Production of the antimicrobial secondary metabolite indigoidine contributes to competitive surface colonization by the marine roseobacter Phaeobacter sp. strain Y4I

Members of the Roseobacter lineage of marine bacteria are prolific surface colonizers in marine coastal environments, and antimicrobial secondary metabolite production has been hypothesized to provide a competitive advantage to colonizing roseobacters. Here, we report that the roseobacter Phaeobacter sp. strain Y4I produces the blue pigment indigoidine via a nonribosomal peptide synthase (NRPS)-based biosynthetic pathway encoded by a novel series of genetically linked genes: igiBCDFE. A Tn5-based random mutagenesis library of Y4I showed a perfect correlation between indigoidine production by the Phaeobacter strain and inhibition of Vibrio fischeri on agar plates, revealing a previously unrecognized bioactivity of this molecule. In addition, igiD null mutants (igiD encoding the indigoidine NRPS) were more resistant to hydrogen peroxide, less motile, and faster to colonize an artificial surface than the wild-type strain. Collectively, these data provide evidence for pleiotropic effects of indigoidine production in this strain. Gene expression assays support phenotypic observations and demonstrate that igiD gene expression is upregulated during growth on surfaces. Furthermore, competitive cocultures of V. fischeri and Y4I show that the production of indigoidine by Y4I significantly inhibits colonization of V. fischeri on surfaces. This study is the first to characterize a secondary metabolite produced by an NRPS in roseobacters.     

Disruption of Cell-to-Cell Signaling Does Not Abolish the Antagonism of Phaeobacter gallaeciensis toward the Fish Pathogen Vibrio anguillarum in Algal Systems

Quorum sensing (QS) regulates Phaeobacter gallaeciensis antagonism in broth systems; however, we demonstrate here that QS is not important for antagonism in algal cultures. QS mutants reduced Vibrio anguillarum to the same extent as the wild type. Consequently, a combination of probiotic Phaeobacter and QS inhibitors is a feasible strategy for aquaculture disease control.     

Production of Two Chitosanases from a Chitosan-Assimilating Bacterium, Acinetobacter sp. Strain CHB101

A bacterial strain capable of utilizing chitosan as a sole carbon source was isolated from soil and was identified as a member of the genus Acinetobacter. This strain, designated CHB101, produced extracellular chitosan-degrading enzymes in the absence of chitosan. The chitosan-degrading activity in the culture fluid increased when cultures reached the early stationary phase, although the level of activity was low in the exponential growth phase. Two chitosanases, chitosanases I and II, which had molecular weights of 37,000 and 30,000, respectively, were purified from the culture fluid. Chitosanase I exhibited substrate specificity for chitosan that had a low degree of acetylation (10 to 30%), while chitosanase II degraded colloidal chitin and glycol chitin, as well as chitosan that had a degree of acetylation of 30%. Rapid decreases in the viscosities of chitosan solutions suggested that both chitosanases catalyzed an endo type of cleavage reaction; however, chitosan oligomers (molecules smaller than pentamers) were not produced after a prolonged reaction.     

Site-Directed Mutagenesis of the Anabaena sp. Strain PCC 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

Cyanobacteria use sunlight and water to produce hydrogen gas (H₂), which is potentially useful as a clean and renewable biofuel. Photobiological H₂ arises primarily as an inevitable by-product of N₂ fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H₂ production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N₂. Most of the 49 variants examined were deficient in N₂-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N₂ atmosphere significantly increased their in vivo rates of H₂ production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H₂ compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H₂ in an aerobic, nitrogen-containing environment.Photobiologically produced hydrogen gas (H₂) is a clean energy source with the potential to greatly supplement our use of fossil fuels (39). Whereas coal and oil are limited, cyanobacteria and eukaryotic microalgae can use inexhaustible sunlight as the energy source and water as the electron donor to produce H₂ (42). This gas is generated either by hydrogenases (52) or as an inevitable by-product of N₂ fixation by nitrogenases (49). In contrast to the reaction of hydrogenases which is reversible, nitrogenases catalyze the unidirectional production of H₂, although with substantial energy input in the form of ATP (47). Under optimal N₂-fixing conditions: N₂ + 8 e⁻ + 8 H⁺ + 16 ATP → H₂ + 2 NH₃ + 16 (ADP + P_i), whereas, in the absence of N₂ (e.g., under Ar), all electrons are allocated to proton reduction: 2 e⁻ + 2 H⁺ + 4 ATP → H₂ + 4 (ADP + P_i). Thus, one expects to be able to increase the H₂ production activity of nitrogenase by decreasing the electron allocation to N₂ fixation.Nitrogenases are sensitive to inactivation by O₂; however, N₂-fixing cyanobacteria have developed mechanisms to protect these enzymes from photosynthetically generated oxygen (5). Of particular interest, Anabaena (also known as Nostoc) sp. strain PCC 7120 and some other filamentous cyanobacteria respond to combined-nitrogen deprivation by undergoing differentiation in which a subset of cells become heterocysts that provide a microaerobic environment, allowing nitrogenase to function in aerobic culture conditions. The nitrogenase-related (nif) genes are specifically expressed in heterocysts which lack O₂-evolving photosystem II activity and are surrounded by a thick cell envelope composed of glycolipids and polysaccharides that impede the entry of O₂ (56). Vegetative cells perform oxygenic photosynthesis and fix CO₂. Heterocysts obtain carbohydrates from those cells and, in turn, provide them with fixed nitrogen.The molybdenum-containing nitrogenase of Anabaena sp. strain PCC 7120 consists of the Fe protein (encoded by nifH) and the MoFe protein (encoded by nifD and nifK). As in other organisms, the Fe protein is a homodimer containing a single [4Fe-4S] cluster and functions as an ATP-dependent electron donor to the MoFe protein. The latter is an α₂β₂ heterotetramer with each nifD-encoded α subunit coordinating the FeMo cofactor (FeMo-co; MoFe₇S₉X-homocitrate) that binds and reduces substrate, while α plus the nifK-encoded β subunits coordinate the [8Fe-7S] P-cluster (14). Additional nif genes are required for the biosynthesis of the metal clusters and maturation of the enzyme (40). The major nif gene cluster of Anabaena sp. strain PCC 7120 undergoes two rearrangements in the heterocyst to yield nifB-fdxN-nifSUHDK-(1 ORF)-nifENX-(2 ORFs)-nifW-hesAB-fdxH (19).One approach to increase H₂ production by nitrogenase is to enhance the electron flux to proton reduction and away from N₂ reduction. Although replacement of N₂ by Ar is effective for increasing H₂ production, this approach increases the operational cost for large-scale generation of H₂. Mutagenesis offers an alternative mechanism to overcome N₂ competition. The amino acid sequences of the MoFe α subunit are highly conserved among different phyla (18). The V75I substitution in the suspected gas channel of NifD2 of Anabaena variabilis (equivalent to V70 in A. vinelandii) resulted in greatly diminished N₂ fixation, while allowing for H₂ production rates (under N₂) that were similar to those of wild-type cells under Ar (55). Significantly, however, the nonheterocyst nitrogenase of this strain, which is expressed mainly in vegetative cells under anaerobic conditions, is incompatible with O₂-evolving photosynthesis and thus requires continuous anaerobic conditions along with a supply of exogenous reducing sugars for H₂ production. Substitutions of selected amino acids in the vicinity of the FeMo-co active site within Azotobacter vinelandii nitrogenase were shown to eliminate or greatly diminish N₂ fixation while, in some cases, allowing for effective proton reduction (2, 10, 17, 27, 36, 44, 45, 48). Therefore, certain amino acid exchanges near FeMo-co might produce variant MoFe proteins in heterocyst-forming Anabaena that redirect the electron flux through the enzyme preferentially to proton reduction so as to synthesize more H₂ in the presence of N₂ in an aerobic environment.To examine whether Anabaena sp. strain PCC 7120 nitrogenase can be modified to increase photobiological H₂ production by effecting such a redirection, we evaluated in vivo H₂ production and acetylene reduction rates of a series of cyanobacterial nifD site-directed mutants. We mutated six NifD residues (Fig. ​(Fig.1)1) predicted to lie within 5 Å of FeMo-co to create 49 variants using an Anabaena ΔNifΔHup (previously denoted ΔhupL) parental strain that lacks both an intact nifD and an uptake hydrogenase (34). In an atmosphere containing N₂ and O₂, several mutants exhibited significantly enhanced rates of in vivo H₂ production and accumulated high levels of H₂ compared to the reference strains.Open in a separate windowFIG. 1.Side-on (left) and Mo end-on (right) views of the predicted active site for nitrogenase of Anabaena sp. strain PCC 7120. The FeMo-co cluster, a [7Fe-8S-Mo-X-homocitrate] complex, where X is a central unidentified light atom (N, C, or O), and its two coordinating residues (C282 and H449) are shown in a ball-and-stick representation. Water molecules near the FeMo-co are indicated by isolated spheres in red. The side chains of the residues targeted for mutagenesis—Q193, H197, Y236, R284, S285, and F388—are shown in stick representation. Residues V362 through P367 are represented by lines. The Anabaena residues were mapped onto the corresponding residues from the crystal structure of the A. vinelandii enzyme (PDB file 1M1N). The figure was generated by using PyMOL (www.pymol.org/), with the following color scheme: Fe, orange; S, yellow; C, gray; N and central atom X, blue; O, red; and Mo, pink.     

Dynamics of Metabolic Activities and Gene Expression in the Roseobacter Clade Bacterium Phaeobacter sp. Strain MED193 during Growth with Thiosulfate

Metagenomic analyses of surface seawater reveal that genes for sulfur oxidation are widespread in bacterioplankton communities. However, little is known about the metabolic processes used to exploit the energy potentially gained from inorganic sulfur oxidation in oxic seawater. We therefore studied the sox gene system containing Roseobacter clade isolate Phaeobacter sp. strain MED193 in acetate minimal medium with and without thiosulfate. The addition of thiosulfate enhanced the bacterial growth yields up to 40% in this strain. Concomitantly, soxB and soxY gene expression increased about 8-fold with thiosulfate and remained 11-fold higher than that in controls through stationary phase. At stationary phase, thiosulfate stimulated protein synthesis and anaplerotic CO₂ fixation rates up to 5- and 35-fold, respectively. Several genes involved in anaplerotic CO₂ fixation (i.e., pyruvate carboxylase, propionyl coenzyme A [CoA], and crotonyl-CoA carboxylase) were highly expressed during active growth, coinciding with high CO₂ fixation rates. The high expression of key genes in the ethylmalonyl-CoA pathway suggests that this is an important pathway for the utilization of two-carbon compounds in Phaeobacter sp. MED193. Overall, our findings imply that Roseobacter clade bacteria carrying sox genes can use their lithotrophic potential to gain additional energy from sulfur oxidation for both increasing their growth capacity and improving their long-term survival.     

Efficient Production of Lumichrome by Microbacterium sp. Strain TPU 3598

Lumichrome is a photodegradation product of riboflavin and is available as a photosensitizer and fluorescent dye. To develop new efficient methods of lumichrome production, we isolated bacterial strains with high lumichrome productivity from soil. The strain with highest productivity was identified as Microbacterium sp. strain TPU 3598. Since this strain inductively produced lumichrome when cultivated with riboflavin, we developed two different methods, a cultivation method and a resting cell method, for the production of large amounts of lumichrome using the strain. In the cultivation method, 2.4 g (9.9 mmol) of lumichrome was produced from 3.8 g (10.1 mmol) of riboflavin at the 500-ml scale (98% yield). The strain also produced 4.7 g (19.4 mmol) of lumichrome from 7.6 g (20.2 mmol) of riboflavin (96% yield) by addition of riboflavin during cultivation at the 500-ml scale. In the resting cell method, 20 g of cells (wet weight) in 100 ml of potassium phosphate buffer, pH 7.0, produced 2.4 g of lumichrome from 3.8 g of riboflavin (98% yield). Since the lumichrome production by these methods was carried out in suspension, the resulting lumichrome was easily purified from the cultivation medium or reaction mixture by centrifugation and crystallization. Thus, the biochemical methods we describe here are a significant improvement in terms of simplicity and yield over the existing chemical, photolytic, and other biochemical methods of lumichrome production.     

Enhancement of the Potential To Utilize Octopine in the Nonfluorescent Pseudomonas sp. Strain 92

The nonfluorescent Pseudomonas sp. strain 92 requires the presence of a supplementary carbon source for growth on octopine, whereas the spontaneous mutant RB100 has acquired the capacity to utilize this opine as the sole carbon and nitrogen source. Insertional mutagenesis of RB100 with transposon Tn5 generated mutants which were unable to grow on octopine and others which grew slowly on this substrate. Both types of mutants yielded revertants that had regained the ability to utilize octopine. Some of the revertants had lost the transposon, whereas in others the transposon was retained but with rearrangements of the insertion site. Genes of octopine catabolism from strain 92 were cloned on a cosmid vector to generate pK3. The clone pK3 conferred the ability to utilize octopine as the sole carbon and nitrogen source on the host Pseudomonas putida KT2440. Although they conferred an equivalent growth phenotype, the mutant genes carried by RB100 and the cloned genes on pK3 differed in their regulation. Utilization of [¹⁴C]octopine was inducible by octopine in RB100 and was constitutive in KT2440(pK3).     

Engineering the Genotype of Acinetobacter sp. Strain ADP1 To Enhance Biosynthesis of Cyanophycin

To study the importance of arginine provision and phosphate limitation for synthesis and accumulation of cyanophycin (CGP) in Acinetobacter sp. strain ADP1, genes encoding the putative arginine regulatory protein (argR) and the arginine succinyltransferase (astA) were inactivated, and the effects of these mutations on CGP synthesis were analyzed. The inactivation of these genes resulted in a 3.5- or 7-fold increase in CGP content, respectively, when the cells were grown on glutamate. Knockout mutations in both genes led to a better understanding of the effect of the addition of other substrates to arginine on CGP synthesis during growth of the cells of Acinetobacter sp. strain ADP1. Overexpression of ArgF (ornithine carbamoyltransferase), CarA-CarB (small and large subunits of carbamoylphosphate synthetase), and PepC (phosphoenolpyruvate carboxylase) triggered synthesis of CGP if amino acids were used as a carbon source whereas it was not triggered by gluconate or other sugars. Cells of Acinetobacter sp. strain ADP1, which is largely lacking genes for carbohydrate metabolism, showed a significant increase in CGP contents when grown on mineral medium supplemented with glutamate, aspartate, or arginine. The Acinetobacter sp. ΔastA(pYargF) strain is unable to utilize arginine but synthesizes more arginine, resulting in CGP contents as high as 30% and 25% of cell dry matter when grown on protamylasse or Luria-Bertani medium, respectively. This recombinant strain overcame the bottleneck of the costly arginine provision where it produces about 75% of the CGP obtained from the parent cells grown on mineral medium containing pure arginine as the sole source of carbon. Phosphate starvation is the only known trigger for CGP synthesis in this bacterium, which possesses the PhoB/PhoR phosphate regulon system. Overexpression of phoB caused an 8.6-fold increase in CGP content in comparison to the parent strain at a nonlimiting phosphate concentration.     

Production and Characterization of a Recombinant Cold-Active Acetyl Xylan Esterase from Psychrophilic Paenibacillus sp. R4 Strain

Applied Biochemistry and Microbiology - Acetyl xylan esterases (AXEs) hydrolyze the specific ester linkages between acetic acid and xylose units, leading to the deacetylation and depolymerization...     

Rhizobium sp. Strain NGR234 Possesses a Remarkable Number of Secretion Systems

Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.Diverse soil bacteria interact with plants in ways that range from symbiotic to pathogenic. Symbiotic Eubacteria (both alpha- and betaproteobacteria, collectively called rhizobia) form nitrogen-fixing associations of tremendous environmental importance (41, 66). Although some rhizobia are able to reduce atmospheric nitrogen to ammonia under saprophytic, free-living conditions, the reduced oxygen tensions found within the intracellular environment of specialized organs called nodules, maximizes this process (16). As legume roots penetrate the soil, they come in contact with rhizobia. Symbiotic interactions are initiated by the exchange of diverse molecules between the partners. Among them, plants liberate flavonoids into the rhizosphere that upregulate rhizobial genes. As a result, lipo-chito-oligo-saccharidic Nod factors are produced that trigger the nodulation pathway in susceptible legumes. Then, in many hosts, rhizobia enter the roots through root hairs, make their way to the cortex, multiply and fill the intracellular spaces of mature nodules. Centripetal progression of rhizobia into the plant and their maturation into nitrogen-fixing symbiosomes depends on the continued exchange of diverse signals. Many, but not all of these signals have been identified; one sure way to take stock of what is necessary for effective symbiosis is to sequence the partners. We began this work by assembling overlapping sets of cosmids (contigs) of the microsymbiont Rhizobium sp. strain NGR234 (hereafter NGR234) (63), which enabled us to elucidate the nucleotide sequence of the symbiotic (pNGR243a) plasmid (29). Similar techniques permitted the assembly of sections of the extremely large megaplasmid pNGR234b (86), and some snapshot genome information was made available earlier (91); however, the use of pyrosequencing methods greatly facilitated this process. We report here the genome sequence of NGR234 that is able to nodulate more than 120 genera of legumes and the nonlegume Parasponia andersonii (69). It seems likely that the vast richness of secretory systems might be a major key to the broad host range.     

Production and Properties of Alkaline Lipase from Alcaligenes sp. Strain No. 679

For the production of extracellular lipase by Alcaligenes species No. 679, NaNO₃, polyoxyethylene alkyl ether, Fe⁺⁺, sodium citrate and fructose were found to be effective. The enzyme was prepared by acetone precipitation from the filtrate of the culture broth of this strain. The enzyme was most active at pH 9.0 and 50°C, while 35% of its activity was lost on heat treatment at 60°C for 10 min. Sodium salts of bile acids stimulated the enzyme activity. This lipase could hydrolyse natural fats and oils as well as olive oil. During the hydrolysis of olive oil, monoglyceride was found to accumulate up to 70 mol percent. This lipase possesses special properties similar to those of pancreatic lipase as shown in the comparative experiments.     

Utilization of Ganglioside-Degrading Paenibacillus sp. Strain TS12 for Production of Glucosylceramide

Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by Paenibacillus sp. strain TS12, which was isolated from soil, as follows: GM1a → asialo GM1 → asialo GM2 → lactosylceramide → glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, β-galactosidases, and β-hexosaminidases. TS12 also produced β-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.     

Characterization of the 4-Hydroxybenzoyl-Coenzyme A Thioesterase from Arthrobacter sp. Strain SU

The Arthrobacter sp. strain SU 4-chlorobenzoate (4-CBA) dehalogenation pathway converts 4-CBA to 4-hydroxybenzoate (4-HBA). The pathway operon contains the genes fcbA, fcbB, and fcbC (A. Schmitz, K. H. Gartemann, J. Fiedler, E. Grund, and R. Eichenlaub, Appl. Environ. Microbiol. 58:4068-4071, 1992). Genes fcbA and fcbB encode 4-CBA-coenzyme A (CoA) ligase and 4-CBA-CoA dehalogenase, respectively, whereas the function of fcbC is not known. We subcloned fcbC and expressed it in Escherichia coli, and we purified and characterized the FcbC protein. A substrate activity screen identified benzoyl-CoA thioesters as the most active substrates. Catalysis of 4-HBA-CoA hydrolysis to 4-HBA and CoA occurred with a k_cat of 6.7 s⁻¹ and a K_m of 1.2 μM. The k_cat pH rate profile for 4-HBA-CoA hydrolysis indicated optimal activity over a pH range of 6 to 10. The amino acid sequence of the FcbC protein was compared to other sequences contained in the protein sequence data banks. A large number of sequence homologues of unknown function were identified. On the other hand, the 4-HBA-CoA thioesterases isolated from 4-CBA-degrading Pseudomonas strains did not share significant sequence identity with the FcbC protein, indicating early divergence of the thioesterase-encoding genes.     

Production of Proteasome Inhibitor Syringolin A by the Endophyte Rhizobium sp. Strain AP16

Syringolin A, the product of a mixed nonribosomal peptide synthetase/polyketide synthase encoded by the syl gene cluster, is a virulence factor secreted by certain Pseudomonas syringae strains. Together with the glidobactins produced by a number of beta- and gammaproteobacterial human and animal pathogens, it belongs to the syrbactins, a structurally novel class of proteasome inhibitors. In plants, proteasome inhibition by syringolin A-producing P. syringae strains leads to the suppression of host defense pathways requiring proteasome activity, such as the ones mediated by salicylic acid and jasmonic acid. Here we report the discovery of a syl-like gene cluster with some unusual features in the alphaproteobacterial endophyte Rhizobium sp. strain AP16 that encodes a putative syringolin A-like synthetase whose components share 55% to 65% sequence identity (72% to 79% similarity) at the amino acid level. As revealed by average nucleotide identity (ANI) calculations, this strain likely belongs to the same species as biocontrol strain R. rhizogenes K84 (formely known as Agrobacterium radiobacter K84), which, however, carries a nonfunctional deletion remnant of the syl-like gene cluster. Here we present a functional analysis of the syl-like gene cluster of Rhizobium sp. strain AP16 and demonstrate that this endophyte synthesizes syringolin A and some related minor variants, suggesting that proteasome inhibition by syrbactin production can be important not only for pathogens but also for endophytic bacteria in the interaction with their hosts.     

Application of Real-Time PCR To Estimate Toxin Production by the Cyanobacterium Planktothrix sp.

Quantitative real-time PCR methods are increasingly being applied for the enumeration of toxic cyanobacteria in the environment. However, to justify the use of real-time PCR quantification as a monitoring tool, significant correlations between genotype abundance and actual toxin concentrations are required. In the present study, we aimed to explain the concentrations of three structural variants of the hepatotoxin microcystin (MC) produced by the filamentous cyanobacterium Planktothrix sp., [Asp, butyric acid (Dhb)]-microcystin-RR (where RR means two arginines), [Asp, methyl-dehydro-alanine (Mdha)]-microcystin-RR, and [Asp, Dhb]-microcystin-homotyrosine-arginine (HtyR), by the abundance of the microcystin genotypes encoding their synthesis. Three genotypes of microcystin-producing cyanobacteria (denoted the Dhb, Mdha, and Hty genotypes) in 12 lakes of the Alps in Austria, Germany, and Switzerland from 2005 to 2007 were quantified by means of real-time PCR. Their absolute and relative abundances were related to the concentration of the microcystin structural variants in aliquots determined by high-performance liquid chromatography (HPLC). The total microcystin concentrations varied from 0 to 6.2 μg liter⁻¹ (mean ± standard error [SE] of 0.6 ± 0.1 μg liter⁻¹) among the samples, in turn resulting in an average microcystin content in Planktothrix of 3.1 ± 0.7 μg mm⁻³ biovolume. Over a wide range of the population density (0.001 to 3.6 mm³ liter⁻¹ Planktothrix biovolume), the Dhb genotype and [Asp, Dhb]-MC-RR were most abundant, while the Hty genotype and MC-HtyR were found to be in the lowest proportion only. In general, there was a significant linear relationship between the abundance/proportion of specific microcystin genotypes and the concentration/proportion of the respective microcystin structural variants on a logarithmic scale. We conclude that estimating the abundance of specific microcystin genotypes by quantitative real-time PCR is useful for predicting the concentration of microcystin variants in water.During the last decade, genetic methods have significantly increased our understanding of the distribution of genes that are involved in the production of toxins within cyanobacteria that occur in fresh and brackish water (45). Although genetic methods can indicate only the potential risk of toxin synthesis and do not provide information about the actual toxin concentrations, quantitative real-time PCR has been increasingly applied for monitoring the toxin-producing genotypes of cyanobacteria in water (26, 33, 44). The development of real-time PCR methods was driven primarily by its potential (i) as an early-warning tool as well as to monitor toxin-producing cyanobacteria and (ii) to identify those factors that lead to a dominance/repression of toxin-producing genotypes versus nontoxic genotypes. For the first aim, it is essential that the abundance of toxin-producing cyanobacteria can be related to the concentration of the respective toxic substance in water. A few studies showed that the concentration of certain toxic genotypes was linearly related to the respective toxin concentrations, e.g., for the most common group of hepatotoxins, the microcystins (MCs) (7, 12, 14), and for the related nodularin (19). Both microcystins and nodularins are known to be potent inhibitors of eukaryotic protein phosphatases 1 and 2A, resulting in a health hazard to humans and the environment (9). In contrast, no correlation was found (37, 50), or even the opposite was reported, by other studies, i.e., that the measurement of microcystin-producing genotypes is not a satisfactory method for use in monitoring programs in order to predict the toxic risk associated with cyanobacterial proliferation (3). For microcystins, these contrasting results may be due to several reasons: (i) several genera producing microcystins frequently coexist in water bodies, and therefore, not all microcystin producers may have been identified; (ii) the semilogarithmic calibration curves limit the accuracy in estimations of genotype numbers and proportions (for example, the only laboratory comparison carried out so far revealed that among the three laboratories tested, the proportions of toxic genotypes were overestimated or underestimated by 0 to 72% and 0 to 50%, respectively [42]); and (iii) inactive mutants that contain the respective genes, however, which have been inactivated in toxin production through the insertion of transposable elements, may co-occur and decrease toxin production in a given population (6). Nevertheless, the real-time PCR technique is the only quantitative technique available for estimating the proportion of potential toxin-producing genotypes in water. The development of automated and field-applicable real-time PCR methods (e.g., see reference 35), in particular, may contribute to a more widespread integration of real-time PCR into routine monitoring programs in the future.In the present study, we attempted to quantify microcystin-producing genotypes in total as well as quantify the specific genotypes that were shown to encode different microcystin structural variants characterized for strains isolated from lakes in the Alps (23): (i) the methyl-dehydro-alanine residue (Mdha) genotype, which was found to synthesize structural variants containing only Mdha in position 7; (ii) the butyric acid (Dhb) genotype, which was found to contain Dhb instead of Mdha in the same position; and (iii) the homotyrosine (Hty) genotype, which was found to contain Hty and Leu in position 2 but never Arg. The Hty variant has always been found to co-occur with Dhb in position 7 of the molecule (24). Consequently, the Hty genotype forms a subgroup of the microcystin-producing population composed of the Mdha and Dhb genotypes. The following hypotheses were tested: (i) as only one microcystin-producing organism (Planktothrix sp.) is of quantitative importance in those lakes (32), the total microcystin concentration should be predictable from the sum of Mdha and Dhb genotypes; (ii) given that all Planktothrix genotypes are amenable to cultivation, all the structural microcystin variants found in the field samples should have been described for the strains isolated previously (23); and (iii) as, on average, the proportion of the inactive microcystin genotypes was found to be low and rather stable (<6.5% [32]), their occurrence should not reduce the ability to predict microcystin concentrations from genotype abundance. For this purpose, the phytoplankton in 12 lakes of the Alps in Austria, Germany, and Switzerland was monitored both with an inverted microscope as well as by means of real-time PCR over the course of 2 years (2005 to 2007). In parallel, microcystin concentrations in aliquots were determined by means of high-performance liquid chromatography (HPLC). We show that the abundance of specific microcystin genotypes can be related to the corresponding microcystin concentrations in water on a logarithmic scale over a range of trophic conditions. The proportion of certain genotypes encoding the synthesis of a specific microcystin variant significantly correlates with the concentration of the respective microcystin variant. We argue that these genotype-toxin concentration relationships are of great importance for the justification of real-time PCR use in monitoring programs.