Bacillus thuringiensis crystal protein (δ-endotoxin) gene expression is independent of early sporulation specific functions

Bacillus thuringiensis produces a parasporal insecticidal crystal protein. The correlation between sporulation and crystal protein production inBacillus thuringiensis var.israelensis was studied. The strain was made resistant’against streptomycin (St^R)-Acrystalliferous (Cry^-) cured derivatives and asporogenous acrystalliferous (Spo⁻ Cry⁻) mutants blocked at an early stage of sporulation were isolated. Plasmid transfer experiments were performed between St^R Spo⁺ Cry⁺ (streptomycin sensitive sporogeneous crystalliferous) and St^RR Spo⁺ Cry⁻ and also between St^s Spo⁺ Cry⁺ and St^R Spo⁻ Cry⁻ strains. StR colonies were selected. Insect toxicity was exhibited by the St^R isolates in both the cases. The process of crystal formation is, therefore, independent of early sporulative events.     

Toxicity of Bacillus thuringiensis Spo− Cr+ Mutants for the European Corn Borer Ostrinia nubilalis

Inclusion bodies isolated from Spo⁻ Cr⁺ mutants of Bacillus thuringiensis were toxic for larvae of the European corn borer. Probit analysis revealed comparable toxicity between wild-type crystals (isolated from B. thuringiensis subsp. kurstaki) and crystals produced from two spore-free mutants of the same subspecies. Death of the larvae was due to starvation, presumably through δ-endotoxin-induced gut paralysis. Inclusion bodies pretreated with α-chymotrypsinogen were equally as toxic as native crystals for the insect larvae.     

Involvement of a transmissible plasmid in heat-stable exotoxin and delta-endotoxin production inBacillus thuringiensis subspeciesdarmstadiensis

A strain ofBacillus thuringiensis subsp.darmstadiensis (serotype 10), which produces heat-stable exotoxin and delta-endotoxin (Exo⁺Cry⁺), was used for curing and conjugation-like transformation experiments. After treatment with sodium dodecyl sulfate, nine independent mutants that lacked exotoxin productivity (Exo^–) were obtained. Agarose gel electrophoresis showed that all Exo^– strains had lost a plasmid, whose size was 62 megadaltons (Mdal). WhenB. thuringiensis was mated with a streptomycin-resistant (Str^r)B. cereus strain, five Exo⁺Str^r transformants that had acquired the 62-Mdal plasmid were isolated. Furthermore, the Cry⁺ phenotype was consistently associated with the Exo⁺ phenotype. These results indicate that a transmissible plasmid is involved in production of both heat-stable exotoxin and delta-endotoxin.     

Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis

Three crystalliferous (Cry⁺) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry^?) mutants, either induced or spontaneously derived from a single Cry⁺ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry⁺ and Cry^? variants were analyzed by both a cleared lysate- and a modified Eckhardt lysateelectrophoresis technique. All of the Cry^? mutants derived from the Cry⁺ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry^? variants. All three Cry⁺ strains, including the parent of the Cry^? strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry⁺ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry^? derivatives differed in other respects from the pattern for their parent strain. The various Cry⁺ and Cry^? strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.     

Correlation between specific plasmids and δ-endotoxin production in Bacillus thuringiensis

Five strains of Bacillus thuringiensis that produce crystalline δ-endotoxin were used as parental strains in an effort to isolate acrystalliferous (Cry⁻) mutants: HD-2 (B. thuringiensis var. thuringiensis, flagellar serotype 1); HD-1 and HD-73 (both var. kurstaki, serotype 3ab); HD-4 (var. alesti, serotype 3a); and HD-8 (var. galleriae, serotype 5ab). The parental strains contain complex plasmid arrays that have been previously characterized (González and Carlton, 1980). The plasmid patterns of both Cry⁻ and Cry⁺ variants were analyzed and compared to the parental strains using a modified Eckhardt (1978) lysate-electrophoresis method. Most Cry⁻ mutants derived from strain HD-2 were found to exhibit a distinctive colony morphology which facilitated their isolation. Loss of crystal production was associated with loss of a 75-Md plasmid. A 50-Md plasmid of strain HD-73 was lost in the Cry⁻ mutants. Crystal production in strain HD-4 appears to be associated with a plasmid about 105 Md in size; in strain HD-1, a smaller plasmid (29 Md in size) seems to be involved. In strain HD-8, a large plasmid (˜130 Md in size) is implicated in crystal production. Direct bioassay of several of the mutant strains has confirmed the loss of δ-endotoxin activity in the acrystalliferous isolates. The evidence obtained supports the notion of a relationship between specific extrachromosomal DNA elements and δ-endotoxin production in B. thuringiensis, and suggests that in each strain only a single plasmid is involved, although the size of the implicated plasmid varies from one strain to another.     

Expression of Bacillus thuringiensis full-length and N-terminally truncated vip184 gene in an acrystalliferous strain of subspecies kurstaki

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. The 3.5 kb full-length vip184 gene was cloned from a wild-type isolate of B. thuringiensis, and the vip184S gene was constructed by deletion of the putative signal peptide encoding sequence. Both genes were expressed in the acrystalliferous strain Cry^–B of B. thuringiensis. Vip184 protein was observed mainly in the centrifuged pellets of B. thuringiensis Cry^–B(pHPT3), which contains the vip184 gene, and was less abundant in the concentrated supernatant. However, Vip184S proteins were not detected in the concentrated supernatant, but only in the pellets of Cry^–B(pHPT3S), which contains vip184S gene. This indicated that Vip184S proteins were not secreted into the culture medium and that the putative signal peptides were essential for the secretion of Vip184. The toxicity of Cry^–B(pHPT3) and Cry^–B(pHPT3S) were demonstrated against the neonate larvae of Spodoptera exigua and S. litura. Pellets and concentrated supernatant of Cry^–B(pHPT3) showed high activity against S. exigua and S. litura, but the Cry^–B(pHPT3S) strain was not toxic to either because of the deletion of N-terminal putative signal peptides. Therefore, this may suggest that the putative signal peptides are required for lethality.     

Genetic evidence for plasmid-encoded lactococcin production inLactococcus lactis subsp.lactis 484

Lactococcus lactis subsp.lactis 484 produced a proteinaceous antibacterial substance designated as lactococcin capable of inhibiting members of theLactococcus group,Bacillus cereus, Staphylococcus aureus, andSalmonella typhi. Growth of this culture in the presence of 2–30 g/ml of ethidium bromide or acriflavin or novobiocin, and at elevated temperature (39° and 41°C), could not produce any lactococcin-negative (Lap^–) variants. However, protoplast-induced curing with lysozyme was successful in developing Lap^– derivatives. Two types of cured derivatives, namely Lac^– Lap⁺ and Lac^– Lap^–, were obtained. Lap^– variants were also lacking sucrose-fermenting ability (Suc⁺) and lactococcin resistance (Lap^r). The lactose-negative (Lac^–) variants and Lap⁺ were clearly lacking the largest (65 Md) plasmid. However, Lap^– Suc^– Lap^s variants lost a 2 Md plasmid.L. lactis subsp.lactis 484 transferred lactose-fermenting ability as well as Lap⁺ Suc⁺ Lap^r phenotypes simultaneously toL. lactis subsp.lactis LM 2306 and LM 0230 by surface mating at a frequency of 10^–4 and 10^–1 per donor respectively. However, cured Lac^– Lap^– transconjugants could not transfer Lac⁺ Lap⁺ Suc⁺ Lap^r phenotypes to any of these recipient strains. Our results indicate that Lac⁺ and Lap⁺ Suc⁺ Lap^r phenotypes are associated with 65 Md and 2 Md plasmids respectively. Conjugal transfer of 2 Md plasmid is possible only in the presence of a conjugative 65 Md plasmid.     

Purification of truncated and mutated Chemotaxis Inhibitory Protein of Staphylococcus aureus--an anti-inflammatory protein

The Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) binds and blocks the C5a receptor (C5aR) and formyl-peptide receptor (FPR). This way, CHIPS is a potent inhibitor of the immune cell recruitment associated with inflammation. Truncation of the protein and the introduction of mutations, shifts the expression towards the insoluble fraction of Escherichia coli, whereas the wild-type protein can be solubly expressed. A protocol for expression and tag independent purification of biologically active CHIPS variants has been established to enable further characterization of an improved CHIPS variant, called ADC-1004. The CHIPS variants were purified by washing of E. coli inclusion bodies followed by refolding and gel filtration. New techniques were utilized to optimize the purification process. Expression in inclusion bodies was increased by the use of Ultra Yield^™ flasks and optimal refolding conditions were determined by the use of the iFOLD Refolding System 2^™.The folding and biological activity of the purified proteins were analyzed by circular dichroism (CD) spectroscopy and flow cytometry, respectively, and compared to solubly produced CHIPS_31–113 and wild-type CHIPS_1–121. We show that the CHIPS variants produced in inclusion bodies can be refolded and purified to achieve equal biological activity as solubly produced CHIPS_31–113 and wild-type CHIPS_1–121. The truncation causes minor structural changes while purification from inclusion bodies or the soluble fraction does not further affect the structure.     

Stabilization and increased production of insecticidal crystal proteins of Bacillus thuringiensis subsp. galleriae in steady- and transient-state continuous cultures

Bacillus thuringiensis subsp. galleriae, grown in continuous cultures, segregated to spontaneous asporogenic variants replacing the wild-type Spo⁺ Cry⁺ strains [Sachidanandham R, Jayaraman K (1993) Appl Microbiol Biotechnol 40:504–507]. Realizing that this was due to specific but unknown nutritional requirements, we undertook further continuous-culture studies to identify growth requirement(s) by pulsing various medium components and growth factors. While carbon, nitrogen and pulses of nutrients exhibited a neutral pulse response, a group of amino acids were shown to improve the stability and volumetric productivity of biomass. The formation of spores and insecticidal crystal proteins was found to be higher with amino acid supplementation. Comparison of carbon-limited steady-state continuous cultures under two different conditions of growth brought forth the stabilizing effects of the amino acid supplementation. Batch experiments carried out with these inputs demonstrated a better carbon utilization, resulting in a higher biomass as well as enhancement of bioinsecticidal activity. Received: 14 May 1996 / Received revision: 9 September 1996 / Accepted: 13 September 1996     

Properties ofPseudomonas aeruginosa exhibiting self-lysis

Spontaneous lysis leading to the production of turbid, iridescent auto-plaques (AP⁺) was noted in 46 out of 50 strains ofPseudomonas aeruginosa. Strain Pa-1 which is mucoid and is a non-auto-plaque former (M⁺AP^–) on rare occasions lyses; the surviving cells are non-mucoid and always exhibited plaques on itself (M^–AP⁺) as well as on the M⁺AP^– culture. In addition, the non-mucoid culture gave rise to a mucoid, auto-plaque producing variant (M⁺AP⁺). Biochemical characterization of the cultures indicated no other qualitative differences, although AP⁺ cultures were more proteolytic, but less hemolytic than the M⁺AP^– strain. All three cultures synthesized the bacteriocin pyocin, but were immune to both their own and each other's agents. In addition, they exhibited the same lysogenic host range when streaked against 18 other cultures ofP. aeruginosa. Treatment of the auto-plaque forming strains with non-inhibitory levels of penicillin, streptomycin, chloromycetin, or polymyxin, stimulated cultures to produce increased numbers of auto-plaques in proportion to antibiotic concentrations, while no lysis was noted with the non-autolytic strain (M⁺AP^–). However, treatment of the three cultures with varying doses of ultra-violet did not stimulate the production of auto-plaques beyond the normal level of non-irradiated cultures, and in some cases suppressed their appearance. Filtrates obtained from the non-mucoid, auto-plaque producing culture (M^–AP⁺) formed iridescent, turbid plaques on M⁺AP^–, M⁺AP⁺, and M^–AP⁺. Similar results were obtained with the M⁺AP⁺ filtrates.     

Characteristics of antibody inhibition of rat kidney (Na+−K+)-ATPase

Summary Antibodies which were raised against highly purified membrane-bound (Na⁺–K⁺)-ATPase from the outer medulla of rat kidneys inhibit the (Na⁺–K⁺)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots.In the purified membrane-bound (Na⁺–K⁺)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na⁺–K⁺)-ATPase activity (n=0.86), (b) for the ATP binding to the enzyme (n=0.58), and (c) for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity (n=0.77). By measuring the Na⁺ dependence of the (Na⁺–K⁺-ATPase reaction, a positive homotropic cooperativity (n=1.67) was found.As reactivation of the antibody-inhibited enzyme proceeds very slowly (t _0.5=5.2hr), it was possible to measure characteristics of the antibody-(Na⁺–K⁺)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na⁺–K⁺)-ATPase reaction and on the ATP binding of the enzyme.V _max of the (Na⁺–K⁺)-ATPase reaction and the number of ATP binding sites were reduced whileK _{0.5 ATP} for the (Na⁺–K⁺)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased theK _0.5 value for the Na⁺ stimulation of the (Na⁺–K⁺)-ATPase activity, but they did not alter the homotropic interactions between the Na⁺-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity was abolished by the antibodies.The data are tentatively explained by the following model: The antibodies bind to the (Na⁺–K⁺)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na⁺–K⁺)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na⁺ dependence of the (Na⁺–K⁺)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.     

A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis

Bacillus thuringiensis var. israelensis (BTI), serotype 14, which produces parasporal crystals toxic to certain dipteran larvae, was analyzed by agarose gel electrophoresis and found to contain a complex plasmid array. Eight plasmids were detected, with approximate sizes of 3.3, 4.2, 4.9, 10.6, 68, 75, 105, and 135 MDa, as well as a plasmidlike linear DNA element of ~10 MDa. Partially cured mutants of BTI implicated the 75-MDa plasmid in crystal production. Fifteen independently isolated acrystalliferous (Cry^?) mutants were found to lack this plasmid. In plasmid transfer experiments, several of the BTI plasmids transferred into a plasmid-free, Cry^? BTI recipient, but only transfer of the 75-MDa plasmid converted the recipient to crystal toxin production. The presence or absence of mosquito-toxic activity in all Cry⁺ and Cry^? variants of BTI was confirmed by bioassay of sporulated cultures against larvae of Aedes aegypti. Southern blot analyses revealed that in one unusual Cry⁺ variant in which no 75-MDa plasmid band was detectable, plasmid sequences were still present, possibly integrated into the chromosome. The 75-MDa plasmid could also apparently recombine with the 68-MDa plasmid, to which it was partially homologous.     

Outer membrane peptides ofYersinia pestis mediating siderophore-independent assimilation of iron

Summary It is established that wild-type cells ofYersinia pestis absorb exogenous hemin or Congo red and thus grow as pigmented colonies at 26° C on media containing these chromatophores (Pgm⁺). Pgm⁺ isolates are known to possess a siderophore-independent mechanism of iron-transport (required for growth in iron-deficient medium) which is absent in avirulent Pgm^– mutants. Production of the bacteriocin pesticin and linked invasins (Pst⁺) is an additional defined virulence factor of yersiniae; mutation of Pgm⁺,Pst^– organisms to pesticin-resistance (Pst^r) results in concomitant conversion to Pgm^–. In this study, autoradiograms of two-dimensional gels of [³⁵S]methionine-labeled outer membranes from Pgm^– mutants were compared to those of the Pgm⁺,Pst⁺ or Pgm⁺,Pst^– parent. An apparently single predominant peptide present in these preparations (> 10% of total membrane protein) existed as a family of iron-modifiable 17.9-kDa molecules focusing down to isoelectric points of about 4.6 and up to 5.89. Expression of eight detectable Pst⁺-specific peptides was not significantly influenced by exogenous iron. Pgm⁺ yersiniae constitutively produced pigmentation-specific peptide F and five iron-repressible peptides termed IrpA to IrpE. Typical spontaneous mutation to Pgm^– resulted in loss of peptide F and IrpB-E. A rare Pgm⁺,Pst^r mutant, selected on Congo red agar containing pesticin, also lost IrpB-E but retained peptide F. This isolate, like Pgm^– mutants, failed to grow in iron-deficient medium. Regardless of phenotype, all yersiniae utilized hemin, hemopexin, myoglobin, hemoglobin, and ferritin, but not transferrin or lactoferrin, as sole sources of iron.This is journal article no. 13025 of the Michigan Agricultural Experiment Station.     

Correlation between alkaline activation of Bacillus thuringiensis var. kurstaki spores and crystal production

The spores of crystal-forming (Cry⁺) and non-crystal-forming (Cry^-) strains of Bacillus thuringiensis var. kurstaki and Bacillus cereus were tested for the ability to be activated by 0.1 m K₂CO₃ (pH 10). Only the spores of crystal-forming strains could be activated, and this phenotype was independent of whether crystals were present with the spores in the activation solution. The spores of a B. thuringiensis var. kurstaki strain that is temperature sensitive for protoxin accumulation could be activated by the alkaline solution when produced at the permissive temperature, whereas spores produced at the nonpermissive temperature were not activated. The results indicate that protoxin in the spore coat is responsible for the alkaline-activation phenotype and may serve an ecological function for the organism.     

Growth of catalase A and catalase T deficient mutant strains of Saccharomyces cerevisiae on ethanol and oleic acid

The parental strain (A⁺T⁺) of Saccharomyces cerevisiae and mutants, deficient in catalase T (A⁺T^–), catalase A (A^–T⁺) or both catalases (A^–T^–), grew on ethanol and oleic acid with comparable doubling times. Specific activities of catalase were low in glucose- and ethanol-grown cells. In the two oleic acid-grown A⁺-strains (A⁺T⁺ and A⁺T^–) high catalase activities were found; catalase activity invariably remained low in the A^–T⁺ strain and was never detected in the A^–T^– strain. The levels of -oxidation enzymes in oleic acid-grown cells of the parental and all mutant strains were not significantly different. However, cytochrome C peroxidase activity had increased 8-fold in oleic acid grown A^– strains (A^–T⁺ and A^–T^–) compared to parental strain cells. The degree of peroxisomal proliferation was comparable among the different strains. Catalase A was shown to be located in peroxisomes. Catalase T is most probably cytosolic in nature and/or present in the periplasmic space.     

Kinetic Characterization of Extracellular Catalases fromPenicillium piceum F-648 and Its Hydrogen Peroxide-Adapted Variants

A comparative kinetic study of extracellular catalases produced by Penicillium piceum F-648 and their variants adapted to H₂O₂ was performed in culture liquid filtrates. The specific activity of catalase, the maximum rate of catalase-induced H₂O₂ degradation (V _max), V _max/K _M ratio, and the catalase inactivation rate constant in the enzymatic reaction (k _in, s^–1) were estimated in phosphate buffer (pH 7.4) at 30°C. The effective constant representing the rate of catalase thermal inactivation (k _in ^*, s^–1) was determined at 45°C. In all samples, the specific activity and K _M for catalase were maximum at a protein concentration in culture liquid filtrates of (2.5–3.5) × 10^–4 mg/ml. The effective constants describing the rate of H₂O₂ degradation (k, s^–1) were similar to that observed in the initial culture. These values reflected a twofold decrease in catalase activity in culture liquid filtrates. We hypothesized that culture liquid filtrates contain two isoforms of extracellular catalase characterized by different activities and affinities for H₂O₂. Catalases from variants 5 and 3 with high and low affinities for H₂O₂, respectively, had a greater operational stability than the enzyme from the initial culture. The method of adaptive selection for H₂O₂ can be used to obtain fungal variants producing extracellular catalases with improved properties.     

The segregation of the SbcA and Rac phenotypes in an Escherichia coli recB mutant

Summary In certain HfrxF^– recB ^– crosses recombinant progeny were examined for their SbcA and Rac phenotypes. Recombinants which inherited either his ⁺ or trp ⁺ from the donor in an Hfr recB21 sbcA8xF^– recB21 Rac^–SbcA⁺ cross acquired the RecB⁺ phenotype in most instances (presumably by inheriting the sbcA8 allele). Several independent Rec⁺ (sbcA8) recombinants from this cross were converted back to the Rec^– (sbcA ⁺) phenotype by mating with a Rac⁺ SbcA⁺ Hfr. Ten out of 14 of these Rec^– recombinants retained the Rac^– phenotype of the original parent. It was concluded that these results were inconsistent with the hypothesis that sbcA is a gene carried by a Rac prophage.     

Inducible nitrate reductase of rice plants as a possible indicator for nitrification in water-logged paddy soils

When following the pattern of the disappearance of NH ₄ ⁺ –N from ammonium sulfate applied to the flooded soil-rice plant system (field and greenhouse experiments) during a growing season, it was observed that the lowest NH ₄ ⁺ –N level coincided with the highest value of NR activity in the leaves. Nitrate was detected in both the root and shoot systems of the rice plants and autotrophic nitrifiers (Nitrosomonas and Nitrobacter) were particularly abundant. Since it was also demonstrated in this work that the NR activity of rice plants grown with nitrate fertilization (growth chamber culture experiments) was inducible by its substrate, it can be assumed that NH ₄ ⁺ –N oxidation takes place in the water-logged soil studied. Therefore, the occurrence of the nitrification process following NH ₄ ⁺ –N fertilizer application can be predicted by thein vitro orin situ evaluation of the NR activity of the rice leaf as an indicator.     

Molecular principles of antigenic variation in Neisseria gonorrhoeae

The genome of Neisseria gonorrhoeae harbours many gene loci for the production of variant pili. Strain MS11 has two expression genes (pilE) with promoter and complete coding sequences. The remaining genes are silent (pilS) lacking the promoter and the conservative amino terminals coding sequences of pilin. The pilus genes consist of six variable minicassettes (mc's), that are flancked by strictly conserved sequences. Upon phase (P⁺ to P⁺) and antigenic (P⁺ to P^–, or vice versa) transitions minicassettes from silent loci are transferred from silent pilus gene copies to the expression gene by gene conversion. P^– variants resulting from such rearrangements still produce pilin mRNA as well as pilin, but only a few are found on the surface of those gonococci.     

Thiol-dependent passive K+Cl− transport in sheep red blood cells: VI. Functional heterogeneity and immunologic identity with volume-stimulated K+(Rb+) fluxes

Summary Ouabain-resistant (OR), volume-or N-ethylmaleimide (NEM)-stimulated K⁺(Rb⁺)Cl^– fluxes were measured in low-K⁺ sheep red cells and found to be functionally separate but immunologically similar. In anisosmotic solutions both K⁺ effluxes and Rb⁺ influxes of NEM-treated and control cells were additive. In contrast to the NEM-stimulated K⁺Cl^– flux, metabolic depletion did not reduce K⁺Cl^– flux of normal or swollen cells. The anion preference of OR K⁺ efflux in NEM-treated cells was Br^–>Cl^–>HCO ₃ ^– =F^–I^–=NO ₃ ^– =CNS^–, and hence consistent with a reported Br^–>Cl^–>NO ₃ ^– sequence of the volume-dependent K⁺Cl^– transport. Alloimmune anti-L_l antibodies known to decrease passive K⁺ fluxes in low K⁺ cells reduced by 51% both volume-and NEM-stimulated, furosemidesensitive Rb⁺Cl^– fluxes suggesting their immunologic identity, a conclusion also supported by anti-L₁ absorption studies. Since pretreatment with anti-L₁ prevented the activation of Rb⁺ influx by NEM, and the impermeant glutathionmaleimide-I did not stimulate Rb⁺Cl^– influx, the NEM reactive SH groups must be located apart from the L₁ antigen either within the membrane or on its cytoplasmic face. A model is proposed consisting of a K⁺Cl^– transport path(s) regulated by a protein with two functional subunits or domains; a chemically (C _s) and a volume (V _s)-stimulated domain, both interfacing with the L₁ surface antigen. Attachment of alloanti-L₁ from the outside reduces K⁺Cl^– transport stimulated throughC _s by NEM orV _s by cell swelling.