Tarragon essential oil as a source of bioactive compounds with anti-quorum sensing and anti-proteolytic activity against Pseudomonas spp. isolated from fish - in vitro, in silico and in situ approaches.

The present study aimed to evaluate the anti-quorum sensing (anti-QS) and anti-proteolytic potentials of tarragon essential oil (TEO) and its major compounds against food-associated Pseudomonas spp. The activities were verified by in vitro, in silico and in situ approaches. In this work, methyl eugenol (ME)- and ß-phellandrene (ß-PH)-rich TEO was investigated. TEO at subMIC increased the percentage of saturated fatty acids in the bacterial membranes (from 7 to 22%) and exhibited anti-quorum sensing via decreasing the efficiency of QS autoinducer synthesis [3-oxo-C12-HSL (from 2.028 µg/mL to

Lyso-DGTS lipid isolated from microalgae enhances PON1 activities in vitro and in vivo, increases PON1 penetration into macrophages and decreases cellular lipid accumulation.

High-density lipoprotein (HDL) plays an important role in preventing atherosclerosis. The antioxidant effect of HDL is mostly associated with paraoxonase 1 (PON1) activity. Increasing PON1 activity using nutrients might improve HDL function and quality and thus, decrease atherosclerotic risk. We previously isolated and identified a novel active compound, lyso-DGTS (C20:5,0) from Nannochloropsis sp. ethanol extract. In the present study, its effect on PON1 activities was examined and the mechanism by which the compound affects PON1 activity was explored. Lyso-DGTS elevated recombinant PON1 (rePON1) lactonase and esterase activities in a dose- and time-responsive manner, and further stabilized and preserved rePON1 lactonase activity. Incubation of lyso-DGTS with human serum for 4 h at 37 °C also increased PON1 lactonase activity in a dose-responsive manner. Using tryptophan-fluorescence-quenching assay, lyso-DGTS was found to interact with rePON1 spontaneously with negative free energy (ΔG = -22.87 kJ mol-1 at 25 °C). Thermodynamic parameters and molecular modeling calculations showed that the main interaction of lyso-DGTS with the enzyme is through a hydrogen bond with supporting van der Waals interactions. Furthermore, lyso-DGTS significantly increased rePON1 influx into macrophages and prevented lipid accumulation in macrophages stimulated with oxidized low-density lipid dose-dependently. In vivo supplementation of lyso-DGTS to the circulation of mice fed a high-fat diet via osmotic mini-pumps implanted subcutaneously significantly increased serum PON1 lactonase activity and decreased serum glucose concentrations to the level of mice fed a normal diet. Our findings suggest a beneficial effect of lyso-DGTS on increasing PON1 activity and thus, improving HDL quality and atherosclerotic risk factors. © 2018 BioFactors, 44(3):299-310, 2018.

A novel screen-printed mast cell-based electrochemical sensor for detecting spoilage bacterial quorum signaling molecules (N-acyl-homoserine-lactones) in freshwater fish.

A novel screen-printed cell-based electrochemical sensor was developed to assess bacterial quorum signaling molecules, N-acylhomoserine lactones (AHLs). Screen-printed carbon electrode (SPCE), which possesses excellent properties such as low-cost, disposable and energy-efficient, was modified with multi-walled carbon nanotubes (MWNTs) to improve electrochemical signals and enhance the sensitivity. Rat basophilic leukemia (RBL-2H3) mast cells encapsulated in alginate/graphene oxide (NaAgl/GO) hydrogel were immobilized on the MWNTs/SPCE to serve as recognition element. Electrochemical impedance spectroscopy (EIS) was employed to record the cell impedance signal as-influenced by Pseudomonas aeruginosa quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC12-HSL). Experimental results show that 3OC12-HSL caused a significant decrease in cell viability in a dose dependent manner. The EIS value decreased with concentrations of 3OC12-HSL in the range of 0.1-1µM, and the detection limit for 3OC12-HSL was calculated to be 0.094µM. These results were confirmed via cell viability, SEM, TEM analysis. Next, the sensor was successfully applied to monitoring the production of AHLs by spoilage bacteria in three different freshwater fish juice samples which efficiently proved the practicability of this cell based method. Therefore, the proposed cell sensor may serve as an innovative and effective approach to the measurement of quorum signaling molecule and thus provides a new avenue for real-time monitoring the spoilage bacteria in freshwater fish production.

Expression of toll-like receptors in T lymphocytes stimulated with N-(3-oxododecanoyl)-L-homoserine lactone from Pseudomonas aeruginosa.

The establishment of chronic Pseudomonas aeruginosa infections is correlated with the disturbance of the host immune system. The P. aeruginosa quorum-sensing molecule N-3-(oxododecanoyl)-L-homoserine lactone (3-O-C12-HSL) has the potential to modulate the host immune system. The immune system recognizes pathogens via toll-like receptors (TLRs). We found that 3-O-C12-HSL induced TLR changes in monocytes. However, the role of T cells in P. aeruginosa infection has not been delineated. In order to understand this activity, we examined whether 3-O-C12-HSL has an effect on the immune function and the expression of TLRs in T lymphocytes. Human peripheral blood mononuclear cells (PBMCs) cells were cultured with 0, 1, 10, 50, or 100 µM 3-O-C12-HSL for 12 h. TLR2/TLR4 expression and T-lymphocyte proliferation were increased in a dose-dependent manner, and 100 µM 3-O-C12-HSL significantly increased TLR2 expression. Moreover, tumor necrosis factor-α production of these PBMCs was inhibited. To conclude, 3-O-C12-HSL can induce lymphocyte cell proliferation. These findings provide a new perspective on our understanding of the persistence of the chronic inflammation that accompanies P. aeruginosa infection.

Anti-Biofilm Activity of a Long-Chain Fatty Aldehyde from Antarctic Pseudoalteromonas haloplanktis TAC125 against Staphylococcus epidermidis Biofilm.

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis.

Quorum sensing signal production and microbial interactions in a polymicrobial disease of corals and the coral surface mucopolysaccharide layer.

Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome.

Detection of quorum sensing signal molecules in the family Vibrionaceae.

AIMS: The aim of this study was to detect the production of three kinds of quorum sensing (QS) signal molecules, i.e. the N-acyl-homoserine lactone (AHL), the autoinducer-2 (AI-2) and the cholerae autoinducer-1-like (CAI-1-like) molecules in 25 Vibrionaceae strains. METHODS AND RESULTS: The QS signal molecules in 25 Vibrionaceae strains were detected with different biosensors. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty-one of the 25 strains were found to produce AHL signal molecules by using Vibrio harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin-layer chromatography with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI-2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. Only six (five Vibrio strains and A. logei VIB414) of the 25 strains possessed the CAI-1-like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules. CONCLUSIONS: The results indicated that the QS signal molecules, especially AHL and AI-2 molecules, were widespread in the family Vibrionaceae. SIGNIFICANCE AND IMPACT OF THE STUDY: In response to a variety of environmental conditions and selection forces, the family Vibrionaceae produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.

Determination of acyl homoserine lactone and tetramic acid concentrations in biological samples.

Within environmental communities, there is a constant struggle for survival, as nutrients are often limited. In response, bacteria have developed elaborate methods to deal with competitors. One such mechanism is the coordination of behaviors and function via the exchange of small chemical signals in a process known as quorum sensing. This process is especially prominent in the pathogenicity of Pseudomonas aeruginosa, an opportunistic human pathogen that forms sessile communities known as biofilms. These biofilms play an important role in the lifestyle of P. aeruginosa, either in their natural environment or during establishment and maintenance of infection in human hosts; thus, they often have grievous effects on human health. As such, a method for the detection of these QS signals may provide insights into the pathogenicity and survival of P. aeruginosa. In this chapter, we present a method for the extraction and quantitation of the P. aeruginosa QS signal N-3-oxo-dodecanoyl-homoserine lactone, and its rearranged tetramic acid product, C12-TA, which itself has implications as a survival tactic used by P. aeruginosa.

Zoosporic plant pathogens produce bacterial autoinducer-2 that affects Vibrio harveyi quorum sensing.

The frequent coisolation of bacteria with Phytophthora and Pythium species suggests possible interspecies communication. Zoospore-free fluids (ZFF) from bacteria-free and nutrient-depleted zoospore suspensions were examined to investigate the production of autoinducer-2 (AI-2), a bacterial interspecies signal molecule, by zoosporic oomycetes. ZFF from Phytophthora nicotianae, Phytophthora sojae, and Pythium aphanidermatum triggered luminescence of the Vibrio harve7yi AI-2 reporter, indicating the presence of AI-2 in zoospore extracellular products and the potential of cross-kingdom communication between oomycetes and bacteria. The production of AI-2 by zoospores was confirmed by chemical assays. These results provide a new insight into the physiology and ecology of oomycetes.

Identification, quantification, and determination of the absolute configuration of the bacterial quorum-sensing signal autoinducer-2 by gas chromatography-mass spectrometry.

SENSING THE SIGNAL: A gas chromatography-mass spectrometry (GC-MS) method for the analysis of the quorum-sensing autoinducer-2 is described. It allows, for the first time, the direct analysis and accurate determination of this highly water soluble signaling compound, which exists in complex equilibria. The application on the caries-causing bacterium Streptococcus mutans is described. Autoinducer-2 (AI-2) is an important, small extracellular signaling molecule that is used by many bacteria. It is part of the AI-2 pool, a group of equilibrium-connected compounds derived from (S)-4,5-dihydroxy-2,3-pentanedione [(S)-DPD, 1]. Currently, these compounds are analyzed by indirect methods relying on the luminescence of sensor strains, the fluorescence of receptor proteins modified with fluorophores, or by isolation procedures not practical for quantitative analysis. Herein, we report a direct analytical procedure that allows for the unambiguous identification and quantification of molecular species by mass spectrometry. Phenylenediamine reacts readily and quantitatively with 1 to form the quinoxalinediol 12 under aqueous conditions. The extraction and silylation of this compound results in the formation of a silyl ether (13), which is amenable for analysis by gas chromatography-mass spectrometry. The use of an isotopically labeled variant (16) of 12 as an internal standard opens the possibility for the accurate quantification of samples containing AI-2 or its equilibrium products. The analysis of cell-free culture supernatants of Vibrio harveyi and Streptococcus mutans allowed for the accurate quantification of the AI-2 concentration above the limit of detection (0.7 ng mL(-1)). No compounds were detected in mutants lacking the capability to produce AI-2. In addition, the absolute configuration of 1 can be analyzed using the derivative 13 by chiral gas chromatography.

AI-2-dependent gene regulation in Staphylococcus epidermidis.

BACKGROUND: Autoinducer 2 (AI-2), a widespread by-product of the LuxS-catalyzed S-ribosylhomocysteine cleavage reaction in the activated methyl cycle, has been suggested to serve as an intra- and interspecies signaling molecule, but in many bacteria AI-2 control of gene expression is not completely understood. Particularly, we have a lack of knowledge about AI-2 signaling in the important human pathogens Staphylococcus aureus and S. epidermidis. RESULTS: To determine the role of LuxS and AI-2 in S. epidermidis, we analyzed genome-wide changes in gene expression in an S. epidermidis luxS mutant and after addition of AI-2 synthesized by over-expressed S. epidermidis Pfs and LuxS enzymes. Genes under AI-2 control included mostly genes involved in sugar, nucleotide, amino acid, and nitrogen metabolism, but also virulence-associated genes coding for lipase and bacterial apoptosis proteins. In addition, we demonstrate by liquid chromatography/mass-spectrometry of culture filtrates that the pro-inflammatory phenol-soluble modulin (PSM) peptides, key virulence factors of S. epidermidis, are under luxS/AI-2 control. CONCLUSION: Our results provide a detailed molecular basis for the role of LuxS in S. epidermidis virulence and suggest a signaling function for AI-2 in this bacterium.

The hydrolysis of unsubstituted N-acylhomoserine lactones to their homoserine metabolites. Analytical approaches using ultra performance liquid chromatography.

Derivatives of N-acylhomoserine lactones (HSLs) with different alkanoyl side chains occur as quorum or diffusion sensing molecules in gram-negative bacteria and their quantitative chemical analysis became important as a possible way to follow regulation processes of their pathogenicity towards plants and animals. The lactone-ring of HSLs is chemically and biologically not stable: the corresponding serines can be formed in alkaline conditions and these may presumably behave inactive for the biological system. A fast and MS compatible liquid chromatographic method applying high pressure (ultra performance liquid chromatography) with diode array detection was optimized for the rapid quantitative determination of HSLs and their corresponding hydrolysis products. The technique was used to follow and model the hydrolysis reactions of HSLs as function of pH under controlled conditions. Moreover, the method could be triggered to allow a confirmation in the assignment of the potential HSLs in real samples by analysis of the real samples before and after hydrolysis. Quantitative performance characteristics and the character of the hydrolysis reaction were studied as well. The optimized method was successfully applied to a bacterial culture supernatant real sample containing HSLs.

L-O-Caffeoylhomoserine from Matteuccia struthiopteris.

A caffeic acid derivative was isolated from Matteuccia struthiopteris (ostrich fern) as a major radical scavenger. The compound consisted of caffeic acid and L-homoserine. NMR and MS analysis revealed the structure as L-O-caffeoylhomoserine.

Determination of the stoichiometry of noncovalent complexes using reverse-phase high-performance liquid chromatography coupled with electrospray ion trap mass spectrometry.

An electrospray mass spectrometry-based methodology has been developed to have a fast and sensitive method for protein-cofactor stoichiometry determination. As model systems, we used two proteins which require the presence of cofactors for activity: TraR, a member of the LuxR family of quorum-sensing transcriptional regulators, which requires an acyl-homoserine lactone molecule called Agrobacterium autoinducer (AAI) as coinducer and the NS3 protease of hepatitis C virus which complexes with a NS4A cofactor peptide. Both TraR/AAI and NS3/NS4A are noncovalent complexes. Our method requires only nanomolar concentration of sample. A calibration curve of the cofactor is determined by high-performance liquid chromatography (HPLC) coupled on-line with an ion trap mass spectrometer operated in selected reaction monitoring mode. Subsequently, the complex is analyzed using the same experimental setup. During the HPLC run, the complex dissociates, and cofactor and protein elute at different retention times. The peak area of the cofactor is integrated and the molar concentration of cofactor in the complex is extrapolated from the calibration curve. The stoichiometry is consequently calculated by dividing the molar concentration of protein injected by that of cofactor measured. Both TraR/AAI and NS3/NS4A complexes have 1:1 stoichiometries, in line with those already reported in the literature.

The Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone has immunomodulatory activity.

Diverse gram-negative bacterial cells communicate with each other by using diffusible N-acyl homoserine lactone (AHL) signal molecules to coordinate gene expression with cell population density. Accumulation of AHLs above a threshold concentration renders the population "quorate," and the appropriate target gene is activated. In pathogenic bacteria, such as Pseudomonas aeruginosa, AHL-mediated quorum sensing is involved in the regulation of multiple virulence determinants. We therefore sought to determine whether the immune system is capable of responding to these bacterial signal molecules. Consequently the immunomodulatory properties of the AHLs N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) were evaluated in murine and human leukocyte immunoassays in vitro. OdDHL, but not OHHL, inhibited lymphocyte proliferation and tumor necrosis factor alpha production by lipopolysaccharide-stimulated macrophages. Furthermore, OdDHL simultaneously and potently down-regulated the production of IL-12, a Th-1-supportive cytokine. At high concentrations (>7 x 10(-5) M) OdDHL inhibited antibody production by keyhole limpet hemocyanin-stimulated spleen cells, but at lower concentrations (<7 x 10(-5) M), antibody production was stimulated, apparently by increasing the proportion of the immunoglobulin G1 (IgG1) isotype. OdDHL also promoted IgE production by interleukin-4-stimulated human peripheral blood mononuclear cells. These data indicate that OdDHL may influence the Th-1-Th-2 balance in the infected host and suggest that, in addition to regulating the expression of virulence determinants, OdDHL may contribute to the pathogenesis of P. aeruginosa infections by functioning as a virulence determinant per se.

A homoserine lactone autoinducer regulates virulence of an insect-pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae).

N-beta-Hydroxybutanoyl homoserine lactone (HBHL), the autoinducer of the luminescent system of Vibrio harveyi, has been identified as the first small compound to restore virulence to avirulent mutants of Xenorhabdus nematophilus. HBHL stimulated the level of lipase activity excreted by avirulent X. nematophilus and lowered the phenoloxidase activity in the hemolymph of insects infected with X. nematophilus, parameters that are both associated with insect pathogenesis. Moreover, mortality of the insects infected with avirulent X. nematophilus was restored upon injection with HBHL. Chloroform extraction of medium conditioned with wild-type but not avirulent X. nematophilus led to the isolation of a compound with the same chromatographic mobility as HBHL as well as the ability to stimulate the luminescence of a dim autoinducer-dependent mutant of V. harveyi. Transfer of the V. harveyi lux operon into avirulent and wild-type X. nematophilus generated dim and bright luminescent strains, respectively, which responded to HBHL and an agonist and antagonist in a manner analogous to their effects on the luminescence of dim autoinducer-deficient and bright wild-type strains of V. harveyi, indicating that similar HBHL-dependent regulatory systems exist in these two bacterial species.

Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones.

Conjugal opines secreted by crown gall tumours induce strains of Agrobacterium tumefaciens that are donors of Ti plasmids to produce a diffusible conjugation factor. This enhances the conjugal transfer efficiency of the Ti plasmid in other strains of A. tumefaciens. This factor behaves as a secondary messenger, transmitting the environmental information to tra genes. Here we report the use of spectrometry to show that this factor is identical to synthetic N-(beta-oxo-octan-1-oyl)-L-homoserine lactone and confirm that the synthetic compound is biologically active. N-(Hexan-1-oyl)-L-homoserine lactone has also been detected. A closely related molecule, N-(beta-oxo-hexan-1-oyl)-L-homoserine lactone, autoinduces bioluminescence in the distantly related bacterium, Vibrio fischeri. N-Acyl-homoserine lactones thus seem to be conserved molecules in which the length and nature of the lipophilic acyl chain determines the biological function to be regulated. Mutants that do not produce the factor fail to conjugate unless supplied with it in the induction medium (our unpublished data). These data indicate that the conjugation factor is an autoinducer and a key signal molecule in the conjugation system of A. tumefaciens. It is, to our knowledge, the first example of a second messenger molecule in a bacterial conjugation system.

Purification and structural identification of an autoinducer for the luminescence system of Vibrio harveyi.

An autoinducer required for the growth-dependent development of luminescence in Vibrio harveyi has been purified, structurally identified, and chemically synthesized. The autoinducer, which is excreted by the cells, was extracted with chloroform from conditioned media in which V. harveyi cells had been grown. The concentrated extract was separated on a silica gel column and the autoinducer activity further purified by thin layer, paper, and high performance liquid chromatography. The structure of the partially purified autoinducer was identified by 1H NMR and mass spectrometry as N-(beta-hydroxybutyryl)homoserine lactone. This compound was chemically synthesized by condensation of beta-hydroxybutyrate with alpha-amino-gamma-butyrolactone hydrobromide using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a carboxyl group activator. The pure synthetic autoinducer gave the characteristic NMR and mass spectra, co-migrated with the natural autoinducer on thin layer plates, and specifically stimulated induction of luminescence of V. harveyi. Light emission of a regulatory dark mutant of V. harveyi could be stimulated over 1000-fold by the addition of N-(beta-hydroxybutyryl)homoserine lactone, reaching intensities comparable to that of the native strain. The similarity in structure of the autoinducer of V. harveyi to that of Vibrio fischeri suggests that the regulation of luminescence induction in these bacteria may be related in spite of their differences in lux gene organization.

Occurrence of low molecular weight O-acetylserine sulfhydrylase in the yeast Saccharomyces cerevisiae.

Studies with crude preparations obtained from a cysteine auxotroph of Saccharomyces cerevisiae showed that O-acetylserine sulfhydrylase could be separated from O-acetylhomoserine sulfhydrylase by chromatography on a DEAE-cellulose column and centrifugation in a sucrose density gradient. On the basis of sedimentation distance, the molecular weights of these enzymes were calculated to be about 99,000 and 182,000, respectively. The former did not react with the amino acid substrate of the latter, and vice versa. The wild-type strain was also demonstrated to possess O-acetylserine sulfhydrylase (molecular weight: about 96,000), in addition to a large amount of O-acetylserine-O-acetylhomoserine sulfhydrylase (Yamagata et al. (1974) J. Biochem. 75, 1221).