Isolation and Characterization of Plasmid DNA in Streptococcus cremoris

Nine industrially important strains of Streptococcus cremoris (HP, AM₂, ML₁, WC, C₃, R₁, E₈, KH, and Wg₂) were shown to possess a diversity of plasmid molecules. Molecular weights of plasmids were determined from their relative mobilities after agarose gel electrophoresis and via electron microscopy. To illustrate the varied plasmid sizes, strain HP contained plasmids of 26, 18, 8.5, 3.3, and 2 megadaltons (Mdal); strain ML₁ contained plasmids of 29, 18, 9, 4, 2.2, and 1.8 Mdal; and strain AM₂ had plasmids of 42, 27, 16, and 8.4 Mdal. The numbers of plasmids observed in the other strains were 6, 5, 5, 7, 5, and 4 for C₃, E₈, KH, R₁, WC, and Wg₂, respectively. A spontaneous proteinase-negative (Prt⁻) mutant of HP was missing the 8.5-Mdal plasmid, which suggests that in this strain proteinase activity could be linked to this particular plasmid. A lactose-negative (Lac⁻) Prt⁻ mutant of ML₁ lacked the 2.2-Mdal plasmid. Under the conditions employed, antibiotic sensitivity and heavy-metal susceptibility did not correlate with the missing plasmid in Prt⁻ HP or in the Lac⁻ Prt⁻ ML₁. Curing experiments with AM₂, using acridine dyes and elevated temperatures, did not yield Lac⁻ variants. AM₂ was also cultured at high dilution rates in a chemostat for 168 h by using a buffered milk or lactic broth at 18 or 32°C with no selection of Lac⁻ derivatives. The inability to obtain Lac⁻ variants under conditions known to facilitate plasmid elimination suggests that lactose metabolism is not plasmid-mediated in AM₂.     

Stabilization of Lactose Metabolism in Streptococcus lactis C2

The integration of the lactose plasmid from lactic streptococci into the host chromosome could stabilize this trait for dairy fermentations. Sixty lactose-positive (Lac⁺) transductants of lactose- and proteinase-negative (Lac⁻ Prt⁻) LM0220 were induced for temperature phage by UV irradiation or mitomycin C. Four of the transductants, designated KB18, KB21, KB54, and KB58, yielded lysates demonstrating less than one Lac⁺ transductant per 0.2 ml of phage lysate. Successive transferring in the presence of acriflavine did not yield Lac⁻ segregants from KB18, KB21, KB54, or KB58, whereas Streptococcus lactis C2 (parent culture) and three other Lac⁺ transductants showed 12 to 88% conversion from Lac⁺ to Lac⁻ within 6 to 10 repetitive transfers. When grown in continuous culture, KB21 did not show any Lac⁻ variants in 168 h, while S. lactis C2 had 96% conversion from Lac⁺ to Lac⁻ in 144 h. Agarose gel electrophoresis of plasmid DNA isolated from KB18, KB21, KB54, and KB58 revealed that the lactose plasmid, pLM2103, normally present in Lac⁺ transductants, was missing. This suggested integration of the transferred lactose plasmid into the chromosome. In contrast to phage lysates induced from S. lactis C2, which exhibited an exponential decrease in the number of Lac⁺ transductants after exposure to small doses of UV irradiation, the transduction frequency for lactose metabolism was stimulated by UV irradiation of lysates from KB58. The latter indicated chromosomal linkage for lac and that integration of the lactose genes plasmid into the chromosome had occurred.     

Plasmid DNA in Streptococcus cremoris Wg2: Influence of pH on Selection in Chemostats of a Variant Lacking a Protease Plasmid

Cleared lysates of a proteolytic (Prt⁺) strain and a naturally occurring non-proteolytic (Prt⁻) variant of Streptococcus cremoris Wg2 contain equal amounts of covalently closed circular plasmid DNA. An analysis of this plasmid DNA by agarose gel electrophoresis revealed the presence of at least five different plasmid species in the Prt⁺ strain and only three plasmid species in the Prt⁻ variant. Curing studies with acriflavine indicated that a 16-megadalton plasmid determined proteolytic activity in the Prt⁺ strain. In energy-limited chemostats inoculated with both strains it was observed that the Prt⁺ strain was replaced by the Prt⁻ variant. This effect was most apparent when the pH of the culture was fixed at a value above 6.3. No selection for the Prt⁻ variant was observed at pH 5.9. Since the two types of organisms contain equal amounts of plasmid DNA, it was concluded that the energy gain of the Prt⁻ variants at pH values above 6.0 probably has to be found in protein synthesis rather than in plasmid DNA synthesis.     

Characterization of Phage-Sensitive Mutants from a Phage-Insensitive Strain of Streptococcus lactis: Evidence for a Plasmid Determinant that Prevents Phage Adsorption

A phage-insensitive strain of Streptococcus lactis, designated ME2, was used as a prototype strain for the study of mechanisms and genetics of phage resistance in the lactic streptococci. Mutants sensitive to a Streptococcus cremoris phage, ϕ18, were isolated at a level of 17% from cultures of ME2 after sequential transfer at 30°C. Phage-sensitive mutants of ME2 were not fully permissive to ϕ18. The efficiency of plating of ϕ18 on the mutants was 5 × 10⁻⁷ as compared with <10⁻⁹ for ϕ18 on ME2. Further characterization of the mutants showed that they efficiently adsorbed ϕ18 at levels of >99.8%, whereas ME2 adsorbed only 20 to 40% of ϕ18. These results suggest that increased phage susceptibility of the mutants may result from the loss of a mechanism that inhibits phage adsorption. Moreover, the high frequency of spontaneous mutation in ME2 indicates the involvement of an unstable genetic determinant in this phage defense mechanism. ME2 was shown to possess 13 plasmids ranging in size from 1.6 to 34 megadaltons. Of 40 mutants examined that had increased efficiencies of plating, all were missing a 30-megadalton plasmid, pME0030. These data suggest that pME0030 codes for a function that prevents phage adsorption. Further phenotypic characterization of the phage-sensitive mutants showed that some mutants were deficient in the ability to ferment lactose (Lac) and hydrolyze milk proteins (Prt). However, the Lac⁺ and Prt⁺ phenotype segregated independently of the phage-sensitivity phenotype. One phage-sensitive adsorption mutant, designated N1, was tested for susceptibility to 14 different phages. N1 showed increased capacity to adsorb 4 and to replicate 2 of these 14 phages, thereby indicating a phage resistance mechanism in ME2 that generalizes to phage interactions other than the specific ϕ18-ME2 phage-host interaction. These data provide evidence for a unique plasmid-linked phage defense mechanism in phage-insensitive strains of lactic streptococci.     

Genetic Evidence for Plasmid-Linked Lactose Metabolism in Streptococcus lactis subsp. diacetylactis

It has been previously observed that loss of plasmid pGK4101 occurred concomitantly with loss of lactose-fermenting ability in Streptococcus lactis subsp. diacetylactis 18-16. Transfer of this 41-megadalton plasmid to LM0230, a lactosenegative (Lac⁻) strain of S. lactis, required cell-to-cell contact and resulted in a conversion of LM0230 to the Lac⁺ phenotype. This confirms the linkage of lactose-fermenting ability to the 41-megadalton plasmid in S. lactis subsp. diacetylactis and, in addition, demonstrates transfer by a process resembling conjugation in the group N streptococci.     

Involvement of a Plasmid in Production of Ropiness (Mucoidness) in Milk Cultures by Streptococcus cremoris MS

Curing and genetic transfer experiments showed that lactose-fermenting ability (Lac⁺) and the ability to produce mucoidness in milk cultures (Muc⁺) in Streptococcus cremoris MS were coded on plasmids. The Lac⁺ phenotype was associated with a 75.8-megadalton plasmid, pSRQ2201. The Muc⁺ phenotype was associated with a 18.5-megadalton plasmid, pSRQ2202. The Lac plasmid, pSRQ2201, was first conjugatively transferred from S. cremoris MS to Lac⁻S. lactis ML-3/2.2. Later, the Muc plasmid, pSRQ2202, was conjugatively transferred from Lac⁻ Muc⁺S. cremoris MS04 to Lac⁺ nonmucoid S. lactis transconjugant ML-3/2.201. Subsequently, pSRQ2201 and pSRQ2202 were cotransferred from Lac⁺ Muc⁺S. lactis transconjugant ML-3/2.202 to Lac⁻, nonmucoid, malty S. lactis 4/4.2 and S. lactis subsp. diacetylactis SLA3.25. Transconjugants showing pSRQ2201 were Lac⁺; those containing pSRQ2202 were Muc⁺. With the transfer of pSRQ2202, the transconjugants S. lactis ML-3/2.202 and S. lactis subsp. diacetylactis SLA3.2501 not only acquired the Muc⁺ phenotype but also resistance to bacteriophages, which were lytic to the respective parent strains S. lactis ML-3/2.201 and S. lactis subsp. diacetylactis SLA3.25.     

Plasmid Profiles of Lactose-Negative and Proteinase-Deficient Mutants of Streptococcus lactis C10, ML(3), and M18

Lactose- and proteinase-negative (Lac Prt) mutants of Streptococcus lactis C10, ML3, and M18 were isolated after treatment with ethidium bromide. The Lac Prt mutants of C10 were missing a 40-megadalton plasmid. A 33-megadalton plasmid was absent in the ML3 mutants, and the M18 variants lacked a 45-megadalton plasmid. The results suggest a linkage of these metabolic traits to the respective plasmids. The possible complexity of the interrelationship between lactose metabolism and proteinase activity is presented.     

Transduction of Lactose Metabolism by Streptococcus cremoris C3 Temperate Phage

Temperate phage was induced from Streptococcus cremoris C3 and morphologically characterized by high-resolution electron micrographic techniques. Interspecies genetic transfer of lactose-fermenting ability by the temperate phage was demonstrated, using two lactose-negative (Lac⁻) S. lactis strains as recipients. Plasmid transfer was confirmed by agarose gel electrophoresis. Transductant plasmid profiles were of three types—those containing no visible plasmid deoxyribonucleic acid, those possessing a 23-megadalton (Mdal) plasmid, and those containing a 23-Mdal plasmid and a 30-Mdal plasmid. A Lac⁺ transductant could serve as a donor of the lac determinants during solid-surface matings. These results add to previously published reports of inter- and intraspecies genetic transfer in dairy starter cultures.     

Conjugal Transfer of Genetic Information in Group N Streptococci

Streptococcus lactis strains ML3 and C₂O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM₄ were found to transfer lactose-fermenting ability to LM0230, an S. lactis C2 lactose-negative (Lac⁻) derivative which is devoid of plasmid deoxyribonucleic acid (DNA). Lactose-positive streptomycin-resistant (Lac⁺ Str^r) recombinants were found when the Lac⁺ Str^s donor was mixed with Lac⁻ Str^r LM0230 in solid-surface matings. Transduction and transformation were ruled out as the mechanism of genetic exchange in strains ML3, DRC3, 11007, and WM₄, nor was reversion responsible for the high number of Lac⁺ Str^r recombinants. Furthermore, chloroform treatment of the donor prevented the appearance of recombinants, indicating that transfer of lactose-fermenting ability required viable cell-to-cell contact. Strain C₂O demonstrated transduction as well as conjugation. Transfer of plasmid DNA during conjugation for all strains was confirmed by demonstrating the presence of plasmid DNA in the transconjugants by using agarose gel electrophoresis. In some instances, a cryptic plasmid was transferred in conjunction with the lactose plasmid by using strains DRC3, 11007, and WM₄. In S. lactis C2 × LM0230 matings, the Str^r marker was transferred from LM0230 to C2, suggesting conjugal transfer of chromosomal DNA. The results confirm conjugation as another mechanism of genetic exchange occurring in dairy starter cultures.     

Construction of Streptococcus lactis subsp. lactis Strains with a Single Plasmid Associated with Mucoid Phenotype

Lactose-fermenting mucoid (Lac⁺ Muc⁺) variants of plasmid-free Streptococcus lactis subsp. lactis MG1614 were obtained by protoplast transformation with total plasmid DNA from Muc⁺S. lactis subsp. cremoris ARH87. By using plasmid DNA from these variants for further transformations followed by novobiocininduced plasmid curing, Lac⁻ Muc⁺ MG1614 strains containing only a single 30-megadalton plasmid could be constructed. This plasmid, designated pVS5, appeared to be associated with the Muc⁺ phenotype.     

Conjugal Transfer of Bacteriophage Resistance Determinants on pTR2030 into Streptococcus cremoris Strains

Agar surface conjugal matings were used to introduce heat-sensitive phage resistance (Hsp⁺) determinants carried on the conjugal plasmid pTR2030 into Streptococcus cremoris KH, HP, 924, and TDM1. Lactose-fermenting (Lac⁺) transconjugants were selected from matings of Lac⁻ variants of S. cremoris KH, HP, 924, and TDM1 with Streptococcus lactis ME2 or a high-frequency donor, S. lactis T-EK1 (pTR1040, Lac⁺; pTR2030, Hsp⁺). For all of the S. cremoris strains examined, select Lac⁺ transconjugants were completely resistant to plaquing by their homologous lytic phages. In all cases the plaquing efficiencies were less than 10⁻⁹. Acquisition of a 30-megadalton plasmid (pTR2030) in the S. cremoris phage-resistant transconjugants was demonstrated by direct plasmid analysis, by hybridization with ³²P-labeled probes, or by conjugal transfer of pTR2030 out of the phage-resistant transconjugants into a plasmid-cured recipient, S. lactis LM2302. Acid production, coagulation ability, and proteolytic activity of phage-resistant transconjugants in milk were comparable to those of their phage-sensitive parents. Further, S. cremoris phage-resistant transconjugants were not attacked by phage in starter culture activity tests, which included a 40°C incubation period. The results demonstrated that phage resistance determinants on pTR2030 could be conjugally transferred to a variety of S. cremoris strains and confer resistance to phage under conditions encountered during cheese manufacture. Phage-resistant transconjugants of S. cremoris M43 and HP were also constructed without the use of antiblotic markers to select conjugal recipients from mating mixtures.     

Transformation of Streptococcus lactis Protoplasts by Plasmid DNA

Polyethylene glycol-treated protoplasts prepared from Streptococcus lactis LM3302, a lactose-negative (Lac⁻) derivative of S. lactis ML3, were transformed to lactose-fermenting ability by a transductionally shortened plasmid (pLM2103) coding for lactose utilization.     

Conjugal Transfer of Lactose-Fermenting Ability Among Streptococcus cremoris and Streptococcus lactis Strains

Streptococcus cremoris C3 was found to transfer lactose-fermenting ability to LM2301, a Streptococcus lactis C2 lactose-negative streptomycin-resistant (Lac⁻ Str^r) derivative which is devoid of plasmid deoxyribonucleic acid (DNA); to LM3302, a Lac⁻ erythromycin-resistant (Ery^r) derivative of S. lactis ML3; and to BC102, an S. cremoris B₁ Lac⁻ Ery^r derivative which is devoid of plasmid DNA. S. cremoris strains R1, EB₇, and Z8 were able to transfer lactose-fermenting ability to LM3302 in solid-surface matings. Transduction and transformation were ruled out as mechanisms of genetic transfer. Chloroform treatment of donor cells prevented the appearance of recombinant clones, indicating that viable cell-to-cell contact was responsible for genetic transfer. Transfer of plasmid DNA was confirmed by agarose gel electrophoresis. Transconjugants recovered from EB₇ and Z8 matings with LM3302 exhibited plasmid sizes not observed in the donor strains. Transconjugants recovered from R1, EB₇, and Z8 matings with LM3302 were able to donate lactose-fermenting ability at a high frequency to LM2301. In S. cremoris R1, EB₇, and Z8 matings with LM2301, streptomycin resistance was transferred from LM2301 to the S. cremoris strains. The results confirm genetic transfer resembling conjugation between S. cremoris and S. lactis strains and present presumptive evidence for plasmid linkage of lactose metabolism in S. cremoris.     

Evidence for Plasmid Linkage of Restriction and Modification in Streptococcus cremoris KH

Restriction and modification have been demonstrated in Streptococcus cremoris KH cells when infected by Streptococcus lactis C2 phage (designated c2) at an efficiency of plating of 2 × 10⁻⁷. The growth of c2 phage through KH cells produces modified progeny phage capable of unrestricted growth on KH cells. The ability of single-colony isolates of S. cremoris KH cultures to restrict and modify c2 phage was found to be variable. From 2 to 6.5% of colonies isolated were partially deficient in restrictive capacity, permitting a greater plaquing ability by c2 phage of 1.8 to 2.9 log cycles. No completely restrictionless mutants were isolated from 1,000 colonies examined. Mutants were shown to be deficient in both restriction and modification capabilities of the same specificity. The frequent occurrence of a genotypic change that resulted in the loss of both restriction and modification capacities indicated the involvement of plasmid deoxyribonucleic acid in genetically determining this specific restriction and modification system. S. cremoris KH was found to harbor 11 plasmid molecules, with molecular weights (×10⁶) estimated to be 50, 41, 24, 18, 10, 7.4, 3.3, 3.0, 2.8, 2.5, and 1.5. Of the 27 mutants examined, 25 were missing the 10-megadalton plasmid. This consistent plasmid difference among the majority of mutants isolated supports the involvement of this plasmid in restriction and modification. Plasmid linkage of restriction and modification systems provides a genetic mechanism for the rapid development of phage-sensitive starter cultures due to the inherent instability of extrachromosomal elements.     

Development of High-Frequency Delivery System for Transposon Tn919 in Lactic Streptococci: Random Insertion in Streptococcus lactis subsp. diacetylactis 18-16

The conjugative transposon Tn919, originally isolated in Streptococcus sanguis FC1, is capable of low-frequency transfer (10⁻⁷ and 10⁻⁸ per recipient) on membrane filters to a wide number of streptococcal recipients including the industrially important lactic streptococci. The introduction of pMG600 (Lac⁺ Lax⁻; a lactose plasmid capable of conjugative transfer at high frequencies and which, in certain hosts, confers an unusual clumping phenotype) into a Streptococcus lactis CH919 donor, generating S. lactis CH001, resulted in a significant improvement in the transfer frequency of Tn919 to S. lactis CK50 (1.25 × 10⁻⁴ per recipient). In addition, these matings could be performed on agar surfaces, allowing the recovery of a greater number of recipients than with filter matings. Tn919 also transferred at high frequency to S. lactis subsp. diacetylactis 18-16S but not to Streptococcus cremoris strains. Insertion in 18-16S transconjugants generated from filter matings with an S. lactis CH919 donor was random, occurring at different sites on the chromosome and also in plasmid DNA. Thus, the conditions necessary for the practical exploitation of Tn919 in the targeting and cloning of genes from a member of the lactic streptococci, namely, high-frequency delivery and random insertion in host DNA, were achieved.     

Saccharomyces cerevisiae BLYAS, a New Bioluminescent Bioreporter for Detection of Androgenic Compounds

A Saccharomyces cerevisiae strain, capable of autonomous bioluminescence, was engineered to respond to androgenic chemicals. The strain, S. cerevisiae BLYAS, contains the human androgen receptor in the chromosome and was constructed by inserting a series of androgen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 that constitutively expressed luxA and luxB to create pUTK420. Cotransformation of this plasmid with a second plasmid (pUTK404), containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp), yielded a bioluminescent bioreporter responsive to androgenic chemicals. Using dihydrotestosterone (DHT) as a standard, the response time and the 50% effective concentration values were 3 to 4 h and (9.7 ± 4.6) × 10⁻⁹ M, respectively. The lower limit of detection in response to DHT was 2.5 × 10⁻⁹ M, and in response to testosterone it was 2.5 × 10⁻¹⁰ M. This strain is suitable for high-throughput screening of chemicals with potential for remote environmental monitoring systems because of the assay speed, sensitivity, and self-containment.     

Effects of Glucose Concentrations on Cadmium, Copper, Mercury, and Zinc Toxicity to a Klebsiella sp

The influence of glucose concentration on Cd, Cu, Hg, and Zn toxicity to a Klebsiella sp. was studied by following the degradation of ¹⁴C-labeled glucose at pH 6.0. Uptake of ¹⁴C into the cells was also determined. The carbon concentrations ranged from 0.01 to 40 mg liter⁻¹, which are equivalent to soluble C concentrations in natural environments. The toxicity of Cu, Cd, and Zn to a Klebsiella sp. was affected considerably by the C concentration. Copper at 10⁻⁵ M was toxic when the carbon concentration was 10 or 40 mg liter⁻¹, while at 0.01 to 1.0 mg liter⁻¹ no toxicity was observed. Cadmium and zinc were toxic at 10⁻² M in media containing 0.01 to 1.0 mg of C liter⁻¹. At C concentrations greater than 1.0 mg liter⁻¹, the inhibition of glucose degradation and carbon assimilation was observed at 10⁻³ M Cd and Zn. The toxicity of mercury seemed to be independent of the C concentration. Results of this study showed that the nutritional state of an organism may have a profound effect on its sensitivity to metals. Metals taken up by an energy-driven transport system may be less toxic under conditions of C starvation. The C concentration should be taken into account when evaluating results from toxicity studies, especially as most microorganisms in nature live under energy-limited conditions.     

IL-10 and NOS2 Modulate Antigen-Specific Reactivity and Nerve Infiltration by T Cells in Experimental Leprosy

Background

Although immunopathology dictates clinical outcome in leprosy, the dynamics of early and chronic infection are poorly defined. In the tuberculoid region of the spectrum, Mycobacterium leprae growth is restricted yet a severe granulomatous lesion can occur. The evolution and maintenance of chronic inflammatory processes like those observed in the leprosy granuloma involve an ongoing network of communications via cytokines. IL-10 has immunosuppressive properties and IL-10 genetic variants have been associated with leprosy development and reactions.

Methodology/Principal Findings

The role of IL-10 in resistance and inflammation in leprosy was investigated using Mycobacterium leprae infection of mice deficient in IL-10 (IL-10−/−), as well as mice deficient in both inducible nitric oxide synthase (NOS2−/−) and IL-10 (10NOS2−/−). Although a lack of IL-10 did not affect M. leprae multiplication in the footpads (FP), inflammation increased from C57Bl/6 (B6)<IL-10−/−<NOS2−/−<10NOS2−/−. While IL-10−/− mice exhibited modest FP induration compared to B6, NOS2−/− and 10NOS2−/− mice developed markedly enlarged FP marking distinct phases: early (1 month), peak (3–4 months), and chronic (8 months). IFN-γ-producing CD4+CD44+ cells responding to M. leprae cell wall, membrane, and cytosol antigens and ML2028 (Ag85B) were significantly increased in the evolved granuloma in NOS2−/− FP compared to B6 and IL-10−/− during early and peak phases. In 10NOS2−/− FP, CD4+CD44+ and especially CD8+CD44+ responses were augmented even further to these antigens as well as to ML0380 (GroES), ML2038 (bacterioferritin), and ML1877 (EF-Tu). Moreover, fragmented nerves containing CD4+ cells were present in 10NOS2−/− FP.

Conclusions/Significance

The 10NOS2−/− strain offers insight on the regulation of granuloma formation and maintenance by immune modulators in the resistant forms of leprosy and presents a new model for investigating the pathogenesis of neurological involvement.     

Use of Saccharomyces cerevisiae BLYES Expressing Bacterial Bioluminescence for Rapid, Sensitive Detection of Estrogenic Compounds

An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17β-estradiol over the concentration range of 1.2 × 10⁻⁸ through 5.6 × 10⁻¹² M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 ± 1.1) × 10⁻¹⁰ and (2.4 ± 1.0) × 10⁻¹⁰ M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 × 10⁻¹¹ to 2.8 × 10⁻⁹ M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.