Novel enzyme-based electrochemical and colorimetric biosensors for tetracycline monitoring in milk.

Recently, there has been a growing demand to develop portable devices for the fast detection of contaminants in food safety, healthcare, and environmental fields. Herein, two biosensing methods were designed by the use of nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent TetX2 enzyme activity and thionine as an excellent electrochemical and colorimetric mediator/probe to monitor tetracycline (TC) in milk. The nanoporous glassy carbon electrode (NPGCE) modified with polythionine was first prepared by electrochemically and then TetX2 was immobilized onto the NPGCE using polyethyleneimine. The prepared biosensor provided a high electrocatalytic response toward NAD(P)H by significantly reducing its overpotential. The proposed biosensor exhibited a detection limit of 40 nM with a linear range of 0.1-0.8 µM for TC determination. Besides, the thionine probe was used to develop a novel colorimetric assay using a simple enzymatic color reaction within a few minutes. The limit of detection for TC was experimentally achieved as 60 nM, which was lower than the safety levels established by the World Health Organization (225 nM). The correlation between change in the color of the solution and the concentration of TC was used for quality control of milk samples, as confirmed by the standard high-performance liquid chromatography method. The results show the great potential of the proposed assays as portable instruments for on-site TC measurements.

Effect of the Nanoclay Treated Streptomyces sp. UTMC 3136 as a Bioformulation on the Growth of Helianthus annuus.

Biofertilizers based on plant growth-promoting actinobacteria are used as potential alternatives to chemical fertilizers for sustainable agricultural systems. However, successful application of PGPA to agricultural land is challenging. The present study was an attempt to develop and evaluate the effect of a low-cost biofertilizer named NCTS (nanoclay-treated-Streptomyces) based on Streptomyces sp. UTMC 3136 spores amalgamated in a hybrid material of nanoclay Na-montmorillonite K10-glycerol-water substrate. In addition, the effect of NCTS on sunflower growth was investigated. In vivo tests showed a statistically significant increase in the agronomic characteristics of sunflowers treated with NCTS. Characterization of NCTS by FTIR, Raman spectroscopy, and scanning electron microscopy testified to the structural alignment and good adhesion of NCTS components. The viability of NCTS was 100% after 72 h of storage at 4 °C. Overall, the present study attempted to validate the efficacy of the formulation of Streptomyces sp. UTMC 3136 in nanoclay for growth improvement of sunflower. It was the first study to show the administration of PGPA in combination with nanomaterials as a growth enhancing biofertilization agent for sunflower.

Optimized bioleaching of copper by indigenous cyanogenic bacteria isolated from the landfill of e-waste.

In this study, indigenous cyanogenic bacterial strains were isolated on nutrient, minimal salt, and soil extract media at various culture conditions from two distinct landfills of e-waste, Iran. Based on their cyanide formation profiles, five most potent isolates were selected for optimization and to this end, the influence of the most effective factors on cyanide production including pH, glycine concentration and temperature were assessed using one-factor at a time method (OFAT). Initial pH of 7, glycine concentration of 2 g/L and temperature of 30°C were obtained as optimal conditions for most of the isolates. Additionally, two bioleaching processes were applied for each bacteria to detect the effect of optimal conditions on bioleaching and to assay their potential in the mobilization of copper. Under optimal conditions and pulp density of 1 g/L, copper recoveries were recorded as 96.73%, 82.49%, 81.17%, 41.72%, and 31.52% by S22, N13, N37, N23, and N41 respectively during 10 days which is approximately 1.5-5 times higher than the recovery obtained without optimization. During the optimization and the bioleaching process, the pH fluctuation of the flasks was monitored which validated the activity of the microorganisms.

Genome mining for ribosomally synthesised and post-translationally modified peptides (RiPPs) reveals undiscovered bioactive potentials of actinobacteria.

One of the most diverse groups of bioactive bacterial metabolites is the ribosomally synthesised and post-translationally modified peptides (RiPPs) with different bioactivities. The process of genome mining has made it possible to predict the presence of such clusters among the huge genomic data available today. Despite the great potential of actinobacteria in producing natural products and the myriad of completely sequenced genomes available, a comprehensive genome mining of these bacteria for RiPPs is lacking. Here, a collection of 629 complete actinobacterial genomes were analysed to explore their RiPP biosynthesis potential. Using BAGEL3 genome mining tool, the presence of 477 RiPP biosynthesis gene clusters (BGCs) was shown, including all known classes of bacterial RiPPs. RiPP-encoding potential was shown to be widespread among different members of actinobacteria especially within the plant and soil-inhabiting strains. The notable presence of LAP BGCs in plant-associating actinobacteria was also illustrated. Streptomyces, Amycolatopsis, Kitasatospora and Frankia showed greater potential in RiPP biosynthesis while lanthipeptides and lasso peptides were the most distributed RiPPs. Three cyanobactin BGCs were also detected. Generally evidence of promising ability of actinobacteria to synthesise diverse classes of RiPPs as well as information needed to rationally select appropriate taxa for rational screening of specific RiPPs are presented.

Effects of microbial volatile organic compounds on Ganoderma lucidum growth and ganoderic acids production in Co-v-cultures (volatile co-cultures).

Co-v-culture (co-cultivations of physically separated microbes that only interact through the air) systems were designed to investigate the effects of microbial volatile organic compounds (mVOCs) from about 20 different microbes, on a medicinal fungus, Ganoderma lucidum. For more accuracy in co-cultivations, a novel synchronized cultivation approach was tested for culturing G. lucidum. The hyphal growth of G. lucidum and the content of its ganoderic acids (GAs) were measured. In almost all of the co-v-cultures, there was an inhibiting effect on hyphal growth and a promoting effect on GAs contents. In inducing GAs production, Bacillus cereus PTCC 1247 and Pseudomonas aeruginosa UTMC 1404 were the most effective ones, as, compared to control cultures, GAs content increased 2.8 fold. Comparing different co-v-cultivations demonstrated that the concentrations of mVOCs, oxygen, and carbon dioxide were the main players in co-v-cultures. No correlation was found between hyphal growth and GAs production. Strains of the same species imposed totally different effects on hyphal growth or GAs production. This study has investigated the effects of mVOCs on G. lucidum for the first time. Moreover, it suggests that co-v-cultivation may be a promising biotechnological approach to improve the production in G. lucidum.

Structural and functional evaluation of recombinant histidine phosphokinase NisK and response regulator NisR: in silico and experimental approach.

In the two-component system of NisRK from Lactococcus lactis, the production of nisin is affected by transmembrane NisK and activation of intracellular NisR. The transcription of nisin structural genes can be induced by derivatives of nisin. NisR activation leads to the activation of nisA/Z transcription, which encodes the nisin maturation machinery, nisin regulation and activation of the nisFEG operon to confer immunity. The aim of this study was to express the Lactococcus lactis histidine phosphokinase NisK and response regulator NisR in E. coli, and to perform activity assays and in silico analysis. In silico methods were applied to study the properties and structures of the NisK and NisR proteins, including prediction of physicochemical characteristics, secondary and tertiary structure, stability and ligand-receptor interactions.pET32a and pET28a vectors containing synthetic nisK and nisR genes were transformed into E. coli followed by IPTG induction. SDS-PAGE and western blotting methods were applied to confirm the presence and identity of the amplified proteins. Following purification, the proteins were dialyzed and then prepared for activity assay. The CAI index showed that the genes was compatible with the E. coli host and that the proteins have effective expression. Also, the mRNA prediction results suggest that there is enough mRNA stability for efficient translation in the new host. NisK and NisR recombinant proteins were expressed in E. coli with half - lives of around 10 h and were confirmed with molecular weights of 27 kDa and 69 kDa, respectively, by SDS-PAGE and western blotting. The secondary structure of the recombinant proteins as predicted by circular dichroism spectroscopy was similar to the in silico protein structures. Activity assay of recombinant NisK was performed by measuring the amount of consumed ATP according to the light produced by luciferase. Because NisK and NisR have a direct impact on each other, they have an essential role in increasing the production of nisin and they can be used in different research fields. Our results demonstrated that recombinant proteins NisK and NisR preserved their structure and function after expression.

Implementing Electric Potential Difference as a New Practical Parameter for Rapid and Specific Measurement of Minimum Inhibitory Concentration of Antibiotics.

New methods to determine antimicrobial susceptibility of bacterial pathogens especially the minimum inhibitory concentration (MIC) of antibiotics have great importance in pharmaceutical industry and treatment procedures. In the present study, the MIC of several antibiotics was determined against some pathogenic bacteria using macrodilution test. In order to accelerate and increase the efficiency of culture-based method to determine antimicrobial susceptibility, the possible relationship between the changes in some physico-chemical parameters including conductivity, electrical potential difference (EPD), pH and total number of test strains was investigated during the logarithmic phase of bacterial growth in presence of antibiotics. The correlation between changes in these physico-chemical parameters and growth of bacteria was statistically evaluated using linear and non-linear regression models. Finally, the calculated MIC values in new proposed method were compared with the MIC derived from macrodilution test. The results represent significant association between the changes in EPD and pH values and growth of the tested bacteria during the exponential phase of bacterial growth. It has been assumed that the proliferation of bacteria can cause the significant changes in EPD values. The MIC values in both conventional and new method were consistent to each other. In conclusion, cost and time effective antimicrobial susceptibility test can be developed based on monitoring the changes in EPD values. The new proposed strategy also can be used in high throughput screening of biocompounds for their antimicrobial activity in a relatively shorter time (6-8 h) in comparison with the conventional methods.

Biosurfactant production by Mucor circinelloides on waste frying oil and possible uses in crude oil remediation.

Biosurfactants are biocompatible surface active agents which many microorganisms produce. This study investigated the production of biosurfactants by Mucor circinelloides. The effects of different factors on biosurfactant production, including carbon sources and concentrations, nitrogen sources, and iron (II) concentration, were studied and the optimum condition determined. Finally, the strain's ability to remove the crude oil and its relationship with biosurfactant production was evaluated. The results showed that M. circinelloides could reduce the surface tension of the culture medium to 26.6 mN/m and create a clear zone of 12.9 cm diameter in an oil-spreading test. The maximum surface tension reduction was recorded 3 days after incubation. The optimum condition for biosurfactant production was achieved in the presence of 8% waste frying oil as a carbon source, 2 g/L yeast extract as a nitrogen source, and 0.01 mM FeSO4. M. circinelloides could consume 8% waste frying oil in 5 days of incubation, and 87.6% crude oil in 12 days of incubation. A direct correlation was observed between oil degradation and surface tension reduction in the first 3 days of fungal growth. The results showed that the waste frying oil could be recommended as an inexpensive oily waste substance for biosurfactant production, and M. circinelloides could have the potential to treat waste frying oil. According to the results, the produced crude biosurfactant or fungal strain could be directly used for the mycoremediation of crude oil contamination in oil fields.

Assessing the biodegradation of polycyclic aromatic hydrocarbons and laccase production by new fungus Trematophoma sp. UTMC 5003.

Environmental pollution caused by petroleum compounds has become a global concern. The aim of this study was to evaluate the indigenous fungal isolates in Iran for biodegradation of crude oil pollutants. In order to isolate fungal strains, the soil samples were enriched in minimal salts medium (MSM) with 1% crude oil and then the crude oil degradation was measured by total petroleum hydrocarbon (TPH) assay. The degradation of hydrocarbons compounds was also analysed by FT-IR and HPLC, and the activity of peroxidase enzyme and biosurfactant production were also measured. We isolated 40 fungal strains and selected the isolate G-05 with 70% degradation ability of petroleum hydrocarbons as a premium isolate after 15 days. Residual crude oil analysis with FT-IR spectrophotometry and HPLC showed that G-05 is able to degrade 90 and 100% of aliphatic compounds and some polycyclic aromatic hydrocarbons (PAH), respectively. Evaluation of enzymatic activity showed that this isolate can produce 4 U L-1 of Laccase enzyme for oil removal; it is capable of producing biosurfactant and reducing the surface tension of the medium to 25.95 ± 0.1 m Nm-1. This strain was identified as a member of Trematophoma genus and the obtained results showed that this strain is a highly potent strain in bioremediation of soils contaminated by crude oil.

Saccharothrix ecbatanensis sp. nov., an actinobacterium isolated from soil.

A novel actinomycete, designated HM 537T, was isolated from soil in Hamedan Province, Iran. Cell-wall hydrolysates of strain HM 537T contained meso-diaminopimelic acid, and whole-cell hydrolysates contained ribose, glucose, galactose, rhamnose and traces of mannose. The main phospholipids were diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol and an unknown phospholipid. MK-9(H4), an unknown MK and MK-10(H4) were the predominant menaquinones. The major fatty acids included iso-C16 : 0, iso-C15 : 0, iso-C16 : 1 G and 9(?)-methyl C16 : 0. Strain HM 537T had the highest 16S rRNA gene sequence similarity to Saccharothrix hoggarensis DSM 45457T (99.5 %) and Saccharothrix saharensis DSM 45456T (99.0 %). DNA-DNA hybridization studies showed relatedness values of 13.8 ± 3.3 % with S. hoggarensis DSM 45457T and 16.3 ± 3.5 % with S. saharensis DSM 45456T. Based on the results of phenotypic and genotypic studies, strain HM 537T represents a novel species of the genus Saccharothrix, for which the name Saccharothrix ecbatanensis sp. nov. is proposed. The type strain is HM 537T ( = DSM 45486T = UTMC 00537T = CCUG 63021T).

Biotechnological application and taxonomical distribution of plant growth promoting actinobacteria.

Plant growth promoting (PGP) bacteria are involved in various interactions known to affect plant fitness and soil quality, thereby increasing the productivity of agriculture and stability of soil. Although the potential of actinobacteria in antibiotic production is well-investigated, their capacity to enhance plant growth is not fully surveyed. Due to the following justifications, PGP actinobacteria (PGPA) can be considered as a more promising taxonomical group of PGP bacteria: (1) high numbers of actinobacteria per gram of soil and their filamentous nature, (2) genome dedicated to the secondary metabolite production (~5 to 10 %) is distinctively more than that of other bacteria and (3) number of plant growth promoter genera reported from actinobacteria is 1.3 times higher than that of other bacteria. Mechanisms by which PGPA contribute to the plant growth by association are: (a) enhancing nutrients availability, (b) regulation of plant metabolism, (c) decreasing environmental stress, (d) control of phytopathogens and (e) improvement of soil texture. Taxonomical and chemical diversity of PGPA and their biotechnological application along with their associated challenges are summarized in this paper.

Streptomyces zagrosensis sp. nov., isolated from soil.

The taxonomic position of a novel actinomycete isolated from soil in Fars Province (Iran) was determined using a polyphasic approach. Phenotypic characterization and 16S rRNA gene sequence analysis of the isolate matched those described for members of the genus Streptomyces. On ISP2 medium, strain HM 1154(T) produced a dark cream, branched substrate mycelium and Retinaculiaperti aerial hyphae that in some images also appeared spiral and that developed into greyish-white spore chains with a smooth surface. The isolate showed optimal growth at 28 °C and pH 6-9 with 0-4% (w/v) NaCl. Whole-cell hydrolysates contained ll-diaminopimelic acid as diagnostic diamino acid, ribose and glucose. The main phospholipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, three unknown phospholipids and an unknown aminophospholipid; MK-9(H4) and MK-9(H2) were the predominant menaquinones. The major cellular fatty acids were the branched saturated iso-C16:0 and anteiso-C15:0. Strain HM 1154(T) exhibited the highest 16S rRNA gene sequence similarities to Streptomyces coerulescens DSM 40146(T) (99.4%), Streptomyces varsoviensis DSM 40346(T) (99.3%), Streptomyces youssoufiensis DSM 41920(T) (99.2%), Streptomyces abikoensis DSM 40831(T) (99.2%), Streptomyces rimosus subsp. rimosus DSM 40260(T) (99.1%), Streptomyces luteireticuli DSM 40509(T) (99.1%), Streptomyces thioluteus DSM 40027(T) (99.1%), Streptomyces blastmyceticus DSM 40029(T) (99.0%) and Streptomyces hiroshimensis DSM 40037(T) (99.0%). DNA-DNA hybridization studies showed relatedness values of 11.0-35.8% with the closest related species. Based on these results, strain HM 1154(T) is considered to represent a novel species within the genus Streptomyces, for which the name Streptomyces zagrosensis sp. nov. is proposed. The type strain is HM 1154(T) ( = DSM 42018(T) = UTMC 1154(T) = CECT 8305(T)).

Promicromonospora iranensis sp. nov., an actinobacterium isolated from rhizospheric soil.

A novel strain of the genus Promicromonospora, designated HM 792(T), was isolated from soil in Fars Province, Iran. On ISP 2 medium, the yellow-pigmented isolate produced long and branched hyphae that developed into a large number of irregularly shaped spores. It showed growth at 25-30 °C and pH 6.0-9.0 with 0-8 % (w/v) NaCl. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Promicromonospora. Whole-cell hydrolysates of strain HM 792(T) contained the amino acids d-glutamic acid, l-alanine and l-lysine along with the sugars glucose and ribose. The main polar lipids were diphosphatidylglycerol, two unknown phospholipids, two unknown glycolipids and two unknown phosphoglycolipids, complemented by minor concentrations of phosphatidylinositol and phosphatidylglycerol. MK-9(H4) was the predominant menaquinone. The fatty-acid pattern was composed mainly of the saturated branched-chain acids anteiso-C15 : 0 and iso-C15 : 0. 16S rRNA gene sequence analysis showed the highest pairwise sequence identity (96.6-99.0 %) with the members of the genus Promicromonospora. Based on phenotypic and genotypic features, strain HM 792(T) is considered to represent a novel species of the genus Promicromonospora, for which the name Promicromonospora iranensis sp. nov. is proposed. Strain HM 792(T) ( = DSM 45554(T) = UTMC00792(T) = CCUG 63022(T)) is the type strain.

Insights into additional lactone-based signaling circuits in Streptomyces: existence of acyl-homoserine lactones and LuxI/LuxR homologs in six Streptomyces species.

Acyl-homoserine lactones (AHLs), mediating pivotal physiological activities through quorum sensing (QS), have conventionally been considered limited to Gram-negative bacteria. However, few reports on the existence of AHLs in Gram-positive bacteria have questioned this conception. Streptomyces, as Gram-positive bacteria already utilizing a lactone-based QS molecule (i.e., gamma-butyrolactones), are yet to be explored for producing AHLs, considering their metabolic capacity and physiological distinction. In this regard, our study examined the potential production of AHLs within Streptomyces by deploying HPLC-MS/MS methods, which resulted in the discovery of multiple AHL productions by S. griseus, S. lavendulae FRI-5, S. clavuligerus, S. nodosus, S. lividans, and S. coelicolor A3(2). Each of these Streptomyces species possesses a combination of AHLs of different size ranges, possibly due to their distinct properties and regulatory roles. In light of additional lactone molecules, we further confirm that AHL- and GBL-synthases (i.e., LuxI and AfsA enzyme families, respectively) and their receptors (i.e., LuxR and ArpA) are evolutionarily distinct. To this end, we searched for the components of the AHL signaling circuit, i.e., AHL synthases and receptors, in the Streptomyces genus, and we have identified multiple potential LuxI and LuxR homologs in all 2,336 Streptomyces species included in this study. The 6 Streptomyces of interest in this study also had at least 4 LuxI homologs and 97 LuxR homologs. In conclusion, AHLs and associated gene regulatory systems could be more widespread within the prokaryotic realm than previously believed, potentially contributing to the control of secondary metabolites (e.g., antibiotics) and their complex life cycle, which leads to substantial industrial and clinical applications.

Kribbella shirazensis sp. nov., isolated from Iranian soil.

The taxonomic position of a strain isolated from soil in Shiraz, Fars province, Iran, was investigated. Strain UTMC 693(T) produced an extensively branched substrate mycelium and aerial hyphae consisting of hyphae that fragment into short to elongated rod-like elements. The chemotaxonomic characteristics of the isolate matched those described for the genus Kribbella. Strain UTMC 693(T) showed the highest 16S rRNA gene sequence similarity to Kribbella karoonensis DSM 17344(T) (98.3%), K. swartbergensis DSM 17345(T) (98.2%), K. hippodromi S1.4(T) (98.0%), K. aluminosa HKI 0478(T) (98.0%) and K. jejuensis HD9(T) (98.0%). DNA-DNA hybridization studies with closely related type strains showed 56.3% relatedness to K. karoonensis, 21.3% to K. swartbergensis, 39.0% to K. jejuensis and 42.0% to K. aluminosa. Thus, strain UTMC 693(T) can be considered to represent a novel Kribbella species. Strain UTMC 693(T) showed the typical morphology found among members of Kribbella, but can be differentiated easily from closely related species by genotypic characteristics, chemotaxonomic results and other phenotypic markers. Based on these results, strain UTMC 693(T) ( =DSM 45490(T) =CCUG 61792(T)) is considered the type strain of a novel species of the genus Kribbella, for which the name Kribbella shirazensis sp. nov. is proposed.

Systematic and biotechnological aspects of halophilic and halotolerant actinomycetes.

More than 70 species of halotolerant and halophilic actinomycetes belonging to at least 24 genera have been validly described. Halophilic actinomycetes are a less explored source of actinomycetes for discovery of novel bioactive secondary metabolites. Degradation of aliphatic and aromatic organic compounds, detoxification of pollutants, production of new enzymes and other metabolites such as antibiotics, compatible solutes and polymers are other potential industrial applications of halophilic and halotolerant actinomycetes. Especially new bioactive secondary metabolites that are derived from only a small fraction of the investigated halophilic actinomycetes, mainly from marine habitats, have revealed the huge capacity of this physiological group in production of new bioactive chemical entities. Combined high metabolic capacities of actinomycetes and unique features related to extremophilic nature of the halophilic actinomycetes have conferred on them an influential role for future biotechnological applications.

Isolation and screening of Thermoactinomycetaceae family members as an extremophilic poor investigated and promising natural source of antimicrobial substances.

Background and Objectives: Recent evidences have shown that methicillin-resistant Staphylococcus aureus (MRSA) can cause severe infections and is resistant to almost all commercially available antibiotics. Therefore, screening unknown sources of biological compounds such as the Thermoactinomycetaceae family as extremophilic bacteria may be helpful to find new antimicrobial agents. Materials and Methods: Various samples were collected from different ecosystems, including desert, volcano, compost, and forest. They were cultured on Soil extract agar and Water agar. The antimicrobial activity of the isolates was evaluated using agar overlay and well diffusion methods. Members of the Thermoactinomycetaceae family were selected for further study: Their ability to grow at different temperatures, NaCl concentrations, and pH values, enzyme production ability, antimicrobial secondary screening, fractionation of their supernatants and so on. Results: According to molecular identification of active isolates against MRSA, three strains, including Laceyella sacchari UTMC 2705, Thermoactinomyces sp. UTMC 2721, and Laceyella sp. UTMC 2731, belonged to Thermoactinomycetaceae were identified. The minimum inhibitory concentrations of their extracts were tested against some pathogenic bacteria, showing their antimicrobial activity with a broad spectrum. The results of TLC bioautography of the extracts showed that the most active fractions were semi-polar. Also, the results of HPLC analysis showed the existence of several UV-active compounds in their extracts. Conclusion: The present study highlighted the importance and potential of Thermoactinomycetaceae members as a less-known source of antibiotics against pathogenic bacteria.

Persipeptides A and B, two cyclic peptides from Streptomyces sp. UTMC 1154.

Two new N-methylated cyclopeptides, persipeptide A (1) and B (2), have been isolated from Streptomyces sp. UTMC1154. Their structures were established using 1D and 2D NMR experiments. 2D TOCSY experiments were applied to identify the amino acid residues, while HMBC correlations were used to determine their sequence. According to Marfey's method, all amino acids had the l-configuration. The two cyclic peptides had the same ring size and amino acid composition, but differed in their sequence; they did not show activity against the tested bacteria, fungi and algae. Molecular identification experiments placed the strain in the genus Streptomyces closely related to Streptomyces coerulescens DSM40146(T) (99.45%) and Streptomyces varsoviensis DSM40346(T) (99.25%).

Prediction of the pho regulon in Streptomyces clavuligerus DSM 738.

Negative control of clavulanic acid by phosphate in Streptomyces clavuligerus DSM 738 suggests that a pho regulon may exist in this bacterium. S. clavuligerus PhoP was expressed with a C-terminal His-tag in Escherichia coli and purified. Binding of PhoP-His 6 to promoter fragments of phoRP/phoU and pstS was demonstrated in gel retardation experiments. These fragments contained direct 11 bp repeats resembling PHO boxes. The tentative consensus sequence, GKTCRHBBNSV, was used to search other potential PhoP target genes in the genomic sequence of S. clavuligerus. In total, the putative PHO binding sequence was found in promoter regions of 31 S. clavuligerus genes. Binding of PhoP- His 6 to the PHO box present in the promoter region of the phosphate transporter gene SSCG_07547 of S. clavuligerus was demonstrated. Furthermore, it was shown by real time PCR that decreased concentrations of phosphate do affect increased expressions of genes to which PhoP binds. These findings confirm that a pho regulon exists in S. clavuligerus.

Identification and determination of extracellular phytate-degrading activity in actinomycetes.

In this study 97 soil samples from different soil ecosystems were collected. The initial screening was performed on modified glycerol arginine agar (MGAA) to isolate common actinomycetes and on modified MGA-SE (MMGA-SE) to isolate rare actinomycetes. Sixty-seven isolates potentially producing extracellular phytate-degrading activity were identified. The potential to dephosphorylate phytate was confirmed in liquid culture for 46.3 % of the isolates. 12 strains were selected for a direct determination of their phytate-degrading capacity. The results highlighted that the selected isolates produced extracellular phytate-degrading activity; however their capacity in InsP(6) degradation was different. In addition the fermentation medium had an effect on the extent of phytate degradation. Some enzymatic properties of the phytases from isolate No. 43 and isolate No. 63 were determined after obtaining phytase-enriched samples. The enzymes had maximum phytate-degrading capability at 55 °C and pH 5 (isolate No. 43) and 37 °C and pH 7 (isolates No. 63), respectively. Due to their properties, the phytase of isolate No. 43 behaves like a histidine acid phytase, whereas the phytase of No. 63 showed similar enzymatic properties to the phytase of lily. To our knowledge, the results from this study demonstrated for the first time that actinomycetes produce extracellular phytate-degrading activity. By 16SrRNA sequencing, the more closely studied phytase producers were identified as Streptomyces sp. Isolate No. 43 showed 98 % identity to Streptomyces alboniger and S. venezuelae, while isolate No. 63 exhibited 98 % sequence identity to S. ambofaciens and S. lienomycini.