Group 2 Sigma Factors are Central Regulators of Oxidative Stress Acclimation in Cyanobacteria.

Regulatory σ factors of the RNA polymerase (RNAP) adjust gene expression according to environmental cues when the cyanobacterium Synechocystis sp. PCC 6803 acclimates to suboptimal conditions. Here we show central roles of the non-essential group 2 σ factors in oxidative stress responses. Cells missing all group 2 σ factors fail to acclimate to chemically induced singlet oxygen, superoxide or H2O2 stresses, and lose pigments in high light. SigB and SigD are the major σ factors in oxidative stress, whereas SigC and SigE play only minor roles. The SigD factor is up-regulated in high light, singlet oxygen and H2O2 stresses, and overproduction of the SigD factor in the ΔsigBCE strain leads to superior growth of ΔsigBCE cells in those stress conditions. Superoxide does not induce the production of the SigD factor but instead SigB and SigC factors are moderately induced. The SigB factor alone in ΔsigCDE can support almost as fast growth in superoxide stress as the full complement of σ factors in the control strain, but an overdose of the stationary phase-related SigC factor causes growth arrest of ΔsigBDE in superoxide stress. A drastic decrease of the functional RNAP limits the transcription capacity of the cells in H2O2 stress, which explains why cyanobacteria are sensitive to H2O2. Formation of RNAP-SigB and RNAP-SigD holoenzymes is highly enhanced in H2O2 stress, and cells containing only SigB (ΔsigCDE) or SigD (ΔsigBCE) show superior growth in H2O2 stress.

Acclimation to High CO2 Requires the ω Subunit of the RNA Polymerase in Synechocystis.

Inactivation of the nonessential ω-subunit of the RNA polymerase core in the ΔrpoZ strain of the model cyanobacterium Synechocystis sp. PCC 6803 leads to a unique high-CO2-sensitive phenotype. Supplementing air in the growth chamber with 30 mL L-1 (3%) CO2 accelerated the growth rate of the control strain (CS) 4-fold, whereas ΔrpoZ did not grow faster than under ambient air. The slow growth of ΔrpoZ during the first days in high CO2 was due to the inability of the mutant cells to adjust photosynthesis to high CO2 The light-saturated photosynthetic activity of ΔrpoZ in high CO2 was only half of that measured in CS, Rubisco content was one-third lower, and cells of ΔrpoZ were not able to increase light-harvesting phycobilisome antenna like CS upon high-CO2 treatment. In addition, altered structural and functional organization of photosystem I and photosystem II were detected in the ΔrpoZ strain compared with CS when cells were grown in high CO2 but not in ambient air. Moreover, respiration of ΔrpoZ did not acclimate to high CO2 Unlike the photosynthetic complexes, the RNA polymerase complex and ribosomes were produced in high CO2 similarly as in CS Our results indicate that the deletion of the ω-subunit specifically affects photosynthesis and respiration, but transcription and translation remain active. Thus, the specific effect of the ω-subunit on photosynthesis but not on all household processes suggests that the ω-subunit might have a regulatory function in cyanobacteria.

In vivo recruitment analysis and a mutant strain without any group 2 σ factor reveal roles of different σ factors in cyanobacteria.

In eubacteria, replacement of one σ factor in the RNA polymerase (RNAP) holoenzyme by another one changes the transcription pattern. Cyanobacteria are eubacteria characterized by oxygenic photosynthesis, and they typically encode numerous group 2 σ factors that closely resemble the essential primary σ factor. A mutant strain of the model cyanobacterium Synechocystis sp. PCC 6803 without functional group 2 σ factors (named as ΔsigBCDE) could not acclimate to heat, high salt or bright light stress, but in standard conditions ΔsigBCDE grew only 9% slower than the control strain. One-fifth of the genes in ΔsigBCDE was differently expressed compared with the control strain in standard growth conditions and several physiological changes in photosynthesis, and pigment and lipid compositions were detected. To directly analyze the σ factor content of RNAP holoenzyme in vivo, a His-tag was added to the γ subunit of RNAP in Synechocystis and RNAPs were collected. The results revealed that all group 2 σ factors were recruited by RNAP in standard conditions, but recruitment of SigB and SigC increased in heat stress, SigD in bright light, SigE in darkness and SigB, SigC and SigE in high salt, explaining the poor acclimation of ΔsigBCDE to these stress conditions.

6S RNA plays a role in recovery from nitrogen depletion in Synechocystis sp. PCC 6803.

BACKGROUND: The 6S RNA is a global transcriptional riboregulator, which is exceptionally widespread among most bacterial phyla. While its role is well-characterized in some heterotrophic bacteria, we subjected a cyanobacterial homolog to functional analysis, thereby extending the scope of 6S RNA action to the special challenges of photoautotrophic lifestyles. RESULTS: Physiological characterization of a 6S RNA deletion strain (ΔssaA) demonstrates a delay in the recovery from nitrogen starvation. Significantly decelerated phycobilisome reassembly and glycogen degradation are accompanied with reduced photosynthetic activity compared to the wild type. Transcriptome profiling further revealed that predominantly genes encoding photosystem components, ATP synthase, phycobilisomes and ribosomal proteins were negatively affected in ΔssaA. In vivo pull-down studies of the RNA polymerase complex indicated that the presence of 6S RNA promotes the recruitment of the cyanobacterial housekeeping σ factor SigA, concurrently supporting dissociation of group 2 σ factors during recovery from nitrogen starvation. CONCLUSIONS: The combination of genetic, physiological and biochemical studies reveals the homologue of 6S RNA as an integral part of the cellular response of Synechocystis sp. PCC 6803 to changing nitrogen availability. According to these results, 6S RNA supports a rapid acclimation to changing nitrogen supply by accelerating the switch from group 2 σ factors SigB, SigC and SigE to SigA-dependent transcription. We therefore introduce the cyanobacterial 6S RNA as a novel candidate regulator of RNA polymerase sigma factor recruitment in Synechocystis sp. PCC 6803. Further studies on mechanistic features of the postulated interaction should shed additional light on the complexity of transcriptional regulation in cyanobacteria.

Roles of Group 2 Sigma Factors in Acclimation of the Cyanobacterium Synechocystis sp. PCC 6803 to Nitrogen Deficiency.

Acclimation of cyanobacteria to environmental conditions is mainly controlled at the transcriptional level, and σ factors of the RNA polymerase have a central role in this process. The model cyanobacterium Synechocystis sp. PCC 6803 has four non-essential group 2 σ factors (SigB, SigC, SigD and SigE) that regulate global metabolic responses to various adverse environmental conditions. Here we show that although none of the group 2 σ factors is essential for the major metabolic realignments induced by a short period of nitrogen starvation, the quadruple mutant without any group 2 σ factors and triple mutants missing both SigB and SigD grow slowly in BG-11 medium containing only 5% of the nitrate present in standard BG-11. These ΔsigBCDE, ΔsigBCD and ΔsigBDE strains lost PSII activity rapidly in low nitrogen and accumulated less glycogen than the control strain. An abnormally high glycogen content was detected in ΔsigBCE (SigD is active), while the carotenoid content became high in ΔsigCDE (SigB is active), indicating that SigB and SigD regulate the partitioning of carbon skeletons in low nitrogen. Long-term survival and recovery of the cells after nitrogen deficiency was strongly dependent on group 2 σ factors. The quadruple mutant and the ΔsigBDE strain (only SigC is active) recovered more slowly from nitrogen deficiency than the control strain, and ΔsigBCDE in particular lost viability during nitrogen starvation. Nitrogen deficiency-induced changes in the pigment content of the control strain recovered essentially in 1 d in nitrogen-replete medium, but little recovery occurred in ΔsigBCDE and ΔsigBDE.

The omega subunit of the RNA polymerase core directs transcription efficiency in cyanobacteria.

The eubacterial RNA polymerase core, a transcription machinery performing DNA-dependent RNA polymerization, consists of two α subunits and ß, ß' and ω subunits. An additional σ subunit is recruited for promoter recognition and transcription initiation. Cyanobacteria, a group of eubacteria characterized by oxygenic photosynthesis, have a unique composition of the RNA polymerase (RNAP) core due to splitting of the ß' subunit to N-terminal γ and C-terminal ß' subunits. The physiological roles of the small ω subunit of RNAP, encoded by the rpoZ gene, are not yet completely understood in any bacteria. We found that although ω is non-essential in cyanobacteria, it has a major impact on the overall gene expression pattern. In ΔrpoZ strain, recruitment of the primary σ factor into the RNAP holoenzyme is inefficient, which causes downregulation of highly expressed genes and upregulation of many low-expression genes. Especially, genes encoding proteins of photosynthetic carbon concentrating and carbon fixing complexes were down, and the ΔrpoZ mutant showed low light-saturated photosynthetic activity and accumulated photoprotective carotenoids and α-tocopherol. The results indicate that the ω subunit facilitates the association of the primary σ factor with the RNAP core, thereby allowing efficient transcription of highly expressed genes.

Oxidative stress and photoinhibition can be separated in the cyanobacterium Synechocystis sp. PCC 6803.

Roles of oxidative stress and photoinhibition in high light acclimation were studied using a regulatory mutant of the cyanobacterium Synechocystis sp. PCC 6803. The mutant strain ΔsigCDE contains the stress responsive SigB as the only functional group 2 σ factor. The ∆sigCDE strain grew more slowly than the control strain in methyl-viologen-induced oxidative stress. Furthermore, a fluorescence dye detecting H2O2, hydroxyl and peroxyl radicals and peroxynitrite, produced a stronger signal in ∆sigCDE than in the control strain, and immunological detection of carbonylated residues showed more protein oxidation in ∆sigCDE than in the control strain. These results indicate that ∆sigCDE suffers from oxidative stress in standard conditions. The oxidative stress may be explained by the findings that ∆sigCDE had a low content of glutathione and low amount of Flv3 protein functioning in the Mehler-like reaction. Although ∆sigCDE suffers from oxidative stress, up-regulation of photoprotective carotenoids and Flv4, Sll2018, Flv2 proteins protected PSII against light induced damage by quenching singlet oxygen more efficiently in ∆sigCDE than in the control strain in visible and in UV-A/B light. However, in UV-C light singlet oxygen is not produced and PSII damage occurred similarly in the ∆sigCDE and control strains. According to our results, resistance against the light-induced damage of PSII alone does not lead to high light tolerance of the cells, but in addition efficient protection against oxidative stress would be required.

Group 2 sigma factor mutant ΔsigCDE of the cyanobacterium Synechocystis sp. PCC 6803 reveals functionality of both carotenoids and flavodiiron proteins in photoprotection of photosystem II.

Adjustment of gene expression during acclimation to stress conditions, such as bright light, in the cyanobacterium Synechocystis sp. PCC 6803 depends on four group 2 σ factors (SigB, SigC, SigD, SigE). A ΔsigCDE strain containing the stress-responsive SigB as the only functional group 2 σ factor appears twice as resistant to photoinhibition of photosystem II (PSII) as the control strain. Microarray analyses of the ΔsigCDE strain indicated that 77 genes in standard conditions and 79 genes in high light were differently expressed compared with the control strain. Analysis of possible photoprotective mechanisms revealed that high carotenoid content and up-regulation of the photoprotective flavodiiron operon flv4-sll0218-flv2 protected PSII in ΔsigCDE, while up-regulation of pgr5-like, hlipB or isiA genes in the mutant strain did not offer particular protection against photoinhibition. Photoinhibition resistance was lost if ΔsigCDE was grown in high CO2, where carotenoid and Flv4, Sll0218, and Flv2 contents were low. Additionally, photoinhibition resistance of the ΔrpoZ strain (lacking the omega subunit of RNA polymerase), with high carotenoid but low Flv4-Sll0218-Flv2 content, supported the importance of carotenoids in PSII protection. Carotenoids likely protect mainly by quenching of singlet oxygen, but efficient nonphotochemical quenching in ΔsigCDE might offer some additional protection. Comparison of photoinhibition kinetics in control, ΔsigCDE, and ΔrpoZ strains showed that protection by the flavodiiron operon was most efficient during the first minutes of high-light illumination.

The SigB σ factor regulates multiple salt acclimation responses of the cyanobacterium Synechocystis sp. PCC 6803.

Changing of principal σ factor in RNA polymerase holoenzyme to a group 2 σ factor redirects transcription when cyanobacteria acclimate to suboptimal environmental conditions. The group 2 sigma factor SigB was found to be important for the growth of the cyanobacterium Synechocystis sp. PCC 6803 in high-salt (0.7 m NaCl) stress but not in mild heat stress at 43°C although the expression of the sigB gene was similarly highly, but only transiently up-regulated at both conditions. The SigB factor was found to regulate many salt acclimation processes. The amount of glucosylglycerol-phosphate synthase, a key enzyme in the production of the compatible solute glucosylglycerol, was lower in the inactivation strain ΔsigB than in the control strain. Addition of the compatible solute trehalose almost completely restored the growth of the ΔsigB strain at 0.7 m NaCl. High-salt conditions lowered the chlorophyll and phycobilin contents of the cells while protective carotenoid pigments, especially zeaxanthin and myxoxanthophyll, were up-regulated in the control strain. These carotenoids were up-regulated in the ΔsigCDE strain (SigB is the only functional group 2 σ factor) and down-regulated in the ΔsigB strain under standard conditions. In addition, the HspA heat shock protein was less abundant and more abundant in the ΔsigB and ΔsigCDE strains, respectively, than in the control strain in high-salt conditions. Some cellular responses are common to heat and salt stresses, but pretreatment with mild heat did not protect cells against salt shock although protection against heat shock was evident.

Roles of Close Homologues SigB and SigD in Heat and High Light Acclimation of the Cyanobacterium Synechocystis sp. PCC 6803.

Acclimation of cyanobacterium Synechocystis sp. PCC6803 to suboptimal conditions is largely dependent on adjustments of gene expression, which is highly controlled by the σ factor subunits of RNA polymerase (RNAP). The SigB and SigD σ factors are close homologues. Here we show that the sigB and sigD genes are both induced in high light and heat stresses. Comparison of transcriptomes of the control strain (CS), ΔsigB, ΔsigD, ΔsigBCE (containing SigD as the only functional group 2 σ factor), and ΔsigCDE (SigB as the only functional group 2 σ factor) strains in standard, high light, and high temperature conditions revealed that the SigB and SigD factors regulate different sets of genes and SigB and SigD regulons are highly dependent on stress conditions. The SigB regulon is bigger than the SigD regulon at high temperature, whereas, in high light, the SigD regulon is bigger than the SigB regulon. Furthermore, our results show that favoring the SigB or SigD factor by deleting other group 2 σ factors does not lead to superior acclimation to high light or high temperature, indicating that all group 2 σ factors play roles in the acclimation processes.

Cd-specific mutants of mercury-sensing regulatory protein MerR, generated by directed evolution.

The mercury-sensing regulatory protein, MerR (Tn21), which regulates mercury resistance operons in Gram-negative bacteria, was subjected to directed evolution in an effort to generate a MerR mutant that responds to Cd but not Hg. Oligonucleotide-directed mutagenesis was used to introduce random mutations into the key metal-binding regions of MerR. The effects of these mutations were assessed using a vector in which MerR controlled the expression of green fluorescent protein (GFP) and luciferase via the mer operator/promoter. An Escherichia coli cell library was screened by fluorescence-activated cell sorting, using a fluorescence-based dual screening strategy that selected for MerR mutants that showed GFP repression when cells were induced with Hg but GFP activation in the presence of Cd. Two Cd-responsive MerR mutants with decreased responses toward Hg were identified through the first mutagenesis/selection round. These mutants were used for a second mutagenesis/selection round, which yielded eight Cd-specific mutants that had no significant response to Hg, Zn, or the other tested metal(loid)s. Seven of the eight Cd-specific MerR mutants showed repressor activities equal to that of wild-type (wt) MerR. These Cd-specific mutants harbored multiple mutations (12 to 22) in MerR, indicating that the alteration of metal specificity with maintenance of repressor function was due to the combined effect of many mutations rather than just a few amino acid changes. The amino acid changes were studied by alignment against the sequences of MerR and other metal-responsive MerR family proteins. The analysis indicated that the generated Cd-specific MerR mutants appear to be unique among the MerR family members characterized to date.

Developing a compound-specific receptor for bisphenol A by directed evolution of human estrogen receptor α.

Directed evolution has become a successful approach to alter ligand binding properties of nuclear receptors. In this study, directed evolution was used to generate a mutant human estrogen receptor α library, which was then used to screen for receptors having enhanced responses to the known endocrine-disrupting chemical, bisphenol A (BPA). A single round of multi-site mutation was combined with an efficient positive/negative library screening method in which positive growth-based selection for the desired activity with BPA was combined with flow cytometric removal of cells having undesired activity with the natural ligand, 17ß-estradiol. The screening steps were performed in a Saccharomyces cerevisiae yeast strain containing a genome-integrated his3-yEGFP reporter gene fusion construct. A single round of mutation and screening yielded nine mutants with enhanced responses towards BPA but no detectable induction by 17ß-estradiol (up to 90 nM). These BPA-specific mutant receptors may prove useful in the field of environmental analytics, where they could be used to monitor and evaluate the proportion of BPA in hormonally active samples.

Analytical strategies for improving the robustness and reproducibility of bioluminescent microbial bioreporters.

Whole-cell bioluminescent (BL) bioreporter technology is a useful analytical tool for developing biosensors for environmental toxicology and preclinical studies. However, when applied to real samples, several methodological problems prevent it from being widely used. Here, we propose a methodological approach for improving its analytical performance with complex matrix. We developed bioluminescent Escherichia coli and Saccharomyces cerevisiae bioreporters for copper ion detection. In the same cell, we introduced two firefly luciferases requiring the same luciferin substrate emitting at different wavelengths. The expression of one was copper ion specific. The other, constitutively expressed, was used as a cell viability internal control. Engineered BL cells were characterized using the noninvasive gravitational field-flow fractionation (GrFFF) technique. Homogeneous cell population was isolated. Cells were then immobilized in a polymeric matrix improving cell responsiveness. The bioassay was performed in 384-well black polystyrene microtiter plates directly on the sample. After 2 h of incubation at 37 °C and the addition of the luciferin, we measured the emitted light. These dual-color bioreporters showed more robustness and a wider dynamic range than bioassays based on the same strains with a single reporter gene and that uses a separate cell strain as BL control. The internal correction allowed to accurately evaluate the copper content even in simulated toxic samples, where reduced cell viability was observed. Homogenous cells isolated by GrFFF showed improvement in method reproducibility, particularly for yeast cells. The applicability of these bioreporters to real samples was demonstrated in tap water and wastewater treatment plant effluent samples spiked with copper and other metal ions.

Mutations Suppressing the Lack of Prepilin Peptidase Provide Insights Into the Maturation of the Major Pilin Protein in Cyanobacteria.

Type IV pili are bacterial surface-exposed filaments that are built up by small monomers called pilin proteins. Pilins are synthesized as longer precursors (prepilins), the N-terminal signal peptide of which must be removed by the processing protease PilD. A mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the PilD protease is not capable of photoautotrophic growth because of the impaired function of Sec translocons. Here, we isolated phototrophic suppressor strains of the original ΔpilD mutant and, by sequencing their genomes, identified secondary mutations in the SigF sigma factor, the γ subunit of RNA polymerase, the signal peptide of major pilin PilA1, and in the pilA1-pilA2 intergenic region. Characterization of suppressor strains suggests that, rather than the total prepilin level in the cell, the presence of non-glycosylated PilA1 prepilin is specifically harmful. We propose that the restricted lateral mobility of the non-glycosylated PilA1 prepilin causes its accumulation in the translocon-rich membrane domains, which attenuates the synthesis of membrane proteins.

Isolation of sensitive nisin-sensing GFP(uv) bioassay Lactococcus lactis strains using FACS.

Fluorescence-activated cell sorting (FACS) was used to isolate mutants of Lactococcus lactis LAC275, an indicator strain in GFP(uv) nisin bioassay. It harbors the GFP(uv) encoding gene under the nisA promoter and the nisin signal transduction nisRK genes whereby nisin concentration can be correlated to GFP(uv) fluorescence. The sorted L. lactis cells, which showed higher fluorescence intensities at low inducer concentration, were analysed for higher responsiveness to low concentration of nisin. Two strains showed lower detection limits (0.2 pg ml(-1)) for nisin than the parent strain (10 pg ml(-1)). This showed that mutants of LAC275 could successfully be isolated using FACS.

Inactivation of group 2 σ factors upregulates production of transcription and translation machineries in the cyanobacterium Synechocystis sp. PCC 6803.

We show that the formation of the RNAP holoenzyme with the primary σ factor SigA increases in the ΔsigBCDE strain of the cyanobacterium Synechocystis sp. PCC 6803 lacking all group 2 σ factors. The high RNAP-SigA holoenzyme content directly induces transcription of a particular set of housekeeping genes, including ones encoding transcription and translation machineries. In accordance with upregulated transcripts, ΔsigBCDE contain more RNAPs and ribosomal subunits than the control strain. Extra RNAPs are fully active, and the RNA content of ΔsigBCDE cells is almost tripled compared to that in the control strain. Although ΔsigBCDE cells produce extra rRNAs and ribosomal proteins, functional extra ribosomes are not formed, and translation activity and protein content remained similar in ΔsigBCDE as in the control strain. The arrangement of the RNA polymerase core genes together with the ribosomal protein genes might play a role in the co-regulation of transcription and translation machineries. Sequence logos were constructed to compare promoters of those housekeeping genes that directly react to the RNAP-SigA holoenzyme content and those ones that do not. Cyanobacterial strains with engineered transcription and translation machineries might provide solutions for construction of highly efficient production platforms for biotechnical applications in the future.

Monitoring promoter activity in a single bacterial cell by using green and red fluorescent proteins.

We investigated the possibility of monitoring promoter activity with flow cytometry by using green fluorescent protein (GFPmut2) and red fluorescent protein (drFP583) in a single bacterial cell. The drFP583 was used as an intrinsic marker of the bacterial cells, because it was expressed constantly in Escherichia coli MC1061 strain. The GFPmut2 expressed under the control of the Hg(2+) ion inducible mer promoter/operator, was used to study promoter activity. Over 75% of the cells were positive for red and green fluorescence in flow cytometric analysis. The average green fluorescence of the whole population increased from 6.7 to 1700 when the mercury concentration was increased from 0 to 1 x 10(-4) M, while the red fluorescence was unaffected by the mercury concentration. These results show that gfpmut2 and drFP583 could be expressed under different promoters in one bacterial cell and measured independently with a flow cytometer.

The ω subunit of RNA polymerase is essential for thermal acclimation of the cyanobacterium Synechocystis sp. PCC 6803.

The rpoZ gene encodes the small ω subunit of RNA polymerase. A ΔrpoZ strain of the cyanobacterium Synechocystis sp. PCC 6803 grew well in standard conditions (constant illumination at 40 µmol photons m(-2) s(-1); 32°C; ambient CO2) but was heat sensitive and died at 40°C. In the control strain, 71 genes were at least two-fold up-regulated and 91 genes down-regulated after a 24-h treatment at 40°C, while in ΔrpoZ 394 genes responded to heat. Only 62 of these heat-responsive genes were similarly regulated in both strains, and 80% of heat-responsive genes were unique for ΔrpoZ. The RNA polymerase core and the primary σ factor SigA were down-regulated in the control strain at 40°C but not in ΔrpoZ. In accordance with reduced RNA polymerase content, the total RNA content of mild-heat-stress-treated cells was lower in the control strain than in ΔrpoZ. Light-saturated photosynthetic activity decreased more in ΔrpoZ than in the control strain upon mild heat stress. The amounts of photosystem II and rubisco decreased at 40°C in both strains while PSI and the phycobilisome antenna protein allophycocyanin remained at the same level as in standard conditions. The phycobilisome rod proteins, phycocyanins, diminished during the heat treatment in ΔrpoZ but not in the control strain, and the nblA1 and nblA2 genes (encode NblA proteins required for phycobilisome degradation) were up-regulated only in ΔrpoZ. Our results show that the ω subunit of RNAP is essential in heat stress because it is required for heat acclimation of diverse cellular processes.

Toxicity detection from EILATox-Oregon Workshop samples by using kinetic photobacteria measurement: the Flash method.

The Flash test, which can be used to measure the toxicity from clear, turbid and coloured samples with identical protocol, was used in the EILATox-Oregon Workshop. During the workshop, 17 synthetic samples and three environmental samples were tested. In the Flash method the photobacteria Vibrio fischeri are initially dispensed on top of the sample and the light output of the bioluminescence reaction is recorded at a rate of several readings per second. The change in the light production was measured at two points: 30 s and 30 min after exposure at room temperature. Toxicity calculations were done by using either the peak height of the sample or the peak height of the control sample (maximum signal at 0-2 s). When the peak height of the sample is used as a reference, the colour and turbidity effect of solid particles are taken into account throughout the whole measurement period. In the EILATox-Oregon Workshop toxicity was seen in samples 1 (chlordimeform), 4 (phosdrin), 5 (HgCl(2)), 6 (sodium arsenite), 8 (metham sodium), 12 (p-chlorophenol), 14 (sodium selenite) and 17 (MNNG). Only samples 1, 4 and 12 showed different ec(50) values between different calculations of toxicity. The ec(50) values were calculated and compared with the ec(50) values of Microtox derived from the literature. The Flash test will not replace the standardized photobacteria test, but is the advanced version of it and is needed when the sample matrix is complex and when speed and low costs are required.

Simultaneous detection of bacteria expressing GFP and DsRed genes with a flow cytometer.

BACKGROUND: In this study, Escherichia coli cells producing red fluorescent protein of Discosoma sp. (drFP583 DsRed) were investigated with flow cytometry by using 488 nm excitation. We also studied whether green fluorescent protein (GFP) and drFP583 could be detected simultaneously from a single bacterial cell. METHODS: Plasmids pDsRed and pEGFP were used for the production of drFP583 and enhanced GFP, respectively, in E. coli MC1061 cells. To produce enhanced GFP and drFP583 simultaneously, plasmids pG9R and pG19R were constructed. These encode tandem fusions of enhanced GFP and drFP583 to ensure similar production levels for both proteins. RESULTS: Bacteria producing enhanced GFP and drFP583 were found to be brightly green and red fluorescent, respectively. Production of enhanced GFP and drFP583 fusion proteins resulted in bacteria that emitted both green and red fluorescence, which was detected easily by a flow cytometer using single laser excitation. Previously reported tetramerization of drFP583 did not restrict its use as a reporter gene, although it maturated significantly slower than enhanced GFP. CONCLUSIONS: The results show that enhanced GFP and drFP583 proteins can be detected simultaneously from single bacteria with a standard flow cytometer with simple optical configuration.