DNA mapping and kinetic modeling of the HrdB regulon in Streptomyces coelicolor.

HrdB in streptomycetes is a principal sigma factor whose deletion is lethal. This is also the reason why its regulon has not been investigated so far. To overcome experimental obstacles, for investigating the HrdB regulon, we constructed a strain whose HrdB protein was tagged by an HA epitope. ChIP-seq experiment, done in 3 repeats, identified 2137 protein-coding genes organized in 337 operons, 75 small RNAs, 62 tRNAs, 6 rRNAs and 3 miscellaneous RNAs. Subsequent kinetic modeling of regulation of protein-coding genes with HrdB alone and with a complex of HrdB and a transcriptional cofactor RbpA, using gene expression time series, identified 1694 genes that were under their direct control. When using the HrdB-RbpA complex in the model, an increase of the model fidelity was found for 322 genes. Functional analysis revealed that HrdB controls the majority of gene groups essential for the primary metabolism and the vegetative growth. Particularly, almost all ribosomal protein-coding genes were found in the HrdB regulon. Analysis of promoter binding sites revealed binding motif at the -10 region and suggested the possible role of mono- or di-nucleotides upstream of the -10 element.

Uptake kinetics of four hydrophobic organic pollutants in the earthworm Eisenia andrei in aged laboratory-contaminated natural soils.

Laboratory studies of pollutant uptake kinetics commonly start shortly after experimental soil contamination when it is not clear if the processes between soil and chemicals are equilibrated and stabilized. For instance, when the concentration in soil quickly decreases due to initial biodegradation, bioaccumulation may show a peak-shape accumulation curve instead of conventional first order kinetics with a plateau at the end. The results of such experiments with soil freshly contaminated in the laboratory are then hardly comparable to bioaccumulation observed in soils from historically contaminated sites. Therefore, our study focused on the uptake kinetics of four hydrophobic organic compounds (pyrene, lindane, p,p'-DDT and PCB 153) in two laboratory-contaminated natural soils with different soil properties (e.g. total organic carbon content of 1.6 and 9.3%) aged for 203 days to mimic long-term contamination. For pyrene, the results surprisingly showed peak-shape accumulation curves despite long aging. It seems compound biodegradation might be significant in aged soils when the conditions change (e.g. by distribution to the experimental vessels) and this should be also considered when testing historically contaminated soils. For lindane, longer aging seems to guarantee stability of the soil-compound-earthworm system and the steady state was reached after 5 days of exposure. Furthermore, although concentrations of p,p'-DDT and PCB 153 in earthworms after 11-15-day exposure did not statistically differ, which is a commonly-used indicator that a steady state was reached, they continuously increased until the end of the exposure. Therefore, despite the aging, longer exposure was probably needed to reach the true equilibrium between concentrations in earthworms and soil. In summary, aging does not warranty the conventional first order kinetic curve with the equilibrium at the end of the exposure but may have diverse effects for compounds with different environmental properties and should be taken into account in the bioaccumulation factor calculation and the risk assessment.

The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti.

Quorum sensing is a cell density-dependent communication system of bacteria relying on autoinducer molecules. During the analysis of the post-transcriptional regulation of quorum sensing in the nitrogen fixing plant symbiont Sinorhizobium meliloti, we predicted and verified a direct interaction between the 5'-UTR of sinI mRNA encoding the autoinducer synthase and a small RNA (sRNA), which we named RcsR1. In vitro, RcsR1 prevented cleavage in the 5'-UTR of sinI by RNase E and impaired sinI translation. In line with low ribosomal occupancy and transcript destabilization upon binding of RcsR1 to sinI, overproduction of RcsR1 in S. meliloti resulted in lower level and shorter half-life of sinI mRNA, and in decreased autoinducer amount. Although RcsR1 can influence quorum sensing via sinI, its level did not vary at different cell densities, but decreased under salt stress and increased at low temperature. We found that RcsR1 and its stress-related expression pattern, but not the interaction with sinI homologs, are conserved in Sinorhizobium, Rhizobium and Agrobacterium. Consistently, overproduction of RcsR1 in S. meliloti and Agrobacterium tumefaciens inhibited growth at high salinity. We identified conserved targets of RcsR1 and showed that most conserved interactions and the effect on growth under salt stress are mediated by the first stem-loop of RcsR1, while its central part is responsible for the species-specific interaction with sinI. We conclude that RcsR1 is an ancient, stress-related riboregulator in rhizobia and propose that it links stress responses to quorum sensing in S. meliloti.

Solid phase microextraction of organic pollutants from natural and artificial soils and comparison with bioaccumulation in earthworms.

The presented study investigates the use of passive sampling, i.e. solid phase microextraction with polydimethylsiloxane fibers (PDMS-SPME), to assess the bioavailability of fiver neutral organic chemicals (phenanthrene, pyrene, lindane, p,p'-DDT, and PCB 153) spiked to natural and artificial soils after different aging times. Contaminant bioavailability was assessed by comparing PDMS concentrations with results from a 10 day bioaccumulation test with earthworms (Eisenia fetida). The hypotheses tested were (i) organic carbon (OC) normalization, which is commonly used to account for sorption and bioavailability of hydrophobic organic chemicals in soil risk assessment, has limitations due to differences in sorptive properties of OC and aging processes (i.e. sequestration and biodegradation) and (ii) PDMS-SPME provides a more reliable measure of soil contaminant bioavailability than OC normalized soil concentrations. The above stated hypotheses were confirmed since the results showed that: (i) the PDMS/soil organic carbon partition ratio (R) accounting for the role that OC plays in partitioning significantly differed between soils and aging times and (ii) the correlation with earthworm concentrations was better using porewater concentrations derived from PDMS concentrations than when organic normalized soil concentrations were used. Capsule: Sorption of organic compounds measured by SPME method and their bioavailability to earthworms cannot be reliably predicted using OC content.

Toxicokinetics of hydrophobic organic compounds in oligochaeta: A critical review.

Toxicokinetic studies appertain to the fundamental research of soil bioavailability. However, the research outcomes of aspects influencing uptake and elimination of hydrophobic organic compounds have not been summarized so far. In our review, a recapitulation of available toxicokinetic data (i.e. experimental conditions, if the steady state was reached, uptake and elimination rate constants, and bioaccumulation factors) is presented in well-arranged tables. Further, toxicokinetic models are overviewed in the schematic form. In the review, the required information could be quickly found and/or the experimental gaps easily identified. Generally a little is known about the effects of soil properties other than soil organic matter. Limited or no data are available about soil treatment, food supply during laboratory exposure, and metabolization in oligochaeta. The impact of these factors might be important especially for arable soils with typically low organic matter content but high consequences on humans. Besides these circumstances, other uncertainties between published studies have been found. Firstly, the scientific results are provided in heterogenous units: bioaccumulation factors as well as the rate constants are reported in dry or wet weight of soil and earthworms. The steady state is another critical factor because the time to reach the equilibrium is influenced not only by soil and compound characteristics but for example also by aging. Nevertheless, toxicokinetic studies bring irreplaceable information about the real situation in soil and our review help to define missing knowledge and estimate the scientific priorities.

Uptake kinetics of pesticides chlorpyrifos and tebuconazole in the earthworm Eisenia andrei in two different soils.

Agriculture is today indispensably connected with enormous use of pesticides. Despite tough regulation, their entrance into soil cannot be excluded and they might enter soil organisms and plants and continue further to terrestrial food chains. This study was conducted to investigate the bioaccumulation of two pesticides currently used in large amounts, the insecticide chlorpyrifos (CLP) and the fungicide tebuconazole (TBZ). Their detailed uptake kinetics in the model earthworm species Eisenia andrei were measured in two arable soils differing in organic carbon content (1.02 and 1.93% respectively). According to our results, a steady state was reached after 3-5 days for both pesticides and soils. The values of bioaccumulation factors calculated at the steady state ranged from 4.5 to 6.3 for CLP and 2.2-13.1 for TBZ. Bioaccumulation factors were also calculated as the ratio of uptake and elimination rate constants with results comparable with steady-state bioaccumulation factors. The results suggested that the degradation and bioaccumulation of tested compounds might be influenced by other factors than only total organic carbon (e.g. clay content). The lower Koc and hydrophobicity of TBZ relative to CLP probably led to higher availability of TBZ through pore water exposure. On the other hand, CLP's higher hydrophobicity probably caused an increase in availability by its additional uptake via ingestion. To enable a proper ecological risk assessment of current pesticides in soils, it is necessary to accurately determine their bioaccumulation in soil invertebrates. We believe that our study not only brings such information for two specific pesticides but also addresses key methodological issues in this area.

A Waking Review: Old and Novel Insights into the Spore Germination in Streptomyces.

The complex development undergone by Streptomyces encompasses transitions from vegetative mycelial forms to reproductive aerial hyphae that differentiate into chains of single-celled spores. Whereas their mycelial life - connected with spore formation and antibiotic production - is deeply investigated, spore germination as the counterpoint in their life cycle has received much less attention. Still, germination represents a system of transformation from metabolic zero point to a new living lap. There are several aspects of germination that may attract our attention: (1) Dormant spores are strikingly well-prepared for the future metabolic restart; they possess stable transcriptome, hydrolytic enzymes, chaperones, and other required macromolecules stabilized in a trehalose milieu; (2) Germination itself is a specific sequence of events leading to a complete morphological remodeling that include spore swelling, cell wall reconstruction, and eventually germ tube emergences; (3) Still not fully unveiled are the strategies that enable the process, including a single cell's signal transduction and gene expression control, as well as intercellular communication and the probability of germination across the whole population. This review summarizes our current knowledge about the germination process in Streptomyces, while focusing on the aforementioned points.

Bioavailability of five hydrophobic organic compounds to earthworms from sterile and non-sterile artificial soils.

Bioaccumulation factors (BAFs) of organic pollutants to soil biota, often required by risk assessment, are mostly obtained in non-sterile laboratory-contaminated artificial soils. However, microbial degradation has been indicated by many authors to influence the fate of hydrophobic organic compounds (HOCs) in soils. A question arises if the microbial community of peat which is used for artificial soil preparation affects the measured values of BAFs. In this study the effect of soil microorganisms on bioavailability of HOCs was studied and a portion of each soil was sterilized by gamma irradiation. Results indicated that the sterilization process significantly affected the fate of polycyclic aromatic hydrocarbons (PAHs; phenanthrene and pyrene) and increased bioavailability of these compounds to earthworms with BAFs several times higher in the sterile soils compared to their non-sterile variants. This suggests that sterilization of soils can be used as the "worst-case scenario" for laboratory tests of toxicity or bioaccumulation of biodegradable HOCs such as PAHs. It represents a situation of limited microbial degradation resulting in higher bioavailable fractions to other organisms (e.g. invertebrates). This may be the case in soils where microbial communities face stresses caused by contamination or land management. The bioavailability of chlorinated HOCs (lindane, 4,4'-DDT and PCB 153) was not affected by sterilization, as their BAFs were similar in the sterile and non-sterile soils during the experiment.

RNase III-Binding-mRNAs Revealed Novel Complementary Transcripts in Streptomyces.

cis-Antisense RNAs (asRNAs) provide very simple and effective gene expression control due to the perfect complementarity between regulated and regulatory transcripts. In Streptomyces, the antibiotic-producing clade, the antisense control system is not yet understood, although it might direct the organism's complex development. Initial studies in Streptomyces have found a number of asRNAs. Apart from this, hundreds of mRNAs have been shown to bind RNase III, the double strand-specific endoribonuclease. In this study, we tested 17 mRNAs that have been previously co-precipitated with RNase III for antisense expression. Our RACE mapping showed that all of these mRNAs possess cognate asRNA. Additional tests for antisense expression uncovered as-adpA, as-rnc, as3983, as-sigB, as-sigH, and as-sigR RNAs. Northern blots detected the expression profiles of 18 novel transcripts. Noteworthy, we also found that only a minority of asRNAs respond to the absence of RNase III enzyme by increasing their cellular levels. Our findings suggest that antisense expression is widespread in Streptomyces, including genes of such important developmental regulators, as AdpA, RNase III, and sigma factors.

Secondary Metabolites Produced during the Germination of Streptomyces coelicolor.

Spore awakening is a series of actions that starts with purely physical processes and continues via the launching of gene expression and metabolic activities, eventually achieving a vegetative phase of growth. In spore-forming microorganisms, the germination process is controlled by intra- and inter-species communication. However, in the Streptomyces clade, which is capable of developing a plethora of valuable compounds, the chemical signals produced during germination have not been systematically studied before. Our previously published data revealed that several secondary metabolite biosynthetic genes are expressed during germination. Therefore, we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are produced during germination of the model streptomycete, S. coelicolor. Interestingly, the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may coordinate the development of the producer (quorum sensing) and/or play a role in competitive microflora repression (quorum quenching) in their nature environments.

Influence of feeding and earthworm density on compound bioaccumulation in earthworms Eisenia andrei.

Earthworm density and feeding during exposure to contaminated soil have been used inconsistently in bioaccumulation studies, which may lead to possible errors in risk assessment and modeling. Hydrophobic organic pollutants with a wide range of environmental properties (phenanthrene, pyrene, lindane, p,p'-DDT, and PCB 153) were used to study the effect of different earthworm densities in combination with the presence or absence of feeding on bioaccumulation factors (BAFs). Similar BAFs were found at various soil-to-worm ratios, with the exception of phenanthrene. We recommend using at least 15 gsoil dw per earthworm. The absence of feeding doubled the BAFs and, thus, using no food ration can be considered as "the worst case scenario". Whenever food is to be applied (i.e. to ensure the validity of the test in earthworm mass loss), we suggest feeding depending on the organic carbon content of the studied soil.

Uptake kinetics of five hydrophobic organic pollutants in the earthworm Eisenia fetida in six different soils.

Hydrophobic organic contaminants in soils may pose toxicity or transfer to food chains after their uptake to soil biota. However, uptake data for earthworms are usually limited, as: (a) only fixed exposure times are studied instead of whole uptake kinetics and (b) studies including compounds with different environmental properties and more than two soils of different properties are quite rare. In our study, five persistent organic pollutants (phenanthrene, pyrene, lindane, p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), and polychlorinated biphenyl congener No. 153 (PCB 153)) were added to six soils of a wide range of soil properties. Detailed kinetics of uptake to earthworms Eisenia fetida were measured. Results indicated that an equilibrium of concentrations for p,p'-DDT and PCB 153 was reached after 11 days of exposure in all soils. Uptake of phenanthrene, pyrene, and lindane was strongly influenced by the decrease in concentrations in the soils, resulting in peak-shaped accumulation curves. Only in soils with the highest total organic carbon content (7.9 and 20.2%), the equilibrium of lindane concentrations was achieved (after 17 and 5 days of exposure, respectively). We recommend calculating bioaccumulation factors as a ratio of the uptake and elimination rate constants to precise the risk assessment.

Supercritical fluid extraction of persistent organic pollutants from natural and artificial soils and comparison with bioaccumulation in earthworms.

Selective supercritical fluid extraction (SSFE) was used as a measurement of compound chemical accessibility and as a predictor of compound bioavailability from three natural soils and artificial analogues prepared to have comparable total organic carbon content. Soils spiked with phenanthrene, pyrene, PCB 153, lindane, and p,p'-DDT were aged for 0, 14, 28, or 56 days and then selectively extracted by supercritical fluid extraction. Compounds exhibited decreasing extractability with increasing pollutant-soil contact time and increasing total organic carbon content in tested soils. However, the different extractability of compounds from artificial and natural pairs having comparable TOC indicates the limitations of using TOC as an extrapolation basis between various soils. The comparison of extractability with bioaccumulation by earthworms (Eisenia fetida) previously published by Vlcková and Hofman (2012) showed that only for PAHs it was possible to predict their bioaccumulation by means of selective SFE.

Fate and bioavailability of ¹4C-pyrene and ¹4C-lindane in sterile natural and artificial soils and the influence of aging.

Soil organic matter is used to extrapolate the toxicity and bioavailability of organic pollutants between different soils. However, it has been shown that other factors such as microbial activity are crucial. The aim of this study was to investigate if sterilization can reduce differences in the fate and bioavailability of organic pollutants between different soils. Three natural soils with increasing total organic carbon (TOC) content were collected and three artificial soils were prepared to obtain similar TOCs. Soils were sterilized and spiked with (14)C-pyrene and (14)C-lindane. Total (14)C radioactivity, HPCD extractability, and bioaccumulation in Eisenia fetida were measured over 56 days. When compared to non-sterile soils, differences between the natural and artificial soils and the influence of soil-contaminant contact time were generally reduced in the sterile soils (especially with middle TOC). The results indicate the possibility of using sterile soils as "the worst case scenario" in soil ecotoxicity studies.