplasmic membrane. The membrane potential represents thereby an important factor for the uptake in vivo. Previous studies demonstrated that adenylate cyclase (AC) delivery into cells requires a negative membrane potential inside the cells. The results of lipid bilayer experiments with ACT presented here indicated that two different types of pore-like structures are formed by ACT dependent on the orientation of the electrical potential across the membranes. Pore formation at a positive potential at the cis side of the membranes, the side of the addition of the toxin, was fast and its conductance had a defined size, whereas at negative potential the pores were not defined, had a reduced pore-forming activity and a very short lifetime. Fluctuations inserted at positive potentials showed asymmetric current-voltage relationships for positive and negative voltages. Positive potentials at the cis side resulted in an increasing current, whereas at negative potentials the current decreased or remained at a constant level. Calcium ions enhanced the voltage dependence of the ACT pores when they were added to the cis side. The single-pore conductance was strongly affected by the variation of the pH value and increased in 1M KCl with increasing pH from about 4 pS at pH 5 to about 60 pS at pH 9. The ion selectivity remained unaffected by pH. Experiments with ACT mutants revealed, that the adenylate cyclase (AC) and repeat (RT) domains were not involved in voltage and pH sensing.     

Escherichia coli haemolysin forms voltage-dependent ion channels in lipid membranes

The action of the 107 kDa hemolysin from Escherichia coli on planar lipid membranes was investigated. We report that a single toxin molecule can form a cation-selective, ion-permeable channel of large conductance in a planar phospholipid bilayer membrane. The conductance of the pore is proportional to that of the bulk solution, indicating that the channel is filled with water. A pore diameter of about 2 nm can be evaluated. The pore formation mechanism is voltage-dependent and essentially resembles that of pore-forming colicins; this implies that opening of the channel is dependent on transfer of an electrical charge through the membrane. We propose that the physiological effects of E. coli hemolysin result from its ability to form ion channels in the membrane of attacked cells, and show that there is quantitative agreement between the effects of this toxin on model membranes and its hemolytic properties.     

Modelling mechanosensitivity in membranes: effects of lateral tension on ionic pores in a microcystin toxin-containing membrane

Lipid bilayers of diphytanoyl lecithin (DPhL) in which a cyanobacterial toxin, microcystin-LR (MC-LR) was incorporated, were found to be a convenient model of natural mechanosensitive membranes. The effects of pressure difference, leading to lateral membrane tension, on artificial membranes formed on the tips of glass micropipettes were investigated using patch-clamp methodology. Emplacement of MC-LR from the bathing solution was enhanced by transmembrane voltage and/or pressure difference. MC-LR pores could be recorded over a wide voltage range, their opening probability being first increased and then reduced at high membrane potential. The pores exhibited several open pore conductance levels, the higher conductance states being more probable at greater lateral tensions. Ion gradient experiments established that the MC-LR pores are cation selective, but discriminate only weakly between K and Na. These results suggest that a lipid liquid crystal matrix containing monomers of multimeric pore-forming molecules could be used as a mechanical sensor and molecular switch. Offprint requests to: P. N. R. Usherwood     

Dimerization constant and single-channel conductance of Gramicidin in thylakoid membranes

Summary The effect of the pore-forming antibiotic gramicidin on pure lipid membranes is well characterized. We studied its action in protein-rich thylakoid membranes that contain less than 25% (wt/wt) acyl lipids. A transmembrane voltage was induced by flashing light, and its decay was measured and interpreted to yield the distribution of gramicidin over thylakoids, its dimerization constant and its single-channel conductance in this membrane. The distribution of gramicidin over the ensemble of thylakoids was immediately homogeneous when the antibiotic was added under stirring, while it became homogeneous only after 20 min in a stirred suspension that was initially heterogeneous. The dimerization constant, 5×10¹⁴ cm²/mol, was about 10 times larger than in pure lipid membranes. This was attributed to the upconcentration of gramicidin in the small fractional area of protein free lipid bilayer and further by a preference of gramicidin for stacked portions of the membrane. The latter bears important consequences with regard to bioenergetic studies with this ionophore. As gramicidin was largely dimerized from a concentration of 1 nm (in the suspension) on, the membrane's conductance then increased linearly as a function of added gramicidin. When the negative surface potential at the thylakoid membrane was screened, the conductance of a single gramicidin dimer agreed well with figures reported for bilayers from neutral lipid (about 0.5 pS at 10 mm NaCl). The modulation of the conductance by the surface potential in spinach versus pea thylakoids and between different preparations is discussed in detail.We would like to thank Ms. H. Kenneweg for photographs. financial support by the DFG (SFB 171/B3) is gratefully acknowledged.This paper is dedicated to the Late Prof. Peter Läger.     

Pore formation by complement in the outer membrane of Gram-negative bacteria studied with asymmetric planar lipopolysaccharide/phospholipid bilayers

Summary The interaction of complement with an asymmetric planar lipopolysaccharide/phospholipid bilayer system as a model for the lipid matrix of the outer membrane of Gram-negative bacteria has been studied. The addition of whole human serum to the aqueous solution at the lipopolysaccharide side of the asymmetric membrane resulted in a rapid increase of the bilayer conductance in discrete steps, indicating the formation of transmembrane pores, which were not observed in the case of pure phospholipid membranes. The amplitudes of the discrete conductance steps varied over a range of more than one order of magnitude. The mean single step conductance was (0.39±0.24) nS for a subphase containing (inmm): 100 KCl, 5 MgCl₂ and 5 HEPES buffer. The steps were grouped into bursts of typically 9±3 events per burst and the conductance change within one burst was (8.25±4.00) nS.The pore-forming activity of serum at the asymmetric membrane system was independent of the presence of specific antibodies against the lipopolysaccharide but was dependent on calcium ions. Furthermore, the pore-forming activity required complement component C9.A model for the mode of pore formation by complement is proposed: The complement pore is generated in discrete steps by insertion of C9 monomers into the membrane and their irreversible aggregation to water-filled channels with a diameter of approximately 7 nm assuming a circular geometry.     

Calorimetric scrutiny of lipid binding by sticholysin II toxin mutants

The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid-protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporin family, have been produced, and their hemolytic and lipid-binding properties were compared to those of the wild-type protein. A thermodynamic approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpy-driven process that occurs with a high affinity constant (1.7 × 10⁸ M^− 1). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation. Membrane binding is achieved through two separate mechanisms, that is, recognition of the lipid-water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholine-binding site. Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation.     

Palmitic Acid Induces the Opening of a Ca2+-Dependent Pore in the Plasma Membrane of Red Blood Cells: The Possible Role of the Pore in Erythrocyte Lysis

Earlier we found that in the presence of Ca²⁺ palmitic acid (Pal) increases the nonspecific permeability of artificial (planar and liposomal) membranes and causes permeabilization of the inner mitochondrial membrane. An assumption was made that the mechanism of Pal/Ca²⁺-induced membrane permeabilization relates to the Ca²⁺-induced phase separation of Pal and can be considered as formation of fast-tightening lipid pores due to chemotropic phase transition in the lipid bilayer. In this article, we continue studying this pore. We have found that Pal plus Ca²⁺ permeabilize the plasma membrane of red blood cells in a dose-dependent manner. The same picture has been revealed for stearic acid (20 μM) but not for myristic and linoleic acids. The Pal-induced permeabilization of erythrocytic membranes can also occur in the presence of Ba²⁺ and Mn²⁺ (200 μM), but other bivalent cations (200 μM Mg²⁺, Sr²⁺, Ni²⁺, Co²⁺) are relatively ineffective. The formation of Pal/Ca²⁺-induced pores in the erythrocytic membranes has been found to result in the destruction of cells.     

Biofilm growth of individual and dual strains of <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> from the dairy industry on ultrafiltration membranes

Formation of biofilms in dairy membrane plants causes membrane pore blocking, product contamination and subsequent economic loss. To investigate the biofilm growth, two Klebsiella oxytoca strains, K. B006 and TR002, previously isolated from New Zealand dairy membrane plants, were grown both individually and combined on three types of ultrafiltration (UF) membranes in different concentrations of whey medium in biofilm reactors (CBR 90, BioSurface Technologies, Bozeman, USA). Biofilms of both the individual and combined strains grew on the membrane surfaces to levels of 4.9–7.99 log colony-forming units (CFU) cm⁻² measured by standard plate counting after removing the cells by sonication. More biofilm grew on used polyethersulfone (PES) membranes than on new PES and polyvinylidene fluoride (PVDF) membranes. Both strains formed good biofilms, although K. B006 formed a denser biofilm than TR002. This corresponded to our previous study on the attachment of these organisms, where K. B006 attached in greater numbers than K. TR002. The dual strains produced a higher biofilm density than single strains on the new membranes. Biofilm density tended to increase with increased whey concentration. The saturated biofilm was approximately 10⁸ CFU cm⁻². PES membranes appeared to support biofilm growth less readily than did PVDF membranes and therefore may be the preferred material for UF membranes to reduce problems with microbial colonisation. Used membranes were more readily colonised with biofilm than were new membranes. Therefore, selecting a membrane type and monitoring membrane age will help manage biofilm development during UF.     

The Pore-Forming Properties of SsoHel308 Helicase from Saccharolobus solfataricus

The pore-forming activity of SsoHel308 helicase from extreme thermophilic archaea Saccharolobus solfataricus has been demonstrated for the first time. This protein embedded in rabbit erythrocyte membranes may cause erythrocyte hemolysis. It has been shown that this enzyme forms pores in a planar artificial bilayer membrane and acts as a transformer. After embedding this enzyme into biolayer lipid membranes, the membrane conductivity is altered. Taken together, our results show that SsoHel308 helicase is able to form pores in artificial bilayer membranes and, in some cases, the current that flows across the membranes shares features typical of ion channels. The short lifetime of the pores in the membrane significantly reduces the toxicity of helicase for a living cell. The possibility of directed translocation of single-stranded DNA in the presence of ATP will enable the use of this enzyme as a molecular syringe for injecting single-stranded DNA into living cells.

     

EtpB Is a Pore-Forming Outer Membrane Protein Showing TpsB Protein Features Involved in the Two-Partner Secretion System

Attachment to host tissues is a critical step in the pathogenesis of most bacterial infections. Enterotoxigenic Escherichia coli (ETEC) remains one of the principal causes of infectious diarrhea in humans. The recent identification of additional ETEC surface molecules suggests that new targets may be exploited in vaccine development. The EtpA protein identified in ETEC H10407 is a large glycosylated adhesin secreted via the two-partner secretion system. EtpA requires its putative partner EtpB for translocation across the outer membrane (OM). We investigated the biochemical and electrophysiological properties of purified EtpB. We showed that EtpB is 65-kDa heat-modifiable protein localized to the OM. Electrophysiological experiments indicated that EtpB is able to form pores in planar lipid bilayer membranes with an asymmetric current, suggesting its functional asymmetry. The pore of EtpB frequently assumes an opened conformation and fluctuates between three well-defined conductance states. In silico analysis of the EtpB amino acid sequence and molecular modeling suggest that EtpB is similar to the well-known TpsB protein FhaC from Bordetella pertussis and has a C-terminal transmembrane β-barrel domain that is occluded by an N-terminal α-helix, an extracellular loop, and two periplasmic polypeptide-transport-associated (POTRA) domains. Together, these data confirm that EtpB is a pore-forming protein mainly folded into a β-barrel conformation and indicate that EtpB presents typical features of the OM TpsB proteins.     

Rapid assembly of a multimeric membrane protein pore

We have observed the assembly of the staphylococcal pore-forming toxin α-hemolysin using single-molecule fluorescence imaging. Surprisingly, assembly from the monomer to the complete heptamer is extremely rapid, occurring in <5 ms. No lower order oligomeric intermediates are detected. Monte Carlo simulation of our experiment shows that assembly is diffusion limited, and pore formation is dependent on the stability of intermediate species. There are close similarities between bacterial pore-forming toxins, such as staphylococcal α-hemolysin, the anthrax protective antigen, and the cholesterol-dependent cytolysins, and their eukaryotic analogs, such as the complement pore membrane attack complex and perforin domain. The assembly mechanism we have observed for α-hemolysin provides a simple model that aids our understanding of these important pore formers.     

Exocytotic fusion pores exhibit semi-stable states

Summary Rapid-freezing/freeze-fracture electron microscopy and whole-cell capacitance techniques were used to study degranulation in peritoneal mast cells of the rat and the mutant beige mouse. These studies allowed us to create a time-resolved picture for fusion pore formation. After stimulation, a dimple in the plasma membrane formed a small contact area with the secretory granule membrane. Within this zone of apposition no ordered proteinaceous specializations were seen. Electrophysiological technique measured a small fusion pore which widened rapidly to 1 nS. Thereafter, the fusion pore remained at semi-stable conductances between 1 and 20 nS for a wide range of times, between 10 and 15,000 msec. These conductances correspond to pore diameters 25–36 nm. Ultrastructural data confirmed small pores of hourglass morphology, composed of biological membrane coplanar with both the plasma and granular membranes. Later, the fusion pore rapidly increased in conductance, consistent with the observed morphology of omega-figures. The hallmarks of channel-like behavior, instantaneous jumps in pore conductance between defined levels, and sharp peaks in histograms of conductance dwell-time, were not seen. Since the morphology of small pores shows contiguous fracture planes, the electrical data represent pores that contain lipid. These combined morphological and electrophysiological data are consistent with a lipid/protein complex mediating both the initial and later stages of membrane fusion.We would like to dedicate this paper to the memory of our friend and mentor, Alex Mauro, who emphasized to us the importance of equivalent circuits. This work was supported by National Institutes of Health grant GM-27367, and National Science Foundation grant IBN-91117509.     

Identification and characterization of the pore-forming protein in the outer membrane of rat liver mitochondria

The proteins of the outer membrane from rat liver mitochondria have been subfractionated by means of density gradient centrifugation. The different polypeptides of the membrane were incorporated into asolectin vesicles and black lipid membranes. It was observed that a polypeptide of M_r 32 000 renders asolectin vesicles permeable to ADP and forms pores in bilayer membrane. These pores showed the same properties as the channels which are formed in the lipid membrane after addition of Triton X-100 solubilized complete outer membrane. The properties of the pore are as follows: (1) The formation of pores depends on the type of phospholipid used for the preparation of the black membranes. (2) The pore is inserted asymmetrically into the membrane. (3) The pore is voltage gated but does not switch off completely at higher voltages. The pore seems to show different conductance states decreasing conductance being observed at increasing voltage. The implications of these findings for the regulation of transport processes across the outer membrane are discussed.     

Ion Channels Induced in Planar Lipid Bilayers by the Bacillus thuringiensis Toxin Cry1Aa in the Presence of Gypsy Moth (Lymantria dispar) Brush Border Membrane

The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm cis/150 mm trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much lower concentrations (1.1–2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to 420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P _Cl/P _NMDG permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of the pores formed by 100–500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P _Cl/P _NMDG permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties. Received: 23 February 2001/Revised: 15 June 2001     

Probing the stability of S-layer-supported planar lipid membranes

Isolated protein subunits of the crystalline bacterial cell surface layer (S-layer) of Bacillus coagulans E38-66 have been recrystallized on one side of planar black lipid membranes (BLMs) and their influence on the electrical properties, rupture kinetics and mechanical stability of the BLM was investigated. The effect on the boundary potential, the capacitance or the conductance of the membrane was negligible whereas the mechanical properties were considerably changed. The mechanical stability was characterized by applying voltage pulses or ramps to induce irreversible rupture. The amplitude of the voltage pulse leading to rupture allows conclusions on the ability of membranes to resist external forces. Surprisingly, these amplitudes were significantly lower for composite S-layer/lipid membranes compared to undecorated BLMs. In contrast, the delay time between the voltage pulse and the appearance of the initial defect was found to be drastically longer for the S-layer-supported lipid bilayer. Furthermore, the kinetics of the rupture process was recorded. Undecorated membranes show a fast linear increase of the pore conductance in time, indicating an inertia-limited defect growth. The attachment of an S-layer causes a slow exponential increase in the conductance during rupture, indicating a viscosity-determined widening of the pore. In addition, the mechanical properties on a longer time scale were investigated by applying a hydrostatic pressure across the BLMs. This causes the BLM to bulge, as monitored by an increase in capacitance. Compared to undecorated BLMs, a significantly higher pressure gradient has to be applied on the S-layer face of the composite BLMs to observe any change in capacitance. Received: 4 May 1999 / Revised version: 1 July 1999 / Accepted: 1 July 1999     

Molecular Uptake of Chitooligosaccharides through Chitoporin from the Marine Bacterium Vibrio harveyi

Background

Chitin is the most abundant biopolymer in marine ecosystems. However, there is no accumulation of chitin in the ocean-floor sediments, since marine bacteria Vibrios are mainly responsible for a rapid turnover of chitin biomaterials. The catabolic pathway of chitin by Vibrios is a multi-step process that involves chitin attachment and degradation, followed by chitooligosaccharide uptake across the bacterial membranes, and catabolism of the transport products to fructose-6-phosphate, acetate and NH₃.

Principal Findings

This study reports the isolation of the gene corresponding to an outer membrane chitoporin from the genome of Vibrio harveyi. This porin, expressed in E. coli, (so called VhChiP) was found to be a SDS-resistant, heat-sensitive trimer. Immunoblotting using anti-ChiP polyclonal antibody confirmed the expression of the recombinant ChiP, as well as endogenous expression of the native protein in the V. harveyi cells. The specific function of VhChiP was investigated using planar lipid membrane reconstitution technique. VhChiP nicely inserted into artificial membranes and formed stable, trimeric channels with average single conductance of 1.8±0.13 nS. Single channel recordings at microsecond-time resolution resolved translocation of chitooligosaccharides, with the greatest rate being observed for chitohexaose. Liposome swelling assays showed no permeation of other oligosaccharides, including maltose, sucrose, maltopentaose, maltohexaose and raffinose, indicating that VhChiP is a highly-specific channel for chitooligosaccharides.

Conclusion/Significance

We provide the first evidence that chitoporin from V. harveyi is a chitooligosaccharide specific channel. The results obtained from this study help to establish the fundamental role of VhChiP in the chitin catabolic cascade as the molecular gateway that Vibrios employ for chitooligosaccharide uptake for energy production.     

Poly-l-lysines and poly-l-arginines induce leakage of negatively charged phospholipid vesicles and translocate through the lipid bilayer upon electrostatic binding to the membrane

Poly-l-lysines (PLL) and poly-l-arginines (PLA) of different polymer chain lengths interact strongly with negatively charged phospholipid vesicles mainly due to their different electrical charges. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and their mixtures (1/1 mol/mol) with the respective phosphatidylcholines of equivalent chain length were chosen as model membrane systems that form at room temperature either the fluid L_α or the gel phase L_β lipid bilayer membranes, respectively. Leakage experiments revealed that the fluid POPG membranes are more perturbed compared to the gel phase DPPG membranes upon peptide binding. Furthermore, it was found that pure PG membranes are more prone to release the vesicle contents as a result of pore formation than the lipid mixtures POPG/POPC and DPPG/DPPC. For the longer polymers (≥ 44 amino acids) maximal dye-release was observed when the molar ratio of the concentrations of amino acid residues to charged lipid molecules reached a value of R_P = 0.5, i.e. when the outer membrane layer was theoretically entirely covered by the polymer. At ratios lower or higher than 0.5 leakage dropped significantly. Furthermore, PLL and PLA insertions and/or translocations through lipid membranes were analyzed by using FITC-labeled polymers by monitoring their fluorescence intensity upon membrane binding. Short PLL molecules and PLA molecules of all lengths seemed to translocate through both fluid and gel phase lipid bilayers. Comparison of the PLL and PLA fluorescence assay results showed that PLA interacts stronger with phospholipid membranes compared to PLL. Isothermal titration calorimetry (ITC) measurements were performed to give further insight into these mechanisms and to support the findings obtained by fluorescence assays. Cryo-transmission electron microscopy (cryo-TEM) was used to visualize changes in the vesicles' morphology after addition of the polypeptides.     

The 35 kDa DCCD-binding protein from pig heart mitochondria is the mitochondrial porin

The protein which can be labelled by low concentrations of dicyclohexylcarbodiimide in the Mr region of 30 000-35 000 has been purified from pig heart mitochondria with a high yield and as a single band of apparent Mr 35 000 in dodecyl sulphate-containing gels. The protein is not identical with the phosphate carrier as suggested before, since the two proteins behave differently during isolation. Incorporation of the isolated 35 kDa dicyclohexylcarbodiimide-binding protein into lipid bilayer membranes causes an increase of the membrane conductance in definite steps, due to the formation of pores. The specific pore-forming activity increases during the purification procedure. The single pore conductance is about 4.0 nS, suggesting a diameter of 1.7 nm of the open pore. The pore conductance is dependent on the voltage across the membrane. Anion permeability of the pore is higher than cation permeability. These properties are similar to those described for isolated mitochondrial and bacterial porins. It is concluded that the 35 kDa dicyclohexylcarbodiimide-binding protein from pig heart mitochondria is identical with porin from outer mitochondrial membrane.     

Prevalence and Heterogeneity of Hemolysin Gene <Emphasis Type="Italic">vhh</Emphasis> Among Hatchery Isolates of <Emphasis Type="Italic">Vibrio harveyi</Emphasis> in India

Vibrio harveyi, pathogenic to fish, harbor a hemolysin gene vhh, the homologues of which are found in many species of the Genus Vibrio. In this study, we investigated the prevalence of vhh gene among V. harveyi isolated from Penaeus monodon hatcheries in India by polymerase chain reaction (PCR). The vhh was detected in 67 of the 70 V. harveyi isolates tested in this study using different combinations of PCR primers. A variant vhh gene detected in a minority of strains was cloned, sequenced, and the recombinant protein was expressed in Escherichia coli. The deduced amino acid sequence of the cloned gene was 86% similar to the previously reported amino acid sequences of VHH. The results of this study suggest that though V. harveyi strains invariably harbor vhh, the sequence variants of the hemolysin gene exist that may impede their detection by PCR.