Mutational analysis of the subunit interface of Vibrio harveyi bacterial luciferase

Bacterial luciferase is a heterodimeric (alphabeta) enzyme which catalyzes a light-producing reaction in Vibrio harveyi. In addition to the alphabeta enzyme, the beta subunit can self-associate to form a stable but inactive homodimer [Sinclair, J. F., Ziegler, M. M., and Baldwin, T. O. (1994) Nat. Struct. Biol. 1, 320-326]. The studies reported here were undertaken to explore the role of the subunit interface in the conformational stability of the enzyme. To this end, we constructed four mutant heterodimers in which residues at the subunit interface were changed in an effort to alter the volume of an apparent solvent accessible channel at the interface or to alter H-bonding groups. Equilibrium unfolding data for the heterodimer have been interpreted in terms of a three-state mechanism [Clark, C. A., Sinclair, J. F., and Baldwin, T. O. (1993) J. Biol. Chem. 268, 10773-10779]. However, we found that unfolding for the wild-type and mutant luciferases is better described by a four-state model. This change in the proposed mechanism of unfolding is based on observation of residual structure in the subunits following dissociation of the heterodimeric intermediate. All of the mutants display modest reductions in activity but, surprisingly, no change in the DeltaG2H2O value for subunit dissociation and no measurable change in the equilibrium dissociation constant relative to that of the wild-type heterodimer. However, the DeltaG1H2O value for the formation of the dimeric intermediate that precedes subunit dissociation is reduced for three of the mutants, indicating that mutations at the interface can alter the stability of a region of the alpha subunit that is distant from the interface. We conclude that the interface region communicates with the distal domains of this subunit, probably through the active center region of the enzyme.     

Relationship between the conserved alpha subunit arginine 107 and effects of phosphate on the activity and stability of Vibrio harveyi luciferase.

The Arg107 of the alpha subunit is a conserved residue for all known bacterial luciferases. The phosphate moiety of the reduced flavin mononucleotide (FMNH(2)) side chain has been hypothesized to be anchored at this site (A. J. Fisher, F. M. Raushel, T. O. Baldwin, and I. Rayment Biochemistry 34, 6581-6586, 1995). Mutations of alphaArg107 of the Vibrio harveyi luciferase to alanine, serine, and glutamate were carried out to test such a hypothesis. These variants were characterized and compared with the wild-type luciferase with respect to their K(m) for decanal, FMNH(2), and reduced riboflavin in both low- (0.01 or 0.05 M) and high- (0.3 M) phosphate buffers at pH 7.0. Results are consistent with the hypothesized binding of the FMNH(2) phosphate group by alphaArg107. Moreover, the alphaArg107 residue was apparently important in the expression of the luciferase maximal activity and aldehyde binding. Phosphate ion is also known to have other effects on luciferase stability. We compared the three luciferase variants with the native enzyme with respect to the decay rate of the FMN 4a-hydroperoxide intermediate II, and rates of inactivation by trypsin digestion, modification by N-ethylmaleimide, and heat treatment in low- and high-phosphate buffers. On the basis of patterns of the phosphate effects, alphaArg107 appeared to be important to the enhancement of luciferase stability against trypsin proteolysis at high phosphate but was not involved in regulating the intermediate II decay or sensitivity to N-ethylmaleimide modification. Differential effects of mutations on luciferase thermal stability were observed. It is uncertain whether alphaArg107 is involved in the enhanced thermal stability of the native luciferase in high phosphate buffer.     

Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the alpha subunit of bacterial luciferase

The nucleotide sequence of the 1.85-kilobase EcoRI fragment from Vibrio harveyi that was cloned using a mixed-sequence synthetic oligonucleotide probe (Cohn, D. H., Ogden, R. C., Abelson, J. N., Baldwin, T. O., Nealson, K. H., Simon, M. I., and Mileham, A. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 120-123) has been determined. The alpha subunit-coding region (luxA) was found to begin at base number 707 and end at base number 1771. The alpha subunit has a calculated molecular weight of 40,108 and comprises a total of 355 amino acid residues. There are 34 base pairs separating the start of the alpha subunit structural gene and a 669-base open reading frame extending from the proximal EcoRI site. At the 3' end of the luxA coding region there are 26 bases between the end of the structural gene and the start of the luxB structural gene. Approximately two-thirds of the alpha subunit was sequenced by protein chemical techniques. The amino acid sequence implied by the DNA sequence, with few exceptions, confirmed the chemically determined sequence. Regions of the alpha subunit thought to comprise the active center were found to reside in two discrete and relatively basic regions, one from around residues 100-115 and the second from around residues 280-295.     

Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase

Vibrio harveyi luciferase is an alphabeta heterodimer containing a single active site, proposed earlier to be at a cleft in the alpha subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe --> Ala mutants, alphaF46, alphaF49, alphaF114, and alphaF117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of alphaF46, alphaF49, alphaF114, and alphaF117 was important to luciferase V(max) and V(max)/K(m), which were reduced by 3-5 orders of magnitude for the Phe --> Asp mutants. Both alphaF46 and alphaF117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by alphaF46D, alphaF49D, alphaF114D, and alphaF117D revealed that their marked reductions in the overall quantum yield (phi( degrees )) were a consequence of diminished yields of luciferase intermediates and, with the exception of alphaF114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed.     

Changes in the kinetics and emission spectrum on mutation of the chromophore-binding platform in Vibrio harveyi luciferase

The recently proposed model for the bacteria luciferase-flavin mononucleotide complex identifies a number of critical intermolecular interactions that define a binding platform for the isoalloxazine ring of flavin [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A. (2001) Protein Sci. 10, 1563-1571]. A key interaction involving van der Waals contact between the isopropyl side chain of alphaVal173 and the 7,8-dimethyl benzene plane of the isoalloxazine chromophore represents an important target to test the validity of the proposed model. Here, structure-function analysis of luciferase variants carrying single point mutations at position alpha173 have verified the functional layout of the active site architecture and implicated this site directly in flavin binding. Moreover, a decrease in the stability of the enzyme-bound C4a-hydroperoxyflavin intermediate in the mutants could account for changes in saturation with the fatty aldehyde substrate. A predicted red-shift on mutation of position alpha173 to increase its polarity confirmed that alphaVal173 was an integral component of the chromophore-binding microenvironment. Introduction of mutations in residues that contact the pyrimidine plane of the isoalloxazine chromophore (alphaA75G/C106V) into the alphaV173A, alphaV173C, alphaV173T, and alphaV173S mutants led to the retention of high levels of enzyme activity (10-40% of wild type) and further red-shifted the emission spectra in the triple mutants. The additivity of the mutation-induced red-shifts in the emission wavelength spectrum provides the basis toward engineering luciferase variants that emit different light colors with the proposed flavin-luciferase model complex as a design reference.     

Functional roles of conserved residues in the unstructured loop of Vibrio harveyi bacterial luciferase

Residues 257-291 of the Vibrio harveyi bacterial luciferase alpha subunit comprise a highly conserved, protease-labile, disordered loop region, most of which is unresolved in the previously determined X-ray structures of the native enzyme. This loop region has been shown to display a time- dependent proteolysis resistance upon single catalytic turnover and was postulated to undergo conformational changes during catalysis ([AbouKhair, N. K., Ziegler, M. M., and Baldwin, T. O. (1985) Biochemistry 24, 3942-3947]. To investigate the role of this region in catalysis, we have performed site-specific mutations of different conserved loop residues. In comparison with V(max) and V(max)/K(m,flavin) of the native luciferase, the bioluminescence activities of alphaG284P were decreased to 1-2% whereas those of alphaG275P and alphaF261D were reduced by 4-6 orders of magnitude. Stopped-flow results indicate that both alphaG275P and alphaF261D were able to form the 4a-hydroperoxy-FMN intermediate II but at lower yields. Both mutants also had enhanced rates for the intermediate II nonproductive dark decay and significantly compromised abilities to oxidize the decanal substrate. Additional mutations were introduced into the alphaG275 and alphaF261 positions, and the activities of the resulting mutants were characterized. Results indicate that the torsional flexibility of the alphaG275 residue and the bulky and hydrophobic nature of the alphaF261 residue were critical to the luciferase activity. Our results also support a functional role for the alpha subunit unstructured loop itself, possibly by serving as a mobile gating mechanism in shielding critical intermediates (including the excited flavin emitter) from exposure to medium.     

Effects of general anesthetics on the bacterial luciferase enzyme from Vibrio harveyi: an anesthetic target site with differential sensitivity

The effects of a diverse range of 36 general anesthetics and anesthetic-like compounds on a highly purified preparation of the bacterial luciferase enzyme from Vibrio harveyi have been investigated. Under conditions where the flavin site was saturated, almost all of the anesthetics inhibited the peak enzyme activity and slowed the rate of decay. However, a small number of the more polar agents only inhibited at high concentrations, while stimulating activity at lower concentrations. The inhibition was found to be competitive in nature, with the anesthetics acting by competing for the binding of the aldehyde substrate n-decanal. The anesthetic binding site on the enzyme could accommodate only a single molecule of a large anesthetic but more than one molecule of a small anesthetic, consistent with the site having circumscribed dimensions. The homologous series of n-alcohols and n-alkanes exhibited cutoffs in inhibitory potency, but these cutoffs occurred at very different chain lengths (about C10 for the n-alkanes and C15 for the n-alcohols), mimicking similar cutoffs observed for general anesthetic potencies in animals. Binding constants determined from peak height measurements showed that the inhibitor binding site was predominantly hydrophobic (with a mean delta delta G CH2 of -5.0 kJ/mol), but fluctuations in the binding constants with chain length revealed regions in the binding site with polar characteristics. Binding constants to an intermediate form of the enzyme (intermediate II) were also determined, and these confirmed the principal features of the binding site deduced from the peak height measurements. The long-chain compounds, however, bound considerably tighter to the intermediate II form of the enzyme, and this was shown to account for the biphasic decay kinetics that were observed with these compounds. Overall, there was poor agreement between the EC50 concentrations for inhibiting the luciferase enzyme from V. harveyi and those which induce general anesthesia in animals, with bulky compounds being much less potent, and moderately long chain alcohols being much more potent, as luciferase inhibitors than as general anesthetics.     

Implications of the reactive thiol and the proximal non-proline cis-peptide bond in the Structure and function of Vibrio harveyi luciferase

The role of a highly reactive cysteine residue, Cys106, in Vibrio harveyi luciferase in modulating the substrate-enzyme interactions and in turn affecting the enzyme activity has been extensively investigated over the past three decades. Replacing Cys106 with valine dramatically hinders the ability of luciferase to stabilize the C4a-hydroperoxyflavin intermediate [Abu-Soud, H. M., Clark, A. C., Francisco, W. A., Baldwin, T. O., and Raushel, F. M. (1993) J. Biol. Chem. 268, 7699-7706] and consume aldehyde substrate [Xi, L., Cho, K.-W., Herndon, M. E., and Tu, S.-C. (1990) J. Biol. Chem. 265, 4200-4203], therefore markedly decreasing enzyme activity. On the basis of the structure-activity relationship of flavin analogues and the location of the phosphate binding site of flavin mononucleotide (FMN) coupled with molecular modeling, the functional part of the isoalloxazine ring of FMN, the thiol side chain of Cys106, the methyl group of Ala75, and the unique non-prolyl cis-peptide bond between Ala74 and Ala75 were found to be closely packed [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A. (2001) Protein Sci. 10, 1563-1571]. Here, by mutating Ala75 to Gly, we restored key wild-type properties to the C106V mutant, in particular, high enzyme activity and a stable C4a-hydroperoxyflavin intermediate, demonstrating that the primary reason for the dark phenotype of the C106V mutant was the unfavorable steric interaction between Val106 and Ala75 side chains, which could in turn disturb the cis-oriented amide linkage of Ala74 and Ala75. Moreover, significant red shifts in light emission of 3-10 nm were measured for luciferases carrying Val106 with the spectrum of the double mutant C106V/A75G now red shifted to that of Photobacterium phosphoreum luciferase, which also has Val and Gly at positions 106 and 75, respectively. These results strengthen the validity of the binding geometry of the modeled flavin with the re-face of the pyrimidine end of the isoalloxazine ring next to Cys106 and implicate the Ala74-Ala75 cis-peptide as a key component in the bioluminescence reaction.     

Probing the functionalities of alphaGlu328 and alphaAla74 of Vibrio harveyi luciferase by site-directed mutagenesis and chemical rescue

This work aimed at identifying essential residues on the alpha subunit of Vibrio harveyi luciferase and elucidating their functional roles. Four conserved alpha-subunit residues at the proposed luciferase active site were initially mutated to Ala. Screening of the in vivo bioluminescence of cells expressing these mutated luciferases allowed the work to focus on alphaGlu328 for additional mutations to Phe, Leu, Gln, His, and Asp. V. harveyi luciferase is known to contain, at the same proposed active site, an unusual cis-peptide linkage between alphaAla74 and alphaAla75. To explore the structure-function relationship, luciferase variants alphaA74F and alphaA74G were constructed. The six alphaGlu328-mutated and the two alphaAla74-mutated luciferase variants were purified and characterized with respect to Vmax, Michaelis constants, light and dark decays, quantum yield, and, for alphaE328F and alphaA74F, yield of the 4a-hydroperoxyFMN intermediate and the ability to oxidize aldehyde substrate. Results indicated that the structural integrities of both alphaGlu328 and alphaAla74 were essential to luciferase bioluminescence activity. Moreover, the essentiality of alphaGlu328 was linked to the acidic nature of its side chain. The low activity of alphaE328A was sensitive to chemical rescue by sodium acetate, an effect that was not reproduced by phosphate. The efficiency of activity rescue by acetate progressively increased at lower pH in the range from 6.0 to 8.0, supporting the interpretation of alphaGlu328 as a catalytic general acid. The rescuing effect of acetate was on a reaction step after the formation of the 4a-hydroperoxyFMN intermediate. The exact catalytic function of alphaGlu328 is unclear, but possibilities are discussed.     

Immunogold labeling of luciferase in the luminous bacterium Vibrio harveyi after fast-freeze fixation and different freeze-substitution and embedding procedures

We studied the ultrastructural localization of luciferase on sections of the bioluminescent bacterium Vibrio harveyi by indirect immunogold staining, using a polyclonal antiluciferase antibody and the usual control tests, after chemical fixation or fast-freeze fixation (FFF) followed by different freeze-substitution (FS) procedures and embedding in either Epon or LR White. After liquid fixation with glutaraldehyde and paraformaldehyde and LR White embedding, labeling occurred over the cytoplasm but not over the condensed nucleoid. Epon embedding almost abolished it. FFF-FS considerably improved the morphological preservation and revealed cytoplasmic "patches" with a complex ultrastructure in Epon sections. The preservation was always less good in LR White. The patches were densely labeled, even in Epon sections, after FS in acetone. However, labeling intensity was 3.7 times greater in LR White than in Epon. With both resins, labeling diminished similarly when fixative agents were present in the FS medium. The localization of luciferase in the cytoplasm and particularly in the patches is discussed.     

Effects of 3' end deletions from the Vibrio harveyi luxB gene on luciferase subunit folding and enzyme assembly: generation of temperature-sensitive polypeptide folding mutants

Ten recombinant plasmids have been constructed by deletion of specific regions from the plasmid pTB7 that carries the luxA and luxB genes, encoding the alpha and beta subunits of luciferase from Vibrio harveyi, such that luciferases with normal alpha subunits and variant beta subunits were produced in Escherichia coli cells carrying the recombinant plasmids. The original plasmid, which conferred bioluminescence (upon addition of exogenous aldehyde substrate) on E. coli carrying it, was constructed by insertion of a 4.0-kb HindIII fragment of V. harveyi DNA into the HindIII site of plasmid pBR322 [Baldwin, T.O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., & Ziegler, M. M. (1984) Biochemistry 23, 3663-3667]. Deletion mutants in the 3' region of luxB were divided into three groups: (A) those with deletions in the 3' untranslated region that left the coding sequences intact, (B) those that left the 3' untranslated sequences intact but deleted short stretches of the 3' coding region of the beta subunit, and (C) those for which the 3' deletions extended from the untranslated region into the coding sequences. Analysis of the expression of luciferase from these variant plasmids has demonstrated two points concerning the synthesis of luciferase subunits and the assembly of those subunits into active luciferase in E. coli. First, deletion of DNA sequences 3' to the translational open reading frame of the beta subunit that contain a potential stem and loop structure resulted in dramatic reduction in the level of accumulation of active luciferase in cells carrying the variant plasmids, even though the luxAB coding regions remained intact.     

Effects of H-bonds on sugar binding to chitoporin from Vibrio harveyi

BackgroundVhChiP is a sugar-specific-porin present in the outer membrane of the marine bacterium Vibrio harveyi and responsible for chitin uptake, with a high selectivity for chitohexaose.MethodsVhChiP and its mutants were expressed and purified from BL21 (DE3) Omp8 Rosetta strain. After reconstitution into planar lipid bilayers, the ion current fluctuations caused by chitohexaose entering the channel were measured in deuterium oxide and in water.ResultsThe role of hydrogen-bonding in sugar binding was investigated by comparing channel occlusion by chitohexaose in buffers containing H₂O and D₂O. The BLM results revealed the significant contribution of hydrogen bonding to the binding of chitohexaose in the constriction zone of VhChiP. Replacing H₂O as solvent by D₂O significantly decreased the on- and off-rates of sugar penetration into the channel. The importance of hydrogen bonding inside the channel was more noticeable when the hydrophobicity of the constriction zone was diminished by replacing Trp¹³⁶ with the charged residues Asp or Arg. The on- and off-rates decreased up to 2.5-fold and 4-fold when Trp¹³⁶ was replaced by Arg, or 5-fold and 3-fold for Trp¹³⁶ replacement by Asp, respectively. Measuring the on-rate at different temperatures and for different channel mutants revealed the activation energy for chitohexaose entrance into VhChiP channel.ConclusionsHydrogen-bonds contribute to sugar permeation.     

Induction of peroxide and superoxide protective enzymes and physiological cross-protection against peroxide killing by a superoxide generator in Vibrio harveyi

Vibrio harveyi is a causative agent of destructive luminous vibriosis in farmed black tiger prawn (Penaeus monodon). V. harveyi peroxide and superoxide stress responses toward elevated levels of a superoxide generated by menadione were investigated. Exposure of V. harveyi to sub-lethal concentrations of menadione induced high expression of genes in both the OxyR regulon (e.g., a monofunctional catalase or KatA and an alkyl hydroperoxide reductase subunit C or AhpC), and the SoxRS regulon (e.g., a superoxide dismutase (SOD) and a glucose-6-phosphate dehydrogenase). V. harveyi expressed two detectable, differentially regulated SOD isozymes, [Mn]-SOD and [Fe]-SOD. [Fe]-SOD was expressed constitutively throughout the growth phase while [Mn]-SOD was expressed at the stationary phase and could be induced by a superoxide generator. Physiologically, pre-treatment of V. harveyi with menadione induced cross-protection against subsequent exposure to killing concentrations of H(2)O(2). This induced cross-protection required newly synthesized proteins. However, the treatment did not induce significant protection against exposures to killing concentrations of menadione itself or cross-protect against an organic hydroperoxide (tert-butyl hydroperoxide). Unexpectedly, growing V. harveyi in high-salinity media induced protection against menadione killing. This protection was independent of SOD induction. Stationary-phase cells were more resistant to menadione killing than exponential-phase cells. The induction of oxidative stress protective enzymes and stress-altered physiological responses could play a role in the survival of this bacterium in the host marine crustaceans.     

Nucleotide sequence of the luxB gene of Vibrio harveyi and the complete amino acid sequence of the beta subunit of bacterial luciferase

The nucleotide sequence of the 1.30-kilobase EcoRI/BglII fragment from Vibrio harveyi carrying the majority of the luciferase beta subunit coding region (luxB gene) has been determined. The EcoRI/BglII fragment was derived from a 4.0-kilobase HindIII fragment carrying both luxA and luxB which was detected in a genomic clone bank based on the expression of bioluminescence from colonies of Escherichia coli carrying V. harveyi HindIII fragments in plasmid pBR322 (Baldwin, T. O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., and Ziegler, M. M. (1984) Biochemistry 23, 3663-3667). The entire alpha subunit coding sequence (luxA gene) and the amino-terminal 13 codons of the beta subunit sequence (luxB gene) were contained on a 1.85-kilobase EcoRI fragment, the sequence of which has been reported (Cohn, D. H., Mileham, A. J., Simon, M. I., Nealson, K. H., Rausch, S. K., Bonam, D., and Baldwin, T. O. (1985) J. Biol. Chem. 260, 6139-6146). The beta subunit coding sequence was found to terminate 972 bases past the start of the luxB coding sequence. The beta subunit had a calculated molecular weight of 36,349 and comprised a total of 324 amino acid residues; the alpha beta dimer had a molecular weight (alpha + beta) of 76,457. There were 27 base pairs separating the stop codon of the beta subunit structural gene and a 340-base open reading frame extending to (and beyond) the distal BglII site. Approximately two-thirds of the beta subunit was sequenced by protein chemical techniques. The amino acid sequence predicted from the DNA sequence, with few exceptions, confirmed the chemically determined sequence, and the measured amino acid composition was in excellent agreement with the composition implied from the DNA sequence.     

哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN-oxidoreductase.

The bioluminescent bacterial enzyme system NAD(P)H:FMN-oxidoreductase-luciferase has been used as a test system for ecological monitoring. One of the modes to quench bioluminescence is the interaction of xenobiotics with the enzymes, which inhibit their activity. The use of endogenous flavin fluorescence for investigation of the interactions of non-fluorescent compounds with the bacterial luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri has been proposed. Fluorescence spectroscopy methods have been used to study characteristics of endogenous flavin fluorescence (fluorophore lifetime, the rotational correlation time). The fluorescence anisotropy behaviour of FMN has been analysed and compared to that of the enzyme-bound flavin. The fluorescence characteristics of endogenous flavin of luciferase and NAD(P)H:FMN-oxidoreductase have been shown to be applicable in studying enzymes' interactions with non-fluorescent compounds.     

Entry of Vibrio harveyi and Vibrio fischeri into the viable but nonculturable state

AIMS: Physiological responses of marine luminous bacteria, Vibrio harveyi (ATCC 14216) and V. fischeri (UM1373) to nutrient-limited normal strength (35 ppt iso-osmolarity) and low (10 ppt hypo-osmolarity) salinity conditions were determined. METHODS AND RESULTS: Plate counts, direct viable counts, actively respiring cell counts, nucleoid-containing cell counts, and total counts were determined. Vibrio harveyi incubated at 22 degrees C in nutrient-limited artificial seawater (ASW) became nonculturable after approximately 62 and 45 d in microcosms of 35 ppt and 10 ppt ASW, respectively. In contrast, V. fischeri became nonculturable at approximately 55 and 31 d in similar microcosms. Recovery of both culturability and luminescence of cells in the viable but nonculturable state was achieved by addition of nutrient broth or nutrient broth supplemented with a carbon source, including luminescence-stimulating compounds. Temperature upshift from 22 degrees C to 30 degrees C or 37 degrees C did not result in recovery from nonculturability. CONCLUSIONS: The study confirms entry of V. harveyi and V. fischeri into the viable but nonculturable state under low-nutrient conditions and demonstrates nutrient-dependent resuscitation from this state. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms loss of luminescence of V. harveyi and V. fischeri on entry into the viable but nonculturable state and suggests that enumeration of luminescent cells in water samples may be a rapid method to deduce the nutrient status of a water sample.