23. " PCR-based detection of mobile genetic elements in total community DNA."
K. Smalla, E. Krogerrecklenfort, H. Heuer, W. Dejonghe, E. Top, M. Osborn, J. Niewint, C. Tebbe, M. Barr, M. Bailey, A. Greated, C. Thomas, S. Turner, P. Young, D. Nikolakopoulou, A.D. Karagouni, A. Wolters, J.D. van Elsas, K. Dronen, R. Sandaa, S. Borin, J. Brabhu, E. Grohnman and P. Sobecky.
Microbiology , vol. 46, pages 1256-1257, (2000).   View at publisher's site   Request copy
Abstract: No abstract available.

22. "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-flow cultures. ".
A.L. Savvides, A. Kallimanis, A. Varsaki, A.I. Koukkou, C. Drainas, M.A. Typas and A.D. Karagouni.
Journal of Applied Microbiology , vol. 89, pages 1002-1008, (2000).   View at publisher's site   Request copy
Abstract: 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- N0-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 pro®le 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.

21. "Amino acids N450 and Q449 are critical for the uptake capacity and specificity of UapA, a prototype of a nucleobase-ascorbate transporter family. "
C. Meintanis, A.D. Karagouni and G. Diallinas.
Molecular Membrane Biology, vol. 17, pages 47-57, (2000).   View at publisher's site   Request copy
Abstract: 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. .

20. "Occurrence and diversity of plasmids in population of streptomycetes in soil. "
T.G. Koraki and A.D. Karagouni.
AAntonie van Leeuwenhooek, vol. 78, pages 323-329, (2000).   View at publisher's site   Request copy
Abstract: 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.

19. "Determination of metabolic activity of streptomycetes in soil microcosms "
E.A. Katsifas, T.G. Koraki and A.D. Karagouni.
Journal of Applied Microbiology, vol. 89, pages 178-184, (2000).   View at publisher's site   Request copy
Abstract: 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.

18. "Diversity of streptomyces species among specific Greek terrestrial ecosystems. Partial characterization of the purified enzyme".
E.A. Katsifas, E.P. Gianoutsou and A.D. Karagouni.
Letters of Applied Microbiology, vol. 29, pages 48-51, (1999).   View at publisher's site   Request copy
Abstract: The diversity of streptomycetes isolated from different Greek terrestrial ecosystems using phenotypic identification, and the relationship between the number of species and the number of isolates as a diversity index, was studied. A total of 344 Streptomyces strains have been isolated and identified from diverse sites in the Greek territory, such as heavily disturbed agricultural areas and preserved forest areas, and from specific rhizosphere ecosystems. According to phenotypic identification, these strains belonged to 19 different cluster groups with a Willcox probability 0·8. Streptomyces cyaneus, Strep. albidoflavus, Strep. diastaticus and Strep. exfoliatus were the most common cluster groups isolated from at least six different habitats. On the other hand, there were cluster groups that appeared in only one or two habitats, such as Strep. griseoflavus, Strep. rimosus, Streptoverticillium blastmyceticum, Nocardia mediterranea and Strep. fulvissimus. The diversity indices among the different cluster groups of each sampling area indicated that the different habitats can be sub-divided into two main groups: rhizosphere habitats and non-rhizosphere habitats, showing that the rhizosphere is one of the most important factors which determines the population structure of a specific soil area.

17. "Selective effects of antibiotics on survival and gene transfer of streptomycetes in soil."
P.R. Herron, I.K. Toth, G.H.J. Heilig, A.D.L. Akkermans, A.D. Karagouni and E.M.H. Wellington E.M.H.
Soil Biology and Biochemistry, vol. 30, pages 673-677, (1998).   View at publisher's site   Request copy
Abstract: No abstract available.

16. "Survival, metabolic activity and conjugative interactions of indigenous and introduced streptomycete strains in soil microcosms."
A.P. Vionis,E.A. Katsifas and A.D. Karagouni.
Antonie Van Leeuwenhoek, vol. 73, pages 103-115, (1996).   View at publisher's site   Request copy
Abstract: 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.

15. "Monitoring chitinolytic activities of streptomycetes in soil amended with crab or fungal chitin."
A.P. Vionis, F. Niemeyer, A.D. Karagouni and H. Schrempf.
Applied and Environmental Microbiology, vol. 62, pages 1774-1780, (1996).   View at publisher's site   Request copy
Abstract: 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 cloned 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.