Greek indigenous streptomycetes as biocontrol agents against the soil-borne fungal plant pathogen Rhizoctonia solani.

AIMS: To examine the biocontrol potential of multiactive Greek indigenous Streptomyces isolates carrying antifungal activity against Rhizoctonia solani that causes damping-off symptoms on beans. METHODS AND RESULTS: A total of 605 Streptomyces isolates originated from 12 diverse Greek habitats were screened for antifungal activity against R. solani DSM843. Almost one-third of the isolates proved to be antagonistic against the fungus. From the above isolates, six were selected due to their higher antifungal activity, identified by analysing their 16S rRNA gene sequence and studied further. The obtained data showed the following: firstly, the isolates ACTA1383 and ACTA1557 exhibited the highest antagonistic activity, and therefore, they were selected for in vivo experiments using bean seeds as target; secondly, in solid and liquid culture experiments under optimum antagonistic conditions, the medium extracts from the isolates OL80, ACTA1523, ACTA1551 and ACTA1522 suppressed the growth of the fungal mycelium, while extracts from ACTA 1383 and ACTA1557 did not show any activity. CONCLUSIONS: These results corresponded important indications for the utility of two Greek indigenous Streptomyces isolates (ACTA1557 and ACTA1383) for the protection of the bean crops from R. solani damping-off symptoms, while four of them (isolates OL80, ACTA1523, ACTA1551 and ACTA1522) seem to be promising producers of antifungal metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on the biocontrol of R. solani using multiactive Streptomyces isolates originated from ecophysiologically special Greek habitats. Our study provides basic information to further explore managing strategies to control this critical disease.

Protein increase and lysine production by a Paecilomyces variotii strain grown on two-phase olive mill waste.

Two-phase olive-mill waste, the so-called "ecological", has been treated with a Paecilomyces variotii isolate in solid state fermentation experiments. The growth of the microorganism was estimated by measuring the production of carbon dioxide, using gas chromatography. A 46% increase of the protein content was achieved at the fermented product, after molasses addition at the initial mixture. The amino acid profile of the produced protein, as far as the essential amino acids are concerned, was significantly improved, resulting in a product that has the potential to be used as animal feed. Furthermore, it contains lysine, one of the essential amino acids that did not exist at the original product and is produced during fermentation. This is the first report on solid state fermentation of the two-phase olive mill waste (TPOMW) as a substrate, using a Paecilomyces variotii strain.

Xanthan production by Xanthomonas campestris using whey permeate medium.

Xanthan gum is a polysaccharide that is widely used as stabilizer and thickener with many industrial applications in food industry. Our aim was to estimate the ability of Xanthomonas campestris ATCC 13951 for the production of xanthan gum by using whey as a growth medium, a by-product of dairy industry. X. campestris ATCC 13951 has been studied in batch cultures using a complex medium for the determination of the optimal concentration of glucose, galactose and lactose. In addition, whey was used under various treatment procedures (de-proteinated, partially hydrolyzed by ß-lactamase and partially hydrolyzed and de-proteinated) as culture medium, to study the production of xanthan in a 2 l bioreactor with constant stirring and aeration. A production of 28 g/l was obtained when partially hydrolysed ß-lactamase was used, which proved to be one of the highest xanthan gum production reported so far. At the same time, an effort has been made for the control and selection of the most appropriate procedure for the preservation of the strain and its use as inoculant in batch cultures, without loss of its viability and its capability of xanthan gum production. The pre-treatment of whey (whey permeate medium hydrolyzed, WPH) was very important for the production of xanthan by the strain X. campestris ATCC 13951 during batch culture conditions in a 2 l bioreactor. Preservation methods such as lyophilization, cryopreservation at various glycerol solution and temperatures have been examined. The results indicated that the best preservation method for the producing strain X. campestris ATCC 13951 was the lyophilization. Taking into account that whey permeate is a low cost by-product of the dairy industry, the production of xanthan achieved under the studied conditions was considered very promising for industrial application.

Identification of OXA-23-producing Acinetobacter baumannii in Greece, 2010 to 2011.

We report on the sequence type and beta-lactamase content of 174 carbapenem-resistant Acinetobacter baumannii isolates recovered from clinical specimens during 2010 and 2011 in a tertiary care hospital in central Greece. Carbapenem resistance was associated mainly with carriage of the bla(OXA-23) gene (in 72.4% of the isolates). To our knowledge, this is the first description of A. baumannii strains producing OXA-23 in Greece. During 2011, in our hospital they rapidly 'replaced' the previously predominant OXA-58- positive A. baumannii strains.

Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus.

AIM: To investigate the applicability of rpoB gene, which encodes the beta subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP- and BOX-polymerase chain reaction) were also used. METHODS AND RESULTS: rpoB DNA (458 bp) were amplified from 21 Geobacillus- and Bacillus type strains, producing different BOX- and REP-PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90-100% (16S rRNA) and 74-100% (rpoB) similarity among examined bacteria. CONCLUSION: BOX- and REP-PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.

Identification of yeast strains isolated from a two-phase decanter system olive oil waste and investigation of their ability for its fermentation.

A dynamic fed-batch microcosm system is described which permits assessment of the progressive growth of yeasts through olive oil waste. We report on its application to measure the effects of the growth of yeast strains upon the chemical composition of "alpeorujo", the waste of a two-phase decanter system used for the extraction of olive oil. Six phenotypically distinct groups of yeasts were isolated. Three selected isolates were identified as being most closely related to Saccharomyces sp., Candida boidinii and Geotrichum candidum using biochemical tests and partial 18S rDNA gene sequence analysis. This is the first report of yeast growth on "alpeorujo" by the use of a fed-batch microcosm system, resulting in the change of the initial chemical composition of "alpeorujo" and in the decrease of the toxic substances such as phenols.

Chromium recycling of tannery waste through microbial fermentation.

An Aspergillus carbonarius isolate, selected from an established microbial culture collection, was used to study the biodegradation of chromium shavings in solid-state fermentation experiments. Approximately 97% liquefaction of the tannery waste was achieved and the liquid obtained from long-term experiments was used to recover chromium. The resulting alkaline chromium sulfate solution was useful in tanning procedures. A proteinaceous liquid was also obtained which has potential applications as a fertilizer or animal feed additive and has several other industrial uses. The A. carbonarius strain proved to be a very useful tool in tannery waste-treatment processes and chromium recovery in the tanning industries.

Gentamicin resistance genes in environmental bacteria: prevalence and transfer.

A comprehensive multiphasic survey of the prevalence and transfer of gentamicin resistance (Gm(r)) genes in different non-clinical environments has been performed. We were interested to find out whether Gm(r) genes described from clinical isolates can be detected in different environmental habitats and whether hot spots can be identified. Furthermore, this study aimed to evaluate the impact of selective pressure on the abundance and mobility of resistance genes. The study included samples from soils, rhizospheres, piggery manure, faeces from cattle, laying and broiler chickens, municipal and hospital sewage water, and coastal water. Six clusters of genes coding for Gm-modifying enzymes (aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6')-II/Ib, ant(2'')-I, aph(2'')-I) were identified based on a database comparison and primer systems for each gene cluster were developed. Gm-resistant bacteria isolated from the different environments had a different taxonomic composition. In only 34 of 207 isolates, mainly originating from sewage, faeces and coastal water polluted with wastewater, were known Gm(r) genes corresponding to five of the six clusters detected. The strains belonged to genera in which the genes had previously been detected (Enterobacteriaceae, Pseudomonas, Acinetobacter) but also to phylogenetically distant bacteria, such as members of the CFB group, alpha- and beta-Proteobacteria. Gm(r) genes located on mobile genetic elements (MGE) could be captured in exogenous isolations into recipients belonging to alpha-, beta- and gamma-Proteobacteria from all environments except for soil. A high proportion of the MGE, conferring Gm resistance isolated from sewage, were identified as IncPbeta plasmids. Molecular detection of Gm(r) genes, and broad host range plasmid-specific sequences (IncP-1, IncN, IncW and IncQ) in environmental DNA indicated a habitat-specific dissemination. A high abundance and diversity of Gm(r) genes could be shown for samples from faeces (broilers, layers, cattle), from sewage, from seawater, collected close to a wastewater outflow, and from piggery manure. In the latter samples all six clusters of Gm(r) genes could be detected. The different kinds of selective pressure studied here seemed to enhance the abundance of MGE, while an effect on Gm(r) genes was not obvious.

Enzymes of the Entner-Doudoroff and pyruvate decarboxylation pathways in Zymomonas mobilis wild-type CP4 and mutant strains grown in continuous culture.

The osmotolerant Zymomonas mobilis strain suc40, (containing plasmid pDS3154-inaZ), which is capable of producing simultaneously ethanol and ice nuclei protein, was cultivated in a chemically defined complete sucrose medium, as well as in a sugar beet molasses medium in continuous culture. The strain exhibited the normal Monod's relationship between biomass and dilution rate, and between growth substrate concentration and dilution rate. Specific activities of a number of enzymes that appear to control important steps in the metabolic flux of the Entner-Doudoroff and pyruvate decarboxylation pathways were investigated over a range of growth rates in steady state cultures. With the exception of glucose-6-phosphate dehydrogenase and gluconate kinase, all of the enzymes exhibited a very similar pattern for the wild type Z. mobilis CP4 and for the osmotolerant mutants, independent of the media used; the enzyme patterns remained relatively constant over the studied growth range. The specific activity of glucose-6-phosphate dehydrogenase was increased 2-fold by decreasing dilution rate in sugar beet molasses. The specific activity of gluconate kinase was 2-fold lower at medium growth rates compared with that at either low or high growth rates. Pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase activities were significantly higher compared with those of the enzymes governing the early steps of the Entner-Doudoroff pathway. The present study, which was designed to determine the behaviour of important enzymes in sucrose metabolism of Z. mobilis suc40/pDS3154-inaZ grown in continuous culture showed that the microorganism required regulation of specific enzyme activities at the transcriptional level when sugar beet molasses were used as the growth medium.

Simultaneous ethanol and bacterial ice nuclei production from sugar beet molasses by a Zymomonas mobilis CP4 mutant expressing the inaZ gene of Pseudomonas syringae in continuous culture.

AIM: The aim of this work was to construct a Zymomonas mobilis mutant capable of simultaneous ethanol and ice nuclei production from agricultural by-product such as sugar beet molasses, in steady-state continuous culture. METHODS AND RESULTS: A sucrose-hypertolerant mutant of Z. mobilis strain CP4, named suc40, capable of growing on 40% (w/v) sucrose medium was isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment. Plasmid pDS3154 carrying the inaZ gene of Pseudomonas syringae was conjugally transferred and expressed in suc40. The potential for simultaneous ethanol and bacterial ice nuclei production was assessed in steady-state continuous cultures over a range of dilution rates from 0.04 to 0.13 h(-1). In addition, the fatty acid and phospholipid profile of the three strains was also investigated. Ethanol production up to 43 g l(-1) was achieved at dilution rates below 0.10 h(-1) in sugar beet molasses. Ice nucleation activity gradually increased with increasing dilution rate and the greatest activity, -3.4 log (ice nuclei per cell), was observed at the highest dilution rate (0.13 h(-1)). Both mutant strains displayed a different fatty acid and phospholipid profile compared with the wild-type strain. CONCLUSIONS: The ability of the mutant and recombinant plasmid-containing strains to grow on high sugar concentrations and in high osmotic pressure environments (molasses) can be attributed to their phospholipid and fatty acid contents. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking into account that sugar beet molasses is a low cost agricultural by-product, the simultaneous ethanol and bacterial ice nucleation production achieved under the studied conditions is considered very promising for industrial applications.

Determination of metabolic activity of streptomycetes in soil microcosms.

Two Streptomyces griseus strains were isolated from different soil types. S. griseus CAG17 strain was isolated from an agricultural area with low organic matter but rich in phosphorus content and S. griseus 26K strain was isolated from a forest area rich in organic matter with a low phosphorus content. The survival and metabolic activity of these isolates were studied in dynamic sterile soil microcosm systems. The fitness of each isolate was studied by re-inoculation in a soil type different from its origin. Maximum percentage of germination and respiration rates occurred within the first 48 h after each soil turnover (removal and addition of certain soil volumes). Data suggested that S. griseus CAG17 survived better independently of the soil type in comparison with S. griseus 26K which sporulated within the first 12 h after inoculation. Incubation temperatures did affect the lifecycles in relation to soil type. For example, the lowest temperature tested, 22 degrees C, was more favourable for extended germination and adaptation in general but revealed lesser spore numbers in the 'foreign' soil environment. Monitoring metabolic activity by estimation of urease, phosphatases and dehydrogenase-specific activities, between 18 and 35 degrees C incubation temperatures, was a reliable method for studying the survival and growth of streptomycete populations in soil. Results also confirmed that respiration rate and enzyme-specific activity corresponded with spore counts in long-term experiments which were designed for the investigation of survival and growth of S. griseus CAG17. Under selective pressure by heavy metals, in soil microcosm systems, metabolic activity proved a useful tool for the investigation of streptomycete activity. These methods could also be applied in agricultural field studies for monitoring microbial populations under conditions where various 'pollutants' are present in soil samples.

Amino acid residues N450 and Q449 are critical for the uptake capacity and specificity of UapA, a prototype of a nucleobase-ascorbate transporter family.

Specific carrier-mediated transport of purine and pyrimidine nucleobases across cell membranes is a basic biological process in both prokaryotes and eukaryotes. Recent in silico analysis has shown that the Aspergillus nidulans (UapA, UapC) and bacterial (PbuX, UraA, PyrP) nucleobase transporters, and a group of mammalian L-ascorbic acid transporters (SVCT1 and SVCT2), constitute a unique protein family which includes putative homologues from archea, bacteria, plants and metazoans. The construction and functional analysis of chimeric purine transporters (UapA-UapC) and UapA-specific missense mutations in A. nidulans has previously shown that the region including amino acid residues 378-446 in UapA is critical for purine recognition and transport. Here, we extend our studies on UapA structure-function relationships by studying missense mutations constructed within a 'signature' sequence motif [(F/Y/S)X(Q/E/P)NXGXXXXT(K/R/G)] which is conserved in the putative functional region of all members of the nucleobase/ascorbate transporter family. Residues Q449 and N450 were found to be critical for purine recognition and transport. The results suggest that these residues might directly or indirectly be involved in specific interactions with the purine ring. In particular, interaction of residue 449 with C-2 groups of purines might act as a critical molecular filter involved in the selection of transported substrates. The present and previous mutagenic analyses in UapA suggest that specific polar or charged amino acid residues on either side of an amphipathic alpha-helical transmembrane segment are critical for purine binding and transport.

Occurrence and diversity of plasmids in populations of streptomycetes in soil.

Studies were made of naturally occurring plasmids hosted in Streptomyces strains isolated from two different terrestrial ecosystems: an agricultural field and a protected forest area. Six out of the 147 screened isolates contained plasmids. The strains containing these plasmids were all isolated from the agricultural soil. Plasmids were not found among the strains isolated from the forest area. Cross hybridization of the six newly isolated plasmids revealed very high similarities between four of them. However, no similarities were found between the six newly isolated plasmids and well studied streptomycete plasmids such as pIJ101 and SCP2*. The host strains of the four similar plasmids belonged to three different species S. anulatus, S. rochei and S. diastaticus. This implies a possible conjugative transfer of these plasmids within the streptomycete population in the agricultural area. The reason for the absence of streptomycete plasmids from the populations derived from the forest area is discussed.

Survival, metabolic activity and conjugative interactions of indigenous and introduced streptomycete strains in soil microcosms.

The growth and activity of introduced (S. lividans TK24 pIJ673 and S. lividans TK23) and indigenous (S. griseus CAG17) streptomycete strains in soil was studied, under controlled conditions. The effect of environmental parameters such as temperature, soil water content and nutrient availability on the growth and activity of these strains, was studied using a highly dynamic fed-batch soil microcosm system. Using this new system, repeated cycles of active streptomycete growth were achieved, allowing long-term investigation of metabolic activity, plasmid stability and conjugative plasmid transfer. In long-term experiments, respiration rates and enzyme activity patterns matched the pattern of germination/sporulation cycles of the inoculants. In situ hybridisation, using fluorescently labelled oligonucleotides, also proved the presence of metabolically active streptomycete mycelia in sterile soil. Plasmid stability under varying temperatures and selective pressure was studied using the above system. In both sterile and non sterile amended antibiotic containing soil, no intraspecific transfer of plasmid pIJ673 from S. lividans TK24 to S. griseus CAG17 was detected. The soil microcosm system used, though, permitted detection of intraspecific conjugative transfer of this plasmid from S. lividans TK24 to S. lividans TK23 in soil.

Production and processing of a 59-kilodalton exochitinase during growth of Streptomyces lividans carrying pCHIO12 in soil microcosms amended with crab or fungal chitin.

Streptomyces lividans (pCHIO12), which carries the previously cloned Streptomyces olivaceoviridis exo-chiO1 gene on a multicopy vector, secretes a 59-kDa exochitinase, consisting of a catalytic domain (40 kDa), a central fibronectin type III-like module, and a chitin-binding domain (12 kDa). The propagation rate of S. lividans (pCHIO12) was higher in soil microcosms amended with fungal mycelia than in those containing crab chitin. Comparative biochemical and immunological studies allowed the following conclusions to be drawn. Within soil microcosm systems amended with crab shell chitin or chitin-containing Aspergillus proliferans mycelia, the strain expressed the clones exo-chiO1 gene and produced high quantities of a 59-kDa exochitinase. The enzyme was preferentially attached via its binding domain to the pellet from soil or liquid cultures. In contrast, truncated forms of 47, 40, and 25 kDa could be easily extracted from soil. The relative proportions of the 59-kDa enzyme and its truncated forms varied depending on the source of chitin and differed in soil and in liquid cultures.