1 VOL. 49 [T. 2 LJUBLJANA 2006 ACTA BIOLOGICA SLOVENICA prej/formerly BIOLO[KI VESTNIK ISSN 1408-3671 izdajatelj/publisher UDK 57(497.4) Dru{tvo biologov Slovenije 2 Acta Biologica Slovenica, 49 (1), 2006 ACTA BIOLOGICA SLOVENICA LJUBLJANA 2006 Vol. 49, [t. 2: 1–36 Acta Biologica Slovenica Glasilo Društva biologov Slovenije – Journal of Biological Society of Slovenia Izdaja – Published by Društvo biologov Slovenije – Biological Society of Slovenia Glavni in odgovorni urednik – Editor in Chief Mihael Jožef Toman, e-mail: mihael.toman@bf.uni-lj.si Tehnični urednik – Managing Editor Branko Vreš, e-mail: branevr@zrc-sazu.si Uredniški odbor – Editorial Board Matija Gogala (SI), Nada Gogala (SI) Peter Maček (SI), Alenka Malej (SI), Andrej Martinčič (SI), Harald Niklfeld (A), Livio Poldini (I), Boris Sket (SI), Robert Zorec (SI), Mitja Zupančič (SI), Thomas F. J. Martin (USA), Mark Tester (UK), Gerhard Thiel (D) Naslov uredništva – Adress of Editorial Offi ce Acta Biologica Slovenica, Večna pot 111, SI-1001 Ljubljana, Slovenija http://bijh.zrc-sazu.si/abs/ Oblikovanje – Design Žare Vrezec ISSN 1408-3671 UDK 57(497.4) Natisnjeno – Printed on: 2007 Tisk – Print: Tiskarna Pleško d.o.o., Ljubljana Naklada: 500 izvodov Cena letnika (dve številki): 15 € za posameznike, 42 € za ustanove Številka poslovnega računa pri Ljubljanski banki: 02083-142508/30 Publikacijo je sofi nancirala Agencija za Raziskovalno dejavnost Republike Slovenije. Acta Biologica Slovenica je indeksirana v – is indexed in: Biological Abstracts, Zoological records. 3 Sprejeto (accepted): 2006-12-20 The RepFIIA replicon of the natural Escherichia coli plasmid pRK100 Replikon RepFIIA naravnega plazmida pRK100 bakterije Escherichia coli Marjanca STARČIČ ERJAVEC & Darja ŽGUR-BERTOK Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia, Fax: 00386 1 257 3390, E-mail: Marjanca.Starcic.Erjavec@bf.uni-lj.si, Darja.Zgur@bf.uni-lj.si. Abstract. The aim of the presented study was to identify the similarity of the plasmid pRK100 RepFIIA replicon (replication region) with similar replicons of other known plasmids of Enterobacteriaceae. For this purpose, within the determined nucleotide sequence of pRK100, the RepFIIA replicon rep genes/regions were identifi ed. The nucleotide sequences of the pRK100 determined rep genes/regions were subsequently compared with the nucleotide sequences of other RepFIIA replicon rep genes/regions deposited in GenBank. Further, the nucleotide divergence between them was calculated. The obtained results clearly demonstrated, that the individual pRK100 rep regions are the same/most similar to rep regions from different plasmids. RepA2 of pRK100 is most similar to repA2 of pCP301, pINV_F6_M1382, pWR501 and R1, copA is the same as copA of plasmids pC15-1a and R100, repA6 of pRK100 is the same as repA6 in plasmids pC15-1a, pCP301, pINV_F6_M1382, pWR501, R1 and R100, repA1 is most similar to repA1 of the plasmid p1658/79, and repA4 of pRK100 is most similar to repA4 of pC15-1a. Hence, the composition of the pRK100 RepFIIA replicon is mosaic and unique among the plasmids. Key words: plasmid, RepFIIA replicon, Enterobacteriaceae, nucleotide divergence Izvleček. Cilj raziskave je bil označiti podobnost replikona (replikacijske regije) RepFIIA z drugimi podobnimi replikoni znanih plazmidov enterobakterij. V ta namen smo v nukleotidnem zaporedju replikona RepFIIA plazmida pRK100 poiskali posamezne gene/regije rep in njihovo nukleotidno zaporedje primerjali z drugimi, deponiranimi, nukleotidnimi zaporedji RepFIIA ter izračunali nukleotidno divergenco. Dobljeni rezultati so jasno pokazali, da so različni geni/regije rep v replikonu RepFIIA plazmida pRK100 enaki/zelo podobni rep različnih plazmidov. RepA2 od pRK100 je najbolj podoben genu repA2 plazmidov pCP301, pINV_F6_M1382, pWR501 in R1, copA je enak genu copA na plazmidih pC15-1a in R100, regija repA6 plazmida pRK100 je enaka regiji repA6 plazmidov pC15-1a, pCP301, pINV_F6_M1382, pWR501, R1 in R100, gen repA1 je najbolj podoben genu repA1 plazmida p1658/79, in regija repA4 plazmida pRK100 je najbolj podobna regiji repA4 plazmida pC15-1a. Povzamemo lahko, da je replikon RepFIIA plazmida pRK100 sestavljen kot mozaik in da ga v takšni sestavi do sedaj še niso našli na no- benem drugem plazmidu. Ključne besede: plazmid, replikon RepFIIA, Enterobacteriaceae, nukleotidna divergenca ACTA BIOLOGICA SLOVENICA LJUBLJANA 2006 Vol. 49, [t. 2: 3–12 4 Acta Biologica Slovenica, 49 (1), 2006 Introduction Plasmids, extrachromosomal DNA elements, can be found in all three domains of the living world, in Archaea, Bacteria and Eukarya (HOLMES & al. 1995, SOLAR & al. 1998, ZILLIG & al. 1998). These elements encode a remarkable array of phenotypic traits of medical, agricultural, environmental and commercial importance (HELINSKI & al. 1996). Encoded traits include resistances to heavy metals, supplementary metabolic pathways and pathways for degradation of xenobiotics, as well as virulence factors and resistances to antibiotics (KADO 1998). Further, plasmids can have the machinery to transfer themselves and other parts of the genome into different species, genera, or sometimes even families (FIRTH & al. 1996). Plasmids can also incorporate and deliver genes by recombination or transposi- tion and by this means increase the genetic exchange in- and between bacterial populations (SOLAR & al. 1998). All plasmids harbour a replicon (replication region), which is needed for the stable propagation and maintenance in the host cell. Regardless of plasmid size, the replicon of a plasmid generally consists of a contiguous set of information that includes a defi nable origin, where DNA replication initiates (ori), a structural gene encoding the plasmid-specifi c protein required for the initiation of replication, and one or more adjoining controlling elements. All this information is often contained within a segment that is 3 kb or less in size (HELINSKI & al. 1996). The replicons are designated and grouped into families (COUTURIER & al. 1988). Replicons belonging to the RepFIIA family typically consist (Fig. 1) of repA2 encoding a repressor, the copA gene that encodes an antisense RNA molecule, a repA1 gene whose protein initiates plasmid replication by binding to the downstream ori, the repA6 region encoding a short leader peptide, and a repA4 region. The RepA2 repressor is assumed to regulate transcription of repA1 mRNA, while the antisense RNA CopA which is complementary to the leader region of repA1 mRNA (CopT), regulates translation. When CopA binds to CopT, repA6, which is necessary for RepA1 synthesis, is not expressed (BLOMBERG & al. 1992). The repA4 appears to be important for the stability of plasmid maintenance (JIANG & al. 1993). Further it is known, that the replicons of this family are mosaic (OSBORN & al. 2000), i.e. individual genes encoded in this replicon originate from different sources. pRK100 is an ~145-kb plasmid isolated from a uropathogenic Escherichia coli strain and it has been to a large extent characterised. It belongs to the IncF incompatibility group and encodes two antibiotic resistances, ampicillin and tetracycline, two colicins, ColV and ColIa, and the aerobactin (iuc) and enterochelin (iro) iron uptake system. Further it was demonstrated that pRK100 harbours two different replicons, a RepFIB and RepFIIA replicon (ŽGUR-BERTOK & al. 1990, AMBROŽIČ & al. 1998, STARČIČ ERJAVEC, 2003). In the presented study the genes/regions of the pRK100 RepFIIA rep- Fig. 1: Map of the RepFIIA replicon. Genes/regions of a typical RepFIIA replicon are depicted. To clearly show the individual studied sequences, some boxes representing genes/regions are offl ine. The direction of mRNA transcription is also marked. ori is the origin of replication, where the RepA1 protein binds and starts the replication. Slika 1: Mapa replikona RepFIIA. Označeni so geni/regije tipičnega replikona RepFIIA. Zaradi razvidnosti lege preučevanih zaporedij, so nekateri okvirčki, ki prikazujejo gene/regije, premaknjeni. Označena je tudi smer prepisa mRNA iz posameznega gena. ori je regija, kjer se veže replikatorski protein RepA1 in prične s podvajanjem plaz- mida. 5M. Starčevič Erjavec & al.: The RepFIIA replicon of the natural Escherichia coli plasmid pRK100 licon were analysed for their similarity with the RepFIIA replicon’s genes/regions of related plasmids p1658/79, pB171, pC15-1a, pCP301, pINV_F6_M1382, pO157, pTUC100, pWR501, R1 and R100. The nucleotide divergence between pRK100 and the other plasmids was determined. The results of our study show that the RepFIIA replicon of pRK100 is mosaic and unique in its composition. Method Sequence analysis for open reading frames (ORF) The determined RepFIIA replicon nucleotide sequence, 2159 bp in length, (GenBank accession number AY234375) was analyzed for open reading frames with the help of the program “ORF Finder” available on the web site http://www.ncbi.nlm.nih.gov/gorf/orfi g.cgi. Sequence analysis for DNA similarity The program Nucleotide-nucleotide BLAST (ALTSCHUL & al. 1997) available on the web site http://www.ncbi.nlm.nih.gov/BLAST/ was used to search for nucleotide sequences similar to the pRK100 RepFIIA nucleotide sequence. The DNA sequences of rep genes/regions were compiled and analysed using the CLUSTAL W (THOMPSON & al. 1994) program for sequence alignment. The program DNADIST in the PHYLIP package (FELSENSTEIN 1993, FELSENSTEIN 1989) was used for calculating distance matrixes. Results Genes/regions of the RepFIIA nucleotide sequence of pRK100 The pRK100 RepFIIA replicon has been deposited in GenBank under the Accession Number AY234377. In this deposited nucleotide sequence, the RepFIIA replicon is harboured from Nt 1401 to Nt 3559. To identify the rep genes/regions in the deposited nucleotide sequence of the pRK100 RepFIIA replicon, the internet program “ORF Finder” was used. To further defi ne the rep genes/regions, the pRK100 RepFIIA sequence was compared to other known rep genes/regions of similar replicons. The RepFIIA replicon was found to harbour repA2, copA, repA6, repA1, and repA4 sequences (Tab. 1). Table 1: Predicted genes/regions in the pRK100 RepFIIA replicon (AY234377). Tabela 1: Predvideni geni/regije replikona RepFIIA (AY234377) plazmida pRK100. Assumed gene/region Frame From (bp) To (bp) Length repA2 +3 1401 1661 261 copA +1 C 1874 1782 93 repA6 +2 1886 1960 75 repA1 +3 1953 2810 858 repA4 +2 3173 3556 384 A pRK100 RepFIIA-like replicon can be found on many other plasmids With the goal to fi nd plasmids with similar replicons, which could be compared with the pRK100 RepFIIA replicon, a BLAST search with the nucleotide sequence of pRK100’s RepFIIA was performed. The search revealed that many other plasmids carry similar replicons (Tab. 2). The most similar RepFIIA replicon was harboured by the Escherichia coli plasmid p1658/9. Most of the plasmids with similar replicon sequences were harboured on plasmids hosted by Escherichia coli or 6 Acta Biologica Slovenica, 49 (1), 2006 Shigella fl exneri however, some plasmids with less similar sequences were harboured also by other enteric bacterial species, as Klebsiella pneumoniae, Shigella sonnei and Salmonella Typhimurium. All plasmids exhibiting similarity with the pRK100 RepFIIA replicon belong to the broad RepFIIA family of replicons, those with higher similarity are members of the same Inc group – the IncFII, and plasmids with lower similarity belong to other Inc groups (IncFIA, IncFIC, IncFIV,..) Table 2: BLAST hits for the nucleotide sequence of the pRK100 RepFIIA replicon with a score higher than 200. Tabela 2: Zadetki z BLAST z rezultatom več kot 200 bitov za replikon RepFIIA plazmida pRK100. GenBank Acc. Number Plasmid Host BLAST score (bits) AF550679 p1658/9 Escherichia coli 3733 V00351 R1(RSC13) Escherichia coli 3527 AF177050 pWR100 Shigella fl exneri 3213 AL391753 pWR100 Shigella fl exneri 3166 AF348706 pWR501 Shigella fl exneri 3166 AF386526 pCP301 Shigella fl exneri 2a strain 301 3152 AY458016 pC15-1a Escherichia coli 3019 AP000342 R100 Shigella fl exneri 2b strain 222, Escherichia coli 2948 AY206448 pINV_F6_M1382 Shigella fl exneri 2843 AB011549 pO157 Escherichia coli 2773 AB024946 pB171 Escherichia coli 2759 AY091607 pTUC100 Escherichia coli 2746 M26937 pSU316 Escherichia coli 2627 V00318 R6-5 Escherichia coli 926 M13472 ColV2-K94 Escherichia coli 924 X55895 pSU212 Escherichia coli 759 M93064 pEI545 Klebsiella pneumoniae 702 AP001918 F Escherichia coli 420 M16167 P307 Escherichia coli 414 M28098 pSU221 Escherichia coli 394 M27528 R124 Escherichia coli 339 AP005147 R64 Salmonella typhimurium 311 AB021078 ColIb-P9 Shigella sonnei (E. coli) 303 K02675 pCG86 Escherichia coli 274 AP002527 R721 Escherichia coli 230 The pRK100 RepFIIA replicon is mosaic and unique in its composition In order to identify the similarity of the pRK100 RepFIIA rep genes/regions with other known rep genes/regions the computer programs CLUSTAL W and DNADIST were used. With these programs nucleotide sequences of rep genes/regions of pRK100 with rep sequences of other similar RepFIIA replicons, which were found with BLAST search and gave a BLAST score of more then 1000 bits, were compiled. For the compilation only those BLAST hits that had a complete RepFIIA replicon nucleotide sequence deposited were used, so that entire RepFIIA replicon could be compared. For the comparison of RepFIIA genes/regions harboured by different plasmids the original genes/regions, if denoted by the submitters of the sequence, were used, otherwise the genes/regions in the deposited sequence were searched for using BLAST (Tab. 3). All together the nucleotide sequences of RepFIIA replicons of 10 plasmids (p1658/79, pB171, pC15-1a, pCP301, pINV_F6_M1382, pO157, pTUC100, pWR501, R1 and R100) were compared with the nucleotide sequence of pRK100 RepFIIA replicon. The fi rst region analysed in the RepFIIA replicon is the repA2 gene, encoding a repressor protein, which by binding to the promoter represses the synthesis of repA1 mRNA (VANOOTEGHEM & CORNELIS 1990). The repA2 gene is in plasmids pRK100, p1658/79, pCP301, pINV_F6_M1382, pWR501, and R1 261 bp long, in plasmids R100, pO157, pC15-1a and pTUC100 it is 255 bp long and in the plasmid 7 Ta bl e 3: Pl as m id s an d th ei r re p se qu en ce s us ed in C L U ST A L W a nd D N A D IS T c om pu te r pr og ra m s. T he s tu di ed s eq ue nc es a re d en ot ed e ith er a cc or di ng to th e da ta in G en B an k or m ar ke d as B L A ST to s ho w th at th e se qu en ce w as s ea rc he d fo r by B L A ST . F or ea ch s tu di ed s eq ue nc e th e po si tio n on th e de po si te d se qu en ce a nd th e le ng th a re g iv en . Ta be la 3 : Pl az m id i i n nj ih ov a za po re dj a re p, k i s m o jih u po ra bi li v ra ču na ln iš ki h pr og ra m ih C L U ST A L W in D N A D IS T. Pr eu če va na z ap or ed ja s o oz na če na ta ko k ot s o po da na v p od at ko vn i b az i G en B an k oz ir om a oz na če na z B L A ST , v p ri m er u če s o bi la p oi sk an a z B L A ST . Z a vs ak o pr eu če va no z ap or ed je je n av ed en a nj eg ov a po zi ci ja n a de po ni ra ne m z ap or ed ju in n je go va d ol ži na . re pA 2 le ng th (b p) co pA le ng th (b p) re pA 6 le ng th (b p) re pA 1 le ng th (b p) re pA 4 le ng th (b p) p1 65 8/ 79 B L A ST : 37 46 9– 37 72 9 26 1 B L A ST : c 37 85 0– 37 94 3 94 re pA 6: 37 95 5– 38 02 9 75 re pA 1: 38 01 1– 38 87 9 86 9 re pA 4: 39 24 3– 39 44 9 20 7 pB 17 1 co pB : c 39 42 0– 39 66 8 24 9 B L A ST : c 39 18 5– 39 27 8 94 B L A ST : 39 09 9– 3 91 73 75 re pA 1: c 38 24 9– 39 10 6 85 8 B L A S T : c 37 50 0– 37 88 7 38 8 pC 15 –1 a re pA 2: 88 55 8– 88 81 2 25 5 B L A ST : c8 89 46 –8 90 38 93 re pA 6: 89 05 0– 89 12 4 75 re pA 1: 89 11 7– 89 97 4 85 8 re pA 4: 90 33 7– 90 72 3 38 7 pC P3 01 re pB : 20 88 97 –2 09 15 7 26 1 B L A ST : c 20 92 78 –2 09 36 9 92 ta pA : 20 93 81 –2 09 45 5 75 re pA : 20 94 36 –2 10 30 5 87 0 B L A S T : 21 06 68 –2 11 04 9 38 3 pI N V _F 6– _M 13 82 re pB : 83 1– 10 91 26 1 B L A ST : c 12 12 –1 30 3 92 ta p: 13 15 –1 38 9 75 re pA : 13 82 –2 23 9 85 8 B L A S T : 26 02 –2 98 8 38 7 pO 15 7 re pA 2: 71 99 9– 72 25 3 25 5 B L A S T : c 72 38 7– 72 47 7 91 B L A ST : 72 48 9– 72 56 3 75 re pA 1: 72 55 6– 73 41 6 85 8 B L A S T : 73 77 5– 74 15 8 38 4 pT U C 10 0 re pA 2: c5 97 6– 62 30 25 5 re pA 3: 57 44 –5 87 2 12 9 re pA 6: c 56 65 –5 73 9 75 re pA 1: c 48 15 –5 67 2 85 8 re pA 4: c 40 66 –4 45 2 38 7 pW R 50 1 re pA 2: 20 86 03 –2 08 86 3 26 1 S0 29 4 (i nc –R N A ): c 2 08 98 0– 20 90 69 90 re pA 6: 20 90 87 –2 09 16 1 75 re pA 1: 20 91 42 –2 10 01 1 87 0 B L A S T : 21 03 74 –2 10 75 6 38 2 R 1 re pA 2: 44 2– 70 2 26 1 B L A ST : c 82 3– 91 5 93 B L A ST : 92 7– 10 01 75 re pA 1: 99 4– 18 51 85 8 re pA 4: 22 14 –2 60 0 38 7 R 10 0 re pA 2: 88 25 3– 88 50 7 25 5 in c: c 88 64 1– 88 73 3 93 re pA 6: 88 74 5– 88 81 9 75 re pA 1: 88 81 2– 89 66 9 85 8 re pA 4: 90 03 2– 90 41 8 38 7 M. Starčevič Erjavec & al.: The RepFIIA replicon of the natural Escherichia coli plasmid pRK100 8 Acta Biologica Slovenica, 49 (1), 2006 pB171 it is only 249 bp long. The sequence analysis showed, that pRK100 repA2 is most similar to repA2 of pCP301, pINV_F6_M1382, pWR501 and R1, since the phylogenetic distance between them is the smallest, namely 0,0038 (Fig. 2A). The second region analysed in the RepFIIA replicon is the copA gene, encoding the antisense RNA regulating the translation of repA1 mRNA (VANOOTEGHEM & CORNELIS 1990). The copA gene is in most of the compared plasmids approximately 90 bp long. The only exception is the copA gene of the plasmid pTUC100, which is 129 bp long. The sequence analysis showed, that there is no nucleotide divergence between pRK100 copA and the copA genes of R100 and pC15-1a (Fig. 2B), and hence the pRK100 repA gene is identical to the appropriate genes in R100 and pC15-1a. The third region analysed in the RepFIIA replicon is the repA6. repA6 encodes a short leader peptide, whose expression is inhibited by CopA binding, preventing translation of RepA and conse- quently preventing plasmid replication. (BLOOMBERG & al. 1992). The repA6 is only 75 nucleotides long and it has the same size in all compared plasmids. The compared nucleotide sequences of pRK100 repA6 was completely identical (no nucleotide divergence) to repA6 of plasmids pC15-1a, pCP301, pINV_F6_M1382, pWR501, R1 and R100 (Fig. 2C). The fourth region analysed in the RepFIIA replicon is repA1 gene, encoding the RepA protein needed for the plasmid’s replication (HELINSKI & al. 1996). The repA1 genes of plasmids pB171, pC15- 1a, pINV_F6_M1382, pO157, pTUC100, R1 and R100 are 858 bp long, the repA1 of p1658/79 is 869 bp long, and the repA1 genes of plasmids pCP301 and pWR501 are 870 bp long. The phylogenetic distance between the pRK100 repA1 nucleotide sequence and the repA1 of plasmid p1658/79 is the smallest, only 0,0154 (Fig. 2D), hence the pRK100 repA1 is most similar to repA1 of plasmid p1658/79. The fi fth and the last region analysed in the RepFIIA replicon, is repA4. Though it encodes no product, it is important for the stability of plasmid maintenance (JIANG & al. 1993). The p1658/79 repA4 is 207 bp long, while the repA4 sequences of other compared plasmids are around 385 bp long. The phylogenetic distance between the pRK100 repA4 nucleotide sequence and repA4 of plasmid pCP15-1a is the smallest, 0,0520 (Fig. 2E), therefore the repA4 nucleotide sequence of pRK100 is most similar to repA4 of plasmid pCP15-1a. Since the individual pRK100 rep genes/regions are similar to rep genes/regions of different plasmids, it can be concluded, that the pRK100 RepFIIA replicon is mosaic in structure and unique among the RepFIIA replicons. Discussion Plasmid replicons are essential for plasmid maintenance in the host cell. The replicons can differ with regard to their replication control mechanisms, origin of replication sequences and replication proteins. Plasmids with very similar origin sequences and replication control mechanisms are as- signed into families. The pRK100 replicon described in this article belongs to the RepFIIA family of replicons. In order to characterise the RepFIIA replicon of pRK100 its nucleotide sequence was fi rst searched for genes/regions. Each RepFIIA replicon consists of fi ve genes/regions, repA2 gene, copA gene, repA6 region, repA1 gene and repA4 region. With the help of the computer programs “ORF Finder” and BLAST the fi ve genes/regions in the pRK100 RepFIIA replicon were determined, however the genes are only putative and more experimental work is needed to confi rm the predicted gene lengths and their functions. To elucidate the similarity of the pRK100 RepFIIA replicon with other known RepFIIA replicons, a BLAST search on the complete pRK100 RepFIIA nucleotide sequence was performed. More then 170 BLAST hits were found, most of them belonging to replicon sequences of plasmids from En- terobacteriaceae. This is not surprising as the broad RepFIIA replicon family is known to be highly prevalent in enteric bacteria (OSBORN & al. 2000). 9M. Starčevič Erjavec & al.: The RepFIIA replicon of the natural Escherichia coli plasmid pRK100 Fig. 2: Nucleotide divergence of pRK100 RepFIIA rep gene/region sequences and related sequences. Nucleotide divergence, as the measurement of the phylogenetic nucleotide distance, was calculated ac- cording to the Kimura-2 parameter. Nucleotide divergences between pRK100 sequences and sequences of other related plasmids are plotted: Panel A – nucleotide divergence of repA2, panel B – nucleotide divergence of copA, panel C – nucleotide divergence of repA6, panel D – nucleotide divergence of repA1, and panel E – nucleotide divergence of repA4. Slika 2: Nukleotidna divergenca dobljenih zaporedij genov/regij rep replikona RepFIIA plazmida pRK100 in sorodnih zaporedij. Nukleotidna divergenca, kot mera za fi logenetsko razdaljo, je bila izračunana po parametru Kimura-2. Nukleotidne divergence med zaporedji plazmida pRK100 in ostalimi sorodnimi zaporedji so prikazana: graf A – nukleotidna divergenca repA2, graf B – nukleotidna divergenca copA, graf C – nukleotidna divergenca repA6, graf D – nukleotidna divergenca repA1 in graf E – nukleotidna divergenca repA4. Nucleotide divergence repA2 0,0154 0,7928 0,8175 0,0038 0,0038 0,8175 0,8175 0,0038 0,0038 0,8175 0 0,2 0,4 0,6 0,8 1 p1658/79 pB171 pC15-1a -pCP301 pINV_F6_M1382 pO157 pTUC100 pWR501 R1 R100 Nucleotide divergence copA 0,0566 0,0572 0,0000 0,0691 0,0691 0,0833 0,0833 0,0742 0,0108 0,0000 0 0,2 0,4 0,6 0,8 1 p1658/79 pB171 -pC15-1a pCP301 pINV_F6_M1382 pO157 pTUC100 pWR501 R1 R100 Nucleotide divergence repA6 0,0134 0,0134 0,0000 0,0000 0,0000 0,0134 0,0134 0,0000 0,0000 0,0000 0 0,2 0,4 0,6 0,8 1 p1658/79 pB171 pC15-1a pCP301 pINV_F6_M1382 pO157 pTUC100 pWR501 R1 R100 Nucleotide divergence repA1 0,0154 0,0546 0,0534 0,0597 0,0597 0,0447 0,0571 0,0597 0,0420 0,0534 0 0,2 0,4 0,6 0,8 1 p1658/79 pB171 pC15-1a pCP301 pINV_F6_M1382 pO157 pTUC100 pWR501 R1 R100 Nucleotide divergence repA4 0,0777 0,0989 0,0520 0,1220 0,1625 0,0841 0,0782 0,1157 0,0664 0,0665 0 0,2 0,4 0,6 0,8 1 p1658/79 pB171 pC15-1a pCP301 pINV_F6_M1382 pO157 pTUC100 pWR501 R1 R100 A B C D E 10 Acta Biologica Slovenica, 49 (1), 2006 The most relevant BLAST hits (BLAST score more then 1000 bits) were used for a detailed similar- ity analysis. The chosen RepFIIA replicons to be compared with the pRK100 replicon were harboured by plasmids p1658/79, pB171, pC15-1a, pCP301, pINV_F6_M1382, pO157, pTUC100, pWR501, R1 and R100. Since it is known, that the RepFIIA replication family is mosaic in its composition (OSBORN & al. 2000), each rep region was analysed separately. Also from our analysis the mosaic structure of the pRK100 RepFIIA replicon is evident, since RepA2 of pRK100 is most similar to repA2 of pCP301, pINV_F6_M1382, pWR501 and R1, copA is the same as copA of plasmids pC15-1a and R100, the repA6 of pRK100 is the same as repA6 in plasmids pC15-1a, pCP301, pINV_F6_M1382, pWR501, R1 and R100, repA1 is the most similar to repA1 of plasmid p1658/79, and repA4 of pRK100 is the most similar to repA4 of pC15-1a. Further, it can also be concluded, that the composition of the pRK100 RepFIIA replicon is unique. Even though all nucleotide sequences of genes/regions incorporated into this study, belong to the same replicon, the RepFIIA, the nucleotide divergence between different genes/regions is not the same, thus the repA6 region is very conserved, while other genes, as copA and repA1, are less conserved. A higher level of nucleotide divergence was observed in the repA4 sequence. This fact is not surprising, since this sequence has no product and hence no product-connected selection can infl uence the evolu- tion of this sequence. However, the greatest differences in nucleotide divergence were observed in the repA2 gene, encoding the repressor. It might be assumed that the observed differences are important for plasmid incompatibility and it would be very interesting to test the ability of the studied plasmids to propagate in the same host. The observed mosaicism of the pRK100 RepFIIA replicon is not the only example of a mosaic sequence of pRK100. In a previous report (STARČIČ ERJAVEC & al. 2002) we also demonstrated, that the pRK100 tra region is mosaic. However, overall the studied tra region genes were most similar to the tra genes of plasmid F, but in the case of the pRK100 RepFIIA replicon, no overall similarity with only one plasmid could be detected. This again illustrates, that plasmid genes are mosaic and formed by multiple recombination events between diverse ancestral genes (BOYD & al. 1996). Conclusions To summarise and conclude: 1. the pRK100 RepFIIA replicon harbours 5 genes/regions repA2, copA, repA6, repA1 and repA4; 2. the individual rep genes/regions of the pRK100 RepFIIA replicon exhibit different nucleotide divergence when compared with different related plasmids; 3. the pRK100 RepFIIA replicon is mosaic and unique; 4. many other plasmids of Enterobacteriaceae carry replicons similar to pRK100 RepFIIA replicon. Povzetek V predstavljeni raziskavi smo na nivoju nukleotidnih zaporedij preučevali podobnost replikona RepFIIA plazmida pRK100 z replikoni RepFIIA ostalih sorodnih plazmidov. S pomočjo računalniških programov “ORF Finder” in BLAST smo na zaporedju replikona RepFIIA plazmida pRK100 poiskali replikacijske gene/zaporedja repA2, copA, repA6, repA1 in repA4. Z računalniškima programoma CLUSTAL W in PHYLIP smo predvidene replikacijske gene/zaporedja primerjali z replikacijskimi geni/zaporedji drugih sorodnih plazmidov (p1658/79, pB171, pC15-1a, pCP301, pINV_F6_M1382, pO157, pTUC100, pWR501, R1 in R100), ki so deponirani v GenBank in smo jih poiskali z računalniškim programom BLAST. Na podlagi dobljenih nukleotidnih divergenc je razvidno, da je gen 11 RepA2 od pRK100 najbolj podoben genu repA2 plazmidov pCP301, pINV_F6_M1382, pWR501 in R1, copA je enak genu copA na plazmidih pC15-1a in R100, regija repA6 plazmida pRK100 je enaka regiji repA6 plazmidov pC15-1a, pCP301, pINV_F6_M1382, pWR501, R1 in R100, gen repA1 je najbolj podoben genu repA1 plazmida p1658/79, in regija repA4 plazmida pRK100 je najbolj podo- bna regiji repA4 plazmida pC15-1a. Če povzamemo vse rezultate, lahko zaključimo, da je replikon RepFIIA plazmida pRK100 sestavljen kot mozaik in da ga v takšni sestavi do sedaj še niso našli na nobenem drugem plazmidu. Literature ALTSCHUL, S. F., T. L. MADDEN, A. A. SCHÄFFER, J. ZHANG, Z. ZHANG, W. MILLER, & D. J. LIPMAN 1997: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389–3402. AMBROŽIČ J., A. OSTROVERŠNIK, M. STARČIČ, I. KUHAR, M. GRABNAR & D. ŽGUR-BERTOK 1998: Escherichia coli ColV plasmid pRK100: genetic organization, stability and conjugal transfer. Microbiology 144: 343–352. BLOMBERG P., K. NORDSTROM & E. G. WAGNER 1992: Replication control of plasmid R1: RepA syn- thesis is regulated by CopA RNA through inhibition of leader peptide translation. EMBO J. 11: 2675–2683. BOYD, E. F., C. W. HILL, S. M. RICH & D. L. HARTL 1996: Mosaic structure of plasmids from natural populations of Escherichia coli. Genetics 143:1091–100. COUTURIER, M., F. BEX, P. L. BERGQUIST, & W. K. MAAS 1988: Identifi cation and classifi cation of bacte- rial plasmids. Microbiol. Rev. 52: 375–395. FELSENSTEIN, J. 1993: PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle. FELSENSTEIN, J. 1989: PHYLIP – Phylogeny Inference Package (Version 3.2) Cladistics 5:164–166. FIRTH, N., K. IPPEN-IHLER, & R. A. SKURRAY 1996: Structure and function of the F factor and mechanism of conjugation, p. 2377–2401. In F. C. Neidhardt, et al. (editors), Escherichia coli and Salmonella, 2nd edition. ASM Press, Washington. HELINSKI, D. R., A. E. TOUKDARIAN, & R. P. NOVICK 1996: Replication control and other stable main- tenance mechanisms of plasmids, p. 2295–2324. In F. C. Neidhardt, et al. (editors), Escherichia coli and Salmonella, 2nd edition. ASM Press, Washington. HOLMES, M. L., F. PFEIFER, & M. L. DYALL-SMITH 1995: Analysis of the halobacterial plasmid pHK2 minimal replicon. Gene 153:117–121. JIANG, T., Y.-N. MIN, W. LIU, D. D. WOMBLE, & R. H. ROWND 1993: Insertion and deletion muta- tions in the repA4 region of the IncFII plasmid NR1 cause unstable inheritance. J. Bacteriol. 175:5350–5358. KADO, C. I. 1998: Origin and evolution of plasmids. Antoine van Leeuwenhoek 73:117–126. OSBORN, A. M., F. M. DA SILVA TATLEY, L. M. STEYN, R. W. PICKUP, & J. R. SAUNDERS 2000: Mosaic plasmids and mosaic replicons: evolutionary lessons form the analysis of genetic diversity in IncFII-related replicons. Microbiology 146: 2267–2275. SOLAR, G. DEL, R. GIRALDO, M. J. RUIZ-ECHEVARRIA, M. ESPINOSA, & R. DIAZ-OREJAS 1998: Replication and control of circular bacterial plasmids. Microbiol. Mol. Biol. Rev. 62:434–464. STARČIČ ERJAVEC, M., W. GAASTRA, D. ŽGUR-BERTOK 2003: tra region of the natural conjugative Es- cherichia coli plasmid pRK100 is F-like. Acta biol Slovenica 45:9–15. STARČIČ ERJAVEC, M. 2003: Mosaic structure and regulation of conjugal transfer of the Escherichia coli plasmid pRK100. PhD thesis, University. of Utrecht, The Netherlands. M. Starčevič Erjavec & al.: The RepFIIA replicon of the natural Escherichia coli plasmid pRK100 12 Acta Biologica Slovenica, 49 (1), 2006 THOMPSON, J. D., D. G. HIGGINS & T. J. GIBSON 1994: CLUSTAL W: improving the sensitivity of pro- gressive multiple sequence alignment through sequence weighting, position-specifi c gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680. VANOOTEGHEM, J.-C. & G. R. CORNELIS 1990: Structural and functional similarities between the replication region of the Yersinia virulence plasmid and the RepFIIA replicons. J. Bacteriol. 172:3600–3608. ZILLIG, W., H. P. ARNOLD, I. HOLZ, D. PRANGISHVILI, A. SCHWEIER, K. STEDMAN, Q. SHE, H. PHAN, R. GARRETT, & J. K. KRISTJANSSON 1998: Genetic elements in the extremely thermophilic archaeon Sulfolobus. Extremophiles 2:131–140. ŽGUR-BERTOK D, E. MODRIČ & M. GRABNAR 1990: Aerobactin uptake system, ColV production, and drug resistance encoded by a plasmid from an urinary tract infection Escherichia coli strain of human origin. Can. J. Microbiol. 36:297–299. 13 ACTA BIOLOGICA SLOVENICA LJUBLJANA 2006 Vol. 49, [t. 2: 13–21 Sprejeto (accepted): 2006-09-28 Colicins of the Escherichia coli uropathogenic strain collection Kolicini zbirke uropatogenih bakterij Escherichia coli Marjanca STARČIČ ERJAVEC, Matija RIJAVEC, Darja ŽGUR-BERTOK Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljub- ljana, Slovenia, Fax: 00386 1 257 3390, E-mail: Marjanca.Starcic.Erjavec@bf.uni-lj.si, r_matija@ yahoo.com, Darja.Zgur@bf.uni-lj.si. Abstract. 110 uropathogenic Escherichia coli (UPEC) strains were screened for colicin production and 42 (38%) of the tested UPEC strains were found to be colicinogenic. The ColA, ColB, ColD, ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColIa, ColIb, ColK, ColN, MccB17, ColS4, MccC7 and ColE6-J colicin producer strains from Pugsley’s collection of colicinogenic strains were lysed by all colicinogenic UPEC strains, the ColM and ColE1 producer strains by 93% of the UPEC colicinogenic strains and the ColV producer strain by only 81% of the UPEC colicinogenic strains. 67% of the colicinogenic UPEC strains were able to lyse all 20 used coli- cin producer strains and 33% of the colicinogenic UPEC strains were able to lyse 19 Pugsley’s strains. Hence, a majority (67%) of the studied UPEC strains encode and produce either more than one colicin, or a colicin not tested. Colicins of UPEC strains producing only one colicin were identifi ed; 8 strains (19% of the colicinogenic strains) produced only ColV, 3 strains (7%) ColM and 3 strains (7%) ColE1. Plasmids were found in 88% of the colicinogenic strains. 11 DL strains were found to harbour conjugative plasmids encoding antibiotic resistance(s) and colicinogenicity. Further, 19% of the haemolytic UPEC strains and 44% of non-haemolytic strains were also colicinogenic, 28% of the cnf encoding strains and 41% of the strains not encoding cnf were colicinogenic, while 40% of ibeA encoding strains and 38% of strains not encoding ibeA were colicinogenic. Key words: colicin, uropathogenic Escherichia coli, UPEC, plasmid, haemolysin, hly, cytotoxic necrotising factor, cnf, invasin, ibeA. Izvleček. 110 uropatogenih sevov bakterije Escherichia coli (UPEC) smo s pomočjo 20 kolicinogenih sevov iz Pugsleyeve zbirke testirali za produkcijo kolicinov. 42 testiranih sevov (38%) je bilo kolicinogenih. Vsi kolicinogeni sevi UPEC so povzročili propad producentskih sevov kolicinov ColA, ColB, ColD, ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColIa, ColIb, ColK, ColN, MccB17, ColS4, MccC7 in ColE6-J iz Pugsleyeve zbirke. 93% kolicinogenih sevov UPEC je povzročilo propad ColM in ColE1 producentskega seva in 81% kolicinogenih sevov UPEC je povzročilo propad producentskega seva kolicina ColV. 67% kolicinogenih sevov UPEC je povzročilo propad vseh 20 kolicinogenih sevov Pugsleyeve zbirke in 33% kolicinogenih sevov UPEC je povzročilo propad 19 sevov iz Pugsleyeve zbirke. Večina kolicinogenih sevov (67%) je sintetizirala vsaj dva kolicina, ali pa kolicin, ki ni bil testiran. Kolicine sevov UPEC, ki sintetizirajo samo 1 kolicin, smo prepoznali; 8 sevov UPEC (19%) je sintetiziralo samo ColV, 3 sevi (7%) so sintetizirali samo ColM in 3 sevi (7%) so sintetizirali samo ColE1. V 88% koli- cinogenih sevov smo našli plazmide. V 11 sevih DL smo našli konjugativne plazmide z zapisi za odpornost proti antibiotikom in za sintezo kolicinov. 19% hemolitičnih sevov UPEC in 44% nehemolitičnih sevov UPEC je bilo kolicinogenih; 28% sevov z zapisom cnf in 41% sevov brez 14 Acta Biologica Slovenica, 49 (1), 2006 zapisa cnf je bilo tudi kolicinogenih, medtem ko je bilo 40% z zapisom ibeA in 38% brez tega zapisa tudi kolicinogenih. Ključne besede: kolicin, uropatogena Escherichia coli, UPEC, plazmid, hemolizin, hly, citotoksičen nekrotizirajoč dejavnik, cnf, invazin, ibeA. Introduction Colicins are bacteriocins produced by Escherichia coli (E. coli) strains. As other bacteriocins, produced by different types of Eubacteria and Archaebacteria (RILEY & GORDON 1999), colicins are extracellular bacterial toxic proteins, that are active against the same species, or closely related species of the producer cell (DAW & FALKINER 1996). The mechanism of action of these compounds involves adsorption to specifi c receptors located on the external surface of sensitive bacteria followed by killing via one of three primary mechanisms: the formation of channels in the cytoplasmic membrane, the degradation of cellular DNA or the inhibition of protein synthesis (RILEY & GORDON 1999). Because of their narrow range of activity, it has been proposed that the primary role of bacteriocins is to mediate intraspecifi c, or population level, interactions (RILEY 1998). However, bacteriocins have been also implicated in virulence determination, since many patho- genic strains harbour plasmid-encoded bacteriocins, for example ColV (WATERS & CROSA 1991). A relatively high frequency of colicin producing strains is found in isolates of pathogenic E. coli (VAN DER WAL & al. 1995), for example approximately 80% of enterohemorrhagic E. coli strains studied by Bradley and Howard were colicinogenic (BRADLEY & HOWARD 1991). In the presented study a collection of 110 uropathogenic E. coli (UPEC) strains, isolated at the Institute of Microbiology and Immunology of the Medical Faculty of Ljubljana, Slovenia, was examined for the ability to produce colicins and lyse other E. coli strains of the Pugsley collection of colicinogenic strains. Further, the association of colicinogenicity and some established virulence factors was also analysed. The results of the study show, that almost 40% of the UPEC strains are colicinogenic and that all colicinogenic strains are very effi cient in lysing E. coli strains of the Pugsley collection. Further, a non-proportional distribution of the ability to produce colicins among the hly and cnf coding versus non-coding strains was determined. In 88% of the colicinogenic strains plasmids were found, 11 colicinogenic strains harboured conjugative plasmids encoding antibiotic resistances and colicin production. Methods Bacterial strains, plasmids and growth conditions The 110 uropathogenic Escherichia coli DL strains used in this study were isolated from urine of patients with urinary tract infections at the Institute of Microbiology and Immunology of the Medical Faculty of Ljubljana. The strain AB1133 [thr1 leuB6 proA2 his argE2 thi ara lacY galK2 xyl mtl rpsL α− supE (B. Bachmann)], which is sensitive to all colicins, was used to identify colicinogenic UPEC strains. The strains of Pugsley’s collection of colicinogenic strains are listed in Tab. 1. For mating experiments the following strains were used: DH5α [Φ80dlacZ∆M15 ∆(lacZYA-argF)U169 endA1 recA1 hsdR17 deoR thi-1 supE44 gyrA96 relA1 (BRL Life)], HB101 [hsdR hsdM recA13 supE44 leuB6 lacZ proA2 (D. Ehrlich)], RU4404 [MM294::Tn1725 Cmr thi endA hsdR (R. Schmitt)], RU4406 [MM294::Tn1732 Knr thi endA hsdR (R. Schmitt)] and TR51 [araD139 ∆(argF-lac)U169 rpsL150 relA1 fl bB5301 ptsF25 deoC1 cpxR::spc Spcr (T. J. Silhavy)]. Bacteria were grown in Luria-Bertani (LB) medium with aeration at 37°C or on LB plates without aeration. Ampicillin (Ap, 100 µg/ml), tetracycline (Tc, 10 µg/ml), kanamycin (Kn, 30 µg/ml), chloramphenicol (Cm, 50 µg/ml), spectinomycin (Spc, 20 µg/ml), streptomycin (Sm, 150 µg/ml), trimethoprim (Tp, 10 µg/ml) and nalidixic acid (Nal, 25 µg/ml) were added to the growth media, when appropriate. 15 Table 1: Bacterial strains of Pugsley’s collection. Tabela 1: Bakterijski sevi Pugsleyeve zbirke. Strain Relevant features BZB2101 ColA producer BZB2102 ColB producer BZB2103 ColD producer BZB2104 ColE1 producer BZB2125 ColE2 producer BZB2106 ColE3 producer BZB2107 ColE4 producer BZB2108 ColE5 producer BZB2109 ColE6 producer BZB2110 ColE7 producer BZB2114 ColIa producer BZB2115 ColIb producer BZB2116 ColK producer BZB2123 ColN producer BZB2283 MccB17 producer PAP1 ColM producer PAP2 ColS4 producer PAP54 MccC7 producer PAP222 ColV producer PAP247 ColE8-J producer Overlay test The overlay test was based on the method described by Pugsley and Oudega (1987). The UPEC strains were inoculated on LB plates (20 colonies grid) using toothpicks. Following overnight incubation the cells were lysed with chloroform, to release the colicins. After aeration, the plates were overlaid with 4 ml of soft agar with 0,2 ml of an overnight culture of either AB1133 or one of the strains from Pugsley’s collection. The plates were then incubated overnight and next day examined for zones of colicin activity (clear zones around the colonies). Plasmid isolation Plasmid DNA was prepared by the alkaline lysis method described in SAMBROOK et al. (1989). Mating assay Conjugation experiments were performed overnight on LB plates. The mating mixture was transferred to LB plates supplemented with appropriate antibiotics to select for transconjugants. The conjugative plasmids encoding antibiotic resistances were initially, depending on the antibiotic resistance profi le, transferred to either DH5α, HB101, RU4404, RU4406 or TR51 and further to a another laboratory strain to confi rm the self conjugative transfer ability. All conjugative plasmids were fi nally transferred to strain DH5α. Results Colicinogenic UPEC strains and their colicins In order to identify the colicinogenic UPEC strains, strain AB1133, which is known to be sensitive to all colicins, was used in an overlay test as described above. 42 UPEC strains (38%) were able to lyse strain AB1133, hence exhibited colicinogenic activity. To attempt to characterise colicin encoded by M. Starčevič Erjavec & al.: Colicins of the Escherichia coli uropathogenic strain collection 16 Acta Biologica Slovenica, 49 (1), 2006 the individual UPEC strains, the 20 strains of Pugsley’s collection of colicin producing strains were used in overlay tests. The tests showed, that most of the colicinogenic UPEC strains (67%) produced either more than one colicin, or a colicin, not harboured by strains of the Pugsley collection. However, 14 UPEC strains were shown to produce only one colicin, namely 8 strains (19% of the colicinogenic strains) produced only ColV, 3 strains (7%) ColM and 3 strains (7%) produced only ColE1 (Tab. 2). Table 2: Colicinogenic DL strains. Tabela 2: Kolicinogeni sevi DL. Strain Number of lysed Pugsley’s strains Identifi ed colicin Plasmid detected Conjugative plasmid DL2 20 + + DL3 20 + – DL6 20 + + DL10 20 + – DL14 20 + + DL22 20 + + DL24 20 + – DL27 19 ColV + – DL35 20 + – DL37 20 + + DL40 19 ColM – – DL46 20 + + DL48 20 + + DL49 20 + – DL51 20 + – DL52 19 ColV + – DL53 20 + – DL56 20 + + DL57 19 ColV + – DL58 20 + – DL59 20 – – DL60 20 + – DL62 19 ColV – – DL63 19 ColE1 + – DL64 19 ColV + – DL66 19 ColV + – DL67 19 ColV + – DL71 19 ColE1 + – DL72 19 ColE1 + – DL75 20 + – DL76 20 + + DL83 19 ColV + – DL89 19 ColM + – DL91 19 ColM + – DL92 20 – – DL93 20 + – DL95 20 – – DL99 20 + – DL104 20 + – DL107 20 + – DL108 20 + + DL110 20 + + 17 Sensitivity of Pugsley’s colicinogenic strains for the colicinogenic UPEC strains The sensitivity of Pugsley’s colicinogenic strains for the colicinogenic UPEC strains was also demonstrated in the overlay tests. The ColA, ColB, ColD, ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColIa, ColIb, ColK, ColN, MccB17, ColS4, MccC7 and ColE6-J colicin producer strain from Pugsley’s collection were lysed by all colicinogenic UPEC strains, the ColM and ColE1 producer strains by 93% of the UPEC colicinogenic strains and the ColV producer strain by only 81% of the UPEC colicinogenic strains (Graph 1).The high sensitivity of the Pugsley colicinogenic strains for the colicinogenic UPEC strains is also evident from Tab. 2, which shows that 67% of the colicinogenic UPEC strains were able to lyse all 20 of the used colicin producer strains and 33% of the colicinogenic UPEC strains were able to lyse 19 of the Pugsley strains. The ability to produce colicins and plasmids Preparation of plasmid DNA with alkaline lysis from colicinogenic strains revealed that 37 (88%) of the colicinogenic strains harboured plasmids. In only 5 strains (DL40, DL59, DL62, DL92 and DL95) no plasmid DNA was detected (Tab. 2). Colicinogenic proprieties encoded on conjugative plasmids In the mating assays, 19 DL strains (DL2, DL6, DL7, DL8, DL14, DL17, DL22, DL37, DL41, DL43, DL46, DL48, DL56, DL76, DL81, DL84, DL108, DL109 and DL110) were found to harbour conjugative plasmids encoding antibiotic resistances. 11 of these strains (DL2, DL6, DL14, DL22, DL37, DL46, DL48, DL56, DL76, DL108 and DL110) were also found to be colicinogenic (Tab. 2). To determine whether the colicinogenicity is encoded on a conjugative plasmid (pDL), the DH5α laboratory strains harbouring the conjugative plasmids from the DL strains were subjected to overlay tests. When the DH5α strains harbouring pDL conjugative plasmids were overlaid with the Pugsley strains, all 20 of the Pugsley strains were lysed. Further, when the original DL strains were overlaid by DH5α strains harbouring pDL conjugative plasmids, the DH5α pDL strains were not lysed by the original DL strain. The obtained results clearly demonstrated that colicinogenicity in all of these strains was encoded by a conjugative plasmid and hence, was transferable to other strains. The ability to produce colicins and some other virulence factors The obtained data concerning colicinogenicity of the tested strains presented in this paper were compared with our unpublished data on the prevalence of virulence factors; namely haemolysin (hly), cytotoxic necrotising factor (cnf) and invasin (ibeA). Our results showed that 19% of the haemolytic UPEC strains and 44% of the non-haemolytic strains were also colicinogenic, 28% of the cnf encoding strains and 41% of the strains not encoding cnf were colicinogenic, while 40% of the ibeA encoding strains and 38% of the strains not encoding ibeA were colicinogenic (Fig. 1). M. Starčevič Erjavec & al.: Colicins of the Escherichia coli uropathogenic strain collection 18 Acta Biologica Slovenica, 49 (1), 2006 Fig. 1: Incidence of colicinogenic strains in correlation with some virulence factors. The incidence of colicino- genic strains is expressed in percentage of strains encoding or not encoding haemolysin (hly), cytotoxic necrotising factor (cnf) and invasin (ibeA). Slika 1: Pogostnost kolicinogenih sevov v povezavi z nekaterimi virulenčnimi dejavniki. Pogostnost kolicinoge- nosti je izražena v odstotkih sevov z oziroma brez zapis za hemolizin (hly), citotoksičen nekrotizirajoč dejavnik (cnf) in invazin (ibeA). Discussion The DL strain collection of uropathogenic E. coli strains was screened for colicin production. 38% of the examined strains exhibited in the performed overlay tests a phenotype consistent with colicin production. Since the overlay tests were performed without mitomycin C treatment, which is known to induce temperate bacteriophage, it can be assumed that the obtained results have not been misin- terpreted due to bacteriophage. However, to completely rule out the possibility of misinterpretation of bacteriophage activity as colicin production further tests would be needed. Typically, 25–50% of E. coli isolates are colicinogenic (RILEY & GORDON 1996). Usually, the percentages are higher in pathogenic than commensal strains, they are also higher in human compa- red to animal strains (RILEY & GORDON 1996). In this study the obtained percentage (38%) of colicin producing strains in the DL collection is comparable to that of other studies, MCGEACHIE (1965) found 36% of colicinogenic strains among uropathogenic E. coli strains and O’BRIEN et al. (1996) found 41% of colicin producers among uropathogenic E. coli strains. The obtained percentage is also com- parable to the incidence of colicinogenicity (41%) in strains from colons of healthy persons (ŠMARDA & OBDRŽALEK 2001). It is established that the frequency of different colicin types varies substantially between populations (RILEY & GORDON 1996), hence the colicin pattern in the DL collection should differ from patterns in other E. coli collections. The applied tests allowed the assignment of colicins of those strains producing only one colicin, namely ColV by 19% of the colicinogenic strains, ColM and ColE1 by 7% of the colicinogenic strains. Colicins of all other colicinogenic strains (67%) could not be assigned as these strains produce more than one colicin or they produce a colicin type not presented in the Pugsley collection of colicinogenic strains. However, the combination of ColV, ColM and ColE1 colicins in one collection is, to our knowledge, unique. High levels of colicin resistance are known to occur in natural E. coli populations (FELDGARDEN & RILEY 1998), hence the high sensitivity of Pugsley’s collection for the colicinogenic strains (the majority, 67%, of colicinogenic strain were able to lyse all 20 strains of Pugsley’s collection), is a bit surprising. But it is explainable with the assumption, that the UPEC strain encode more than one colicin or a colicin not produced by the Pugsley strains collection. 19 It is known, that the genetic determinants of most of the colicins are located on the plasmids, apart from few, which are chromosomally encoded (DAW & FALKINER 1996). Hence, it is not surprising that in 88% of colicinogenic strains we were able to detect plasmid DNA. A very similar percentage, 86%, of plasmid harbouring colicin producing strains was found by Riley and Gordon (1992) in the ECOR collection, a collection of strains representing clinical and non-clinical isolates from man, domestic and zoo animals. To determine whether the colicins are really encoded on the detected plasmids, the plasmids should be transferred to laboratory strains and subsequently tested for colicin production. Unfortunately, the capabilities of transferring the plasmids to laboratory strains are limited due to large plasmid size and lack of selection possibilities. However, several conjugative plasmids encoding antibiotic resistances were found in the DL collection and were successfully transferred. The overlay tests performed on these strains showed that all such plasmids (11) also encoded colicin production. The role of colicins in microbial communities is still not clear (RILEY & WERTZ 2002). Bacteriocins may serve as anti-competitors enabling the invasion of a strain into an established microbial community or they may play a defensive role and act to prohibit the invasion of other strains or species into an occupied niche or limit the advance of neighbouring cells (RILEY & WERTZ 2002). Further it was also suggested that colicins, at least ColV, more exactly the virulence factors also encoded by ColV pla- smids might play a role in pathogenesis (WATERS & CROSA 1991). In a study of UPEC strains ŠMARDA & OBDRŽALEK (2001) observed that the incidence of colicinogenicity signifi cantly differs between haemolytic and non-haemolytic strains (42% of non-haemolytic and only 22% of haemolytic strains were colicinogenic). A comparable difference was also observed in the presented study, as 44% of the non-haemolytic and 19% of the haemolytic strains were colicinogenic. Further, a similar even though less distinctive difference, was observed with the cnf encoding (28%) and cnf non encoding strains (41%). These results suggest, that the production of colicins is associated with some virulence factors, since otherwise the incidence of colicinogenic strains should be equal in both groups, as it is in the presented study for the ibeA encoding (40%) and non ibeA encoding strains (38%). Even though the obtained results could indicate that colicinogenic strains are less virulent, further studies are needed to establish the role colicins play in natural populations. Conclusions To summarise and conclude: 1. 110 uropathogenic Escherichia coli (UPEC) strains were screened for colicin production; 2. 38% of the tested UPEC strains have been found to be colicinogenic; 3. 67% of the colicinogenic UPEC strains were able to lyse all 20 used colicin producer strains, hence these UPEC strains encode and produce either more than one colicin, or a colicin not tested; 4. 19% of the colicinogenic strains produced only ColV, 7% only ColM and 7% only ColE1; 5. plasmids were found in 88% of the colicinogenic strains; 6. 26% of colicinogenic strains harboured conjugative plasmids encoding antibiotic resistance(s) and colicinogenic proprieties and 7. 19% of haemolytic UPEC strains and 44% of non-haemolytic strains were also colicinogenic, 28% of cnf encoding strains and 41% of strains not encoding cnf were colicinogenic, while 40% of ibeA encoding strains and 38% of strains not encoding ibeA were colicinogenic. M. Starčevič Erjavec & al.: Colicins of the Escherichia coli uropathogenic strain collection 20 Acta Biologica Slovenica, 49 (1), 2006 Acknowledgement The authors thank B. Bachmann for providing the strain AB1133, A. P. Pugsley for providing the colicinogenic strain collection, D. Ehrlich for strain HB101, R. Schmitt for strains RU4404 and RU4406 and T. J. Silhavy for strain TR51. Povzetek V predstavljeni raziskavi smo 110 uropatogenih sevov bakterije Escherichia coli (UPEC), ki so jih na Inštitutu za mikrobiologijo in imunologijo Medicinske fakultete v Ljubljani izolirali iz urina bolnikov z urinarno infekcijo, s pomočjo 20 kolicinogenih sevov iz Pugsleyeve zbirke testirali za produkcijo kolicinov. Testiranje je pokazalo, da je bilo 42 sevov UPEC (38%) kolicinogenih. Ker smo testiranje izvedli brez indukcije z mitomicinom C, za katero je znano, da sproži lizogene bakteriofage, lahko predpostavljamo, da dobljeni rezultati temeljijo samo na aktivnosti kolicinov in ne bakteriofagov. Dobljen odstotek kolicinogenih sevov (38%) se ujema tudi z rezultati objavljenimi v podobnih študijah. Vsi kolicinogeni sevi UPEC so povzročili propad producentskih sevov kolicinov ColA, ColB, ColD, ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColIa, ColIb, ColK, ColN, MccB17, ColS4, MccC7 in ColE6-J iz Pugsleyeve zbirke. 93% kolicinogenih sevov UPEC je povzročilo propad ColM in ColE1 producentskega seva in 81% kolicinogenih sevov UPEC je povzročilo propad producentskega seva kolicina ColV. 67% kolicinogenih sevov UPEC je povzročilo propad vseh 20 kolicinogenih sevov Pugsleyeve zbirke in 33% kolicinogenih sevov UPEC je povzročilo propad 19 sevov iz Pugsleyeve zbirke. Večina kolicinogenih sevov (67%) sintetizira vsaj dva kolicina, ali pa kolicin, ki ni bil zastopan v Pugsleyevi zbirki kolicinogenih sevov. Kolicine sevov UPEC, ki sintetizirajo samo 1 kolicin, smo prepoznali; 8 sevov UPEC (19%) je sintetiziralo samo ColV, 3 sevi (7%) so sintetizirali samo ColM in 3 sevi (7%) so sintetizirali samo ColE1. Sicer je znano, da različne populacije E. coli sintetizirajo različne kombinacije kolicinov, a kombinacija ColV, ColM in ColE1, do sedaj še ni bila ugotovljena. Iz 88% kolicinogenih sevov smo uspeli izolirati plazmidno DNA po metodi alkalne lize. S poskusi konjugacije smo v 11 sevih DL našli konjugativne plazmide z zapisi za odpornost proti antibiotikom in za sintezo kolicinov. Vloga kolicinov v naravnih populacijah še ni popolnoma razjasnjena. Za plazmid ColV je znano, da ima poleg zapisa za kolicin V tudi zapise za virulenčne dejavnike. V naši raziskavi smo ugotovili, da je bilo 19% hemolitičnih sevov UPEC in 44% nehemolitičnih sevov UPEC tudi kolicinogenih; 28% sevov z zapisom cnf in 41% sevov brez zapisa cnf je bilo kolicinoge- nih, medtem ko je 40% z zapisom ibeA in 38% brez tega zapisa bilo kolicinogenih. Čeprav dobljeni rezultati nakazujejo, da so kolicinogeni sevi manj virulentni kot nekolicinogeni sevi, so za potrditev te domneve potrebne še nadaljnje raziskave. Literature BRADLEY, D. E. & S. P. HOWARD 1991. Colicinogeny of O157:H7 enterohemorrhagic Escherichia coli and the shielding of colicin and phage receptors by their O-antigenic side chains. Can. J. Microbiol. 37:97–104. DAW, M. A. & F. R. FALKINER 1996: Bacteriocins: nature, function and structure. Micron 27:467– 479. FELDGARDEN, M. & M. A. RILEY 1998. High levels of colicin resistance in Escherichia coli. Evolution 52:1270–1276. MCGEACHIE, J. 1965: Bacteriocin typing in urinary infection: Zentralbl. Bakt. I Orig. 196:377–384. O’BRIEN, G. J., S. T. CHAMBERS, B. PEDDIE & H. K. MAHANTY 1996: The association between colicino- genicity and pathogenesis among uropathogenic isolates of Escherichia coli. Microb. Pathogen. 20:185–190. 21 PUGSLEY, A. P. & B. OUDEGA 1987: Methods for studying colicins and their plasmids. In Hardy, K. G. Plasmids, a practical approach. IRL Press, Oxford, Washington DC. RILEY, M. A. & D. M. GORDON 1992: A survey of Col plasmids in natural isolates of the Esche- richia coli and an investigation into the stability of Col-plasmid lineages. J. Gen. Microbiol. 138:1345–1352. RILEY, M. A. & D. M. GORDON 1996: The ecology and evolution of bacteriocins. J. Industrial. Micro- biol. 17:151–158. RILEY, M. A. 1998: Molecular mechanisms of bacteriocin evolution. Annu. Rev. Genet. 32:255– 278. RILEY, M. A. & D. M. GORDON 1999: The ecological role of bacteriocins in the bacterial competition. Trends in Microbiology 7:129–133. RILEY, M. A. & J. E. WERTZ 2002: Bacteriocin diversity: ecological and evolutionary perspectives. Biochemie 84:357–364. SAMBROOK, J., E. F. FRITSCH & T. MANIATIS 1989. Molecular cloning: a laboratory manual, 2nd edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. ŠMARDA, J. & V. OBDRŽALEK 2001: Incidence of colicinogenic strains among human Escherichia coli. J. Basic Microbiol. 41:367–374. VAN DER WAL, F. J., J. LUIRINK & B. OUDEGA 1995: Bacteriocin release proteins: mode of action, struc- ture, and biotechnological application. FEMS Microbiol Rev 17:318–399. WATERS, V. L. & J. H. CROSA 1991: Colicin V virulence plasmids. Microbiol. Rev. 55:437–450. M. Starčevič Erjavec & al.: Colicins of the Escherichia coli uropathogenic strain collection 22 Acta Biologica Slovenica, 49 (1), 2006 23 ACTA BIOLOGICA SLOVENICA LJUBLJANA 2006 Vol. 49, [t. 2: 23–32 Sprejeto (accepted): 2007-01-29 Quantitative analysis of the macroinvertebrate community in the river Temenica (SE Slovenia) Kvantitativna analiza združbe makroinvertebratov v reki Temenici (JV Slovenia) Mojca PUST, Mihael J. TOMAN Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia; E-mail: mihael.toman@bf.uni-lj.si. Abstract. Macroinvertebrate community of the river Temenica was investigated in order to asses the ecological quality of the stream. Different approaches were used in order to compare their usefulness. Surber sampler methodology was used and all together16 quantitative sam- ples were taken at four stream reaches every three months from October 2003 until July 2004. Saprobic and diversity indices showed severe deterioration of water quality longitudinally and a strong negative infl uence of the town Trebnje on the ecological state of the river Temenica. Multivariate methods DCA and CCA confi rmed those results, but also showed greater differences in community structure at sampling site 1 compared to sites 2 and 3 which was not detected using the indices. Key words: macroinvertebrates, water quality assesment, saprobic index, diversity indices, DCA, CCA Izvleček. Za oceno ekološke kakovosti reke Temenice smo raziskovali združbo makroin- vertebratov. Uporabili smo različne pristope, da bi primerjali njihovo uporabnost. Uporabljena je bila metodologija Surberjevega vzorčevalnika, vzorčili smo na štirih vzorčnih mestih vsake tri mesece od oktobra 2004 do julija 2004, skupno smo pobrali 16 kvantitativnih vzorcev. Saprobni in diverzitetni indeksi so pokazali resno poslabšanje kakovosti vode po toku navzdol in močan negativen vpliv mesta Trebnje na ekološko stanje reke Temenice. Multivariatni metodi DCA in CCA sta potrdili te rezultate, pokazali pa sta tudi večje razlike v strukturi združbe na vzorčnem mestu 1 v primerjavi z mestoma 2 in 3, ki jih z uporabo indeksov nismo zaznali. Ključne besede: makroinvertebrati, ocena kakovosti vode, saprobni indeks, diverzitetni indeksi, DCA, CCA Introduction Macroinvertebrates are extensively used in water quality assessment as they are considered good indicators of environmental pollution. There are various methods for assessing water quality which have different conceptual basis and may therefore provide different information about the aquatic environment. Many authors have compared different methods of summarising responses to pollution (CAO et al. 1996, ROSSARO & PIETRANGELO 1993). Water quality indices have been developed as a rou- tine technique for water monitoring. Saprobic index is often used as a measure of organic pollution (URBANIČ 2004) and is also in use by the Slovenian Ministry of the Environment for biomonitoring of surface waters. Diversity indices are known to be inaccurate at headwater reaches and were also reported to be insensitive to slight and moderate pollution (CAO et al. 1996). However, they can still be used as a complementary method. 24 Acta Biologica Slovenica, 49 (1), 2006 Multivariate methods are increasingly used in biological monitoring of water quality (CAO et al. 1996, ROSSARO & PIETRANGELO 1993). Their main advantage is that they can detect more subtile changes in community structure than the indices. They can also provide information on the responses of taxa to different environmental variables. The aim of this study was to asses the water quality of the stream Temenica using different methods (saprobic index, different diversity indices and multivariate methods DCA and CCA) and to compare the effectiveness of those methods. Description of study sites Our study area was the fi rst part of the disappearing stream Temenica in the south-eastern part of Slovenia. Its length is 25 km and the catchment area comprises 91 km2. The upper part of the stream (approx. 5 km) lies in a forested area with little human impact whereas the lowland part with meandering watercourse runs through cultivated land and by the town Trebnje, which is the largest settlement in the area. Elevations range from 560 m at the source to 260 m at the sinking point. Temperatures fl uctuate between 2,2–3 oC (winter) and 14,2–18,2 oC (summer). Stream pH lies within the range 8,1–8,7 and conductivity between 504 and 604 μScm–1. Sampling site 1 was established in the headwaters, site 2 at the beginning of lowland watercourse, and sites 3 and 4 upstream and downstream from Trebnje. Materials and methods Macroinvertebrates were sampled on four occasions between October 2003 and July 2004 with 3 month-intervals. At each sampling site 6 random sampling units were taken with a Surber sampler (500 cm2 sapling area and 0,5 mm mesh size). Several physical and chemical characteristics (Table 1) were measured at the same time. Table 1: Morphometric and physical characteristics of sampling sites on the river Temenica. Tabela 1: Morfometrične in fi zikalne značilnosti vzorčnih mest v reki Temenici. Stream reach Location Stream order* Distance from source (km) Altitude (m) Mean width (m) Mean depth (cm) Maximum temperature (C) 1 Pusti Javor 2 2 360 1,2 6 14,2 2 Stranje pri Velikem Gabru 3 10 300 3,1 31 14,1 3 Štefan 3 18 280 7,3 23 16,9 4 Gorenje Ponikve 3 23 270 5,9 22 18,2 * After Strahler (1952) All samples were fi xed in 4 % formaldehyde in the fi eld. In the laboratory, organisms were sorted, enumerated, identifi ed under a stereomicroscope and stored in 70 % ethyl alcohol. Macroinvertebrates were determined at least to the family level (except groups Hydrachnida and Collembola) using the keys of BAUERNFEIND & HUMPESCH (2001), BRINKHURST (1971), ELLIOT (1977), GERKEN & STERNBERG (1999), GLOER (2002), KARAMAN & PINKSTER (1977), NESEMANN (1997), REYNOLDSON (1987), SCHME- DTJE & KOHMANN (1992), TACHET (2000), TRONTELJ & SKET (2000), URBANIČ et al. (2003), WARINGER & GRAF (1997), ŽIŠKO (2000). Saprobic index was calculated using saprobic and indicative taxa values after WEGL (1983), MOOG (1995) and URBANIČ (2004) (Trichoptera). Number of taxa per sampling unit (S) and diversity indices (Shannon-Wiener index H’ = – Σ (p i * ln p i ), Eveness index E = H’/ln S and Simpson’s index D = 1 – Σ 25 p i 2) were calculated using PC-ORD (MCCUNE & MEFFORD 1999). One way ANOVAs were performed (using statistical package SPSS) to determine whether the differences in diversity among sampling sites were signifi cant. Multivariate methods used in this study included detrended correspondence analysis (DCA) and canonical correspondence analysis (CCA). Both were performed using statistical package CANOCO. Data on taxa abundances were log transformed. CCA analysis was performed with reduced data matrix which included only suffi ciently common taxa (occurrence in at least 2 samples or at least 4 individuals present) and 5 statistically signifi cant variables (forward selection). Results A total of 76550 individuals, representing 120 taxa were collected (Table 2). Saprobic index (Fig. 1) indicates strong organic pollution at sampling site 4 where Tubifi cidae and Chironomidae were the most abundant. There is an apparent decrease in the value in summertime when Chironomidae (which have a lower saprobic value) outnumbered Tubifi cidae. Values of the saprobic index at the other three sites were very similar in all seasons and indicate low level of pollution. Box-plot presentations of taxa number (S) and values of Shannon-Wiener index (H’), Eveness index (E) and Simpson’s index (D) are shown in Figure 2. There is a signifi cant decrease in all the values at sampling site 4, which confi rms the results of the saprobic index. The number of taxa is also lower at site 1, but diversity indices at this site are equal or even higher (E) than at sites 2 and 3. Using one-way ANOVA we confi rmed satistically signifi cant differences in values S, H’ and E among sampling sites. DCA ordination of 16 samples is shown in Figure 3. The fi rst axis explains 40,5 % of the total variance and axis 2 further explains 12,5 %. Samples from each sampling site are clearly separated by the fi rst axis which represents the upstream-downstream gradient and presumably also the pollution gradient. There is a considerable gap between the samples from site 1 and the other samples which indicates signifi cant differences in community structure between the headwaters and the rest of the stream. Axis 2 is apparently related to the seasonal changes in community structure. M. Pust, M. Toman: Quantitative analysis of the macroinvertebrate community in the river ... Figure 1: Values of saprobic index at different seasons at four sampling sites of theTemenica river. Slika 1: Vrednosti saprobnega indeksa v različnih letnih časih na štirih vzorčnih mestih v reki Temenici. 26 Acta Biologica Slovenica, 49 (1), 2006 Ta bl e 2: T he a bu nd an ce o f ta xa a t t he s am pl in g si te s of T em en ic a. A bb re vi at io ns a re a dd ed o nl y fo r ta xa u se d in th e C C A Sa m pl in g si te 1 2 3 4 Sa m pl in g si te 1 2 3 4 Ta xa A bb re vi at io n Ta xa A bb re vi at io n Po ly ce lis fe lin a Po ly _f el 30 0 0 0 C ol le m bo la 3 0 0 0 D ug es ia sp . D ug e_ sp 0 37 8 17 8 Ep he m er a da ni ca E ph e_ da n 35 6 6 2 0 Pr os to m a sp . Pr os _s p 0 4 0 0 Ep he m er el la m aj or E ph e_ m aj 0 14 0 0 M er m ith id ae M er _i da e 0 23 3 15 4 Ep he m er el la ig ni ta E ph e_ ig n 2 37 6 15 3 17 H ol an dr ia na h ol an dr i H ol a_ ho l 3 22 32 38 1 0 C ae ni s m ac ru ra 0 0 0 1 Es pe ri an a es pe ri E sp e_ es p 0 8 36 7 4 Ec dy on ur us sp . E cd y_ sp 0 4 1 0 Es pe ri an a da ud eb ar tii E sp e_ da u 0 0 83 2 El ec tro ge na sp . E le c_ sp 49 5 0 0 Th eo do xu s d an ub ia lis T he o_ da n 0 0 17 4 0 Rh ith ro ge na sp . R hi t_ sp 54 32 1 0 Sa dl er ia na fl um in en si s 0 2 0 0 Ba et is sp . B ae t_ sp 39 71 2 24 4 18 7 Be lg ra nd ie lla sp . 0 1 0 0 C en tro pt ilu m lu te ol um 9 0 0 0 An cy lu s fl u vi at ili s A nc y_ fl u 0 17 2 0 3 Ac en tre lla si na ic a 0 4 0 0 Ra di x ba lth ic a R ad i_ ba l 0 1 3 3 H ab ro le pt oi de s c on fu sa H ab r_ co n 35 0 17 1 Ph ys a fo nt in al is Ph ys _f on 0 0 0 12 H ab ro ph le bi a fu sc a H ap h_ fu s 67 0 28 1 Pi si di um sp . Pi si _s p 1 90 65 86 H ab ro ph le bi a la ut a 2 0 0 0 H el ob de lla st ag na lis H el o_ st a 1 0 0 88 6 Pa ra le pt op hl eb ia su bm ar gi na ta Pa ra _s ub 0 0 6 0 Al bo gl os si ph on ia h ia lin a 0 0 1 0 Pe rl od es sp . P er l_ sp 20 10 5 55 2 G lo ss ip ho ni a co nc ol or G lo ss _c on 0 0 0 2 Is op er la sp 1 1 0 0 Tr oc he ta b yk ow sk ii 0 2 0 0 N em ou ra sp . N em o_ sp 33 0 0 0 D in a sp . D in a_ sp 0 0 0 9 Pr ot on em ur a sp . P ro t_ sp 16 0 0 0 Ei se ne lla te tr ae dr a E is en _t et 2 41 21 7 Le uc tr a sp . L eu c_ sp 9 0 3 0 L um br ic ul id ae L um _i da e 0 7 1 0 Br ac hy pt er a sp . B ra c_ sp 20 4 3 0 St yl od ri lu s h er in gi an us St yl _h er 21 7 69 9 24 9 22 19 C al op te ry x vi rg o C al o_ vi r 0 4 1 0 T ub ifi ci da e T ub _i da e 4 14 3 55 25 15 5 C al op te ry x sp le nd en s 3 10 0 0 N ai di da e N ai _i da e 2 32 16 44 Pl at yc ne m is p en ni pe s Pl at _p en 0 0 25 2 St yl ar ia la cu st ri s St yl _l ac 0 0 12 3 2 Le st es v ir id is 0 0 0 1 H yd ra ch ni da 1 0 0 0 L es tid ae ( ju v. ) 0 0 2 0 G am m ar us fo ss ar um G am m _f os 29 6 86 3 25 6 6 G om ph us v ul ga tis si m us G om p_ vu l 0 22 14 0 Sy nu re lla a m bu la ns Sy nu _a m b 0 0 3 4 O ny ch og om ph us fo rc ip at us O ny c_ fo r 0 11 48 0 As el lu s a qu at ic us A se l_ aq u 1 0 1 47 O ny ch og om ph us u nc at us O ny c_ un c 0 0 17 0 Au st ro po ta m ob iu s to rr en tiu m A us t_ to r 0 0 4 0 C or du le ga st er b ol to ni co m pl ex C or d_ bo l 10 0 0 0 27 Sa m pl in g si te 1 2 3 4 Sa m pl in g si te 1 2 85 0 Ta xa A bb re vi at io n Ta xa A bb re vi at io n 0 2 Ap he lo ch ei ru s a es tiv al is A ph e_ ae s 0 20 85 0 Le pi do st om a hi rt um 0 1 0 0 Si al is lu ta ri a 0 0 0 2 Po ta m op hy la x sp . 0 0 5 0 El m is sp . E lm i_ sp 14 39 8 13 61 1 O do nt oc er um a lb ic or ne 1 0 0 0 Es ol us sp . E so l_ sp 61 41 67 13 79 18 W or m al di a su bn ig ra W or m _s ub 0 0 19 0 Li m ni us sp . L im n_ sp 5 64 3 13 1 0 W or m al di a oc ci pi ta lis 1 0 0 0 O ul im ni us sp . O ul i_ sp 0 34 6 24 74 33 Pl ec tro cn em ia sp . 3 0 0 0 Ri ol us sp . R io l_ sp 7 65 37 7 1 Ps yc ho m ia k la pa le ki Ps yc _k la 0 5 0 0 St en el m is sp . 0 0 2 0 Ps yc ho m iid ae 3 0 0 0 H yd ra en a sp . H yd n_ sp 61 90 14 5 5 Ti no de s r os to ck i T in o_ ro s 7 0 0 0 H al ip lu s s p. 0 0 1 0 Ly pe re du ct a 1 0 0 0 D yt is ci da e 0 0 1 0 Ry ac op hi la tr is tis 2 1 0 0 Sc ir tid ae Sc i_ id ae 27 6 0 0 0 Ry ac op hi la sp . ( s.s tr. ) R ya c_ sp 2 3 2 0 Eu br ia sp . 0 1 0 0 N ot id ob ia c ili ar is 0 0 2 0 H yd ro ps yc he sa xo ni ca H yd r_ sa x 9 0 0 0 H yd ro pt ila sp . H yp t_ sp 1 1 26 5 H yd ro ps yc he p el lu ci du la H yd r_ pe l 0 71 8 0 At he ri x m ar gi na ta A th e_ m ar 46 14 7 15 0 H yd ro ps yc he a ng us tip en ni s H yd r_ an g 0 0 1 13 At he ri x ib is A th e_ ib i 0 6 0 0 H yd ro ps yc he si lta la i H yd r_ si l 0 11 0 0 A nt ho m yi da e A nt _i da e 0 0 39 1 H yd ro ps yc he sp . ( ju v.) H yd r_ sp 0 59 4 0 C hi ro no m in ae C hi _n ae 22 56 3 10 73 13 18 1 Si lo p ic eu s Si lo _p ic 0 57 1 7 0 O rt ho cl ad in ae O rt _i na e 18 4 29 2 33 9 38 18 Si lo p al lip es 0 1 0 0 Ta ny po di na e Ta n_ in ae 14 23 16 9 84 3 Ag ap et us d el ic at ul us A ga p_ de l 0 54 0 0 D ol ic ho po di da e D ol _i da e 0 1 4 0 G lo ss os om a bi fi d um 0 1 0 0 C er at op og on in i C er _i ni 0 7 4 12 At hr ip so de s c in er eu s A th r_ ci n 0 8 0 0 E m pi di da e E m p_ id ae 1 13 25 38 At hr ip so de s b ili ne at us A th r_ bi l 0 5 0 0 L im on iid ae L im _i da e 64 37 21 2 Le pt oc er us in te rr up tu s L ep t_ in t 0 0 24 0 Ps yc ho di da e Ps d_ id ae 0 1 21 6 Ad ic el la sp . 0 0 2 0 Si m ul iu m sp . Si m u_ sp 54 15 68 21 28 13 8 Se to de s s p. 0 0 1 0 Pr os im ul iu m sp . Pr os i_ sp 41 0 0 0 M ys ta ci de s a zu re a 0 10 0 0 St ra tio m yi da e St r_ id ae 2 2 1 0 Li m ne ph ilu s l un at us 0 0 1 2 Ta ba ni da e Ta b_ id ae 0 15 2 0 C ha et op te ry x m aj or C ha e_ m aj 17 0 0 0 T ip ul id ae 0 1 1 1 To ta l n um be r of in di vi du al s 21 85 14 87 5 12 33 4 47 15 6 To ta l n um be r of ta xa 55 69 73 46 M. Pust, M. Toman: Quantitative analysis of the macroinvertebrate community in the river ... 28 Acta Biologica Slovenica, 49 (1), 2006 With 5 selected environmental variables (Fig. 4a) used in the CCA, we explained 64 % of the variance of the taxa-environment relation. Eigenvalues of axes 1 and 2 are 0,38 and 0,28 and display 42 % of the variance. Axis 1 shows high correlation with stream order (R2 = –0,95), and stream width (R2 = –0,74). Taxa which appear on the extreme right side of the biplot (Fig. 4b) therefore prefer headwaters of the stream. Axis 2 is mostly correlated with saprobic index (R2 = 0,84) and may thus present the gradient of organic pollution. Taxa showing affi nity (in the upper part of the biplot) to this factor were mostly found at the sampling site 4. Figure 2: Box-plots of taxa number/sampling unit (S), Shannon-Wiener index (H’), Eveness index (E) and Sim- pson’s index (D) in different seasons at four sampling sites of the Temenica river. Slika 2: Diagrami kvartilov števila taksonov/vzorčno enoto (S), Shannon-Wienerjevega indeksa (H’), indeksa stalnosti (E) in Simpsonovega indeksa (D) v različnih letnih časih na štirih vzorčnih mestih v reki Temenici. 29M. Pust, M. Toman: Quantitative analysis of the macroinvertebrate community in the river ... Discussion Macroinvertebrate fauna in the fi rst part of Temenica is relatively diverse with signifi cant differen- ces in community structure among the sampling sites. The fourth site, which lies downstream from Trebnje (population approx. 3000), is especially dissimilar from the rest due to its high saprobic index, low diversity values of indices and the number of taxa. This indicates that the town of Trebnje is a major pollutant of the stream. On the other hand, neither the saprobic index nor the diversity indices showed signifi cant differences among the other three sampling sites. The values of saprobic index at the fourth sampling site are markedly varying between seasons as a result of changes in abundance of two most abundant groups – Tubifi cidae (Oligochaeta) and Chironomidae (Diptera). There were also signifi cant seasonal differences in diversity. This indicates the importance of monitoring over a longer period of time. The fi rst DCA axis can be interpreted both as the upstream-downstream gradient as in the study of ROSSARO & PIETRANGELO (1993) and the pollution gradient, as suggested by Cao et al. (1996). In comparison with the results of saprobic and diversity indices the samples of the sampling site 1 are here clearly separated from the other samples while the differences in community structure between site 4 and the other sites in much less pronounced than in the case of the indices. The reason for this distribution lies in the small number of individuals and different taxonomic structure at site 1, where the morphometric parameters (depth, width etc.) are very different from those at the other sampling sites. The second DCA axis represents seasonal differences among the samples which are very distinct at all four sampling sites. Group of taxa, found at sampling site 1 (correlated with stream order), is pointing out in the CCA biplot, as well as the taxa, characteristic for site 4 (correlation with saprobic index). This is in agreement with both the indices and the DCA results. Although indices did not show the difference in community structure between the fi rst and the two next sampling sites, they appear to be better at detecting severe pollution (site 4) than multivariate methods. Complementary use of different methods is therefore the most reasonable approach in water quality assessment. Figure 3: DCA ordination of samples. Slika 3: DCA ordinacija vzorcev. 30 Acta Biologica Slovenica, 49 (1), 2006 Figure 4: F1 X F2 plane of Canonical correspondence analysis (CCA) between 84 taxa and 5 selected environ- mental variables. See Table 2 for taxa abbreviations. Slika 4: F1 x F2 ravnina CCA ordinacijskega diagrama med 84 taksoni in 5 izbranimi okoljkimi spremenljivkami. Glej Tabelo 2 za okrajšave taksonov. 31 Conclusions 1. High values of saprobic index and low values of diversity indices at sampling site 4 indicate that the town Trebnje as the major pollutant of the Temenica river. Differences among other three sampling sites are not detectable with the use of indices. 2. Axis 1 of DCA biplot, representing the upstream-downstream gradient, revealed great diffe- rences in macroinvertebrate community structure between the fi rst and the second sampling site, whereas the distinction of the fourth sampling site is much less pronounced. Seasonal changes in community structure are distinct at all sampling sites. 3. Stream order and saprobic index were the most prominent factors affecting the longitudinal distribution of taxa. Taxa signifi cant for sites 1 and 4 were highly correlated with these two factors. Povzetek Makroinvertebrati se kot dobri pokazatelji obremenjevanja okolja veliko uporabljajo pri ocenje- vanju kakovosti voda. Metode, ki se pri tem uporabljajo pa temeljijo na različnih principih in lahko dajejo različne informacije o vodnem okolju. Zaradi številnih pomanjkljivosti indeksov, ki se običajno uporabljajo za rutinski biomonitoring celinskih voda, se v vrednotenju kakovosti čedalje pogosteje uporabljajo multivariatne metode. S temi lahko zaznamo manjše spremembe v združbi kot z indeksi, dajo pa nam lahko tudi informacije o odzivu taksonov na posamezne okoljske spremenljivke. V naši raziskavi smo z analizo združbe makroinvertebratov ocenjevali ekološko stanje prvega dela ponikalne reke Temenice, pri čemer smo uporabili različne metode, da bi preverili njihovo uporabnost pri ocenjevanju kakovosti voda. Kvantitativno vzorčenje s Surberjevim vzorčevalnikom smo izvajali vsake tri mesece od oktobra 2003 do julija 2004 na štirih vzorčnih mestih. Vrednosti saprobnega indeksa, diverzitetnih indeksov ter število taksonov na vzorčno enoto kažejo na močno poslabšanje kakovosti reke Temenice na zadnjem vzorčnem mestu v primerjavi s prvimi tremi in s tem na močan negativen vpliv mesta Trebnje na njeno ekološko stanje. Razvrstitev vzorcev po prvi DCA osi potrjuje našo domnevo o naraščanju obremenjevanja reke po toku navzdol, pri čemer pa četrto vzorčno mesto v primerjavi z rezultati indeksov ni tako izstopajoče. Večje razlike v združbi makroinvertebratov so po tej analizi med prvim vzorčnim mestom in ostalimi tremi. Na vseh vzorčnih mestih so izrazite tudi sezonske razlike v strukturi združbe. Najpomembnejši spremenljivki za razporeditev taksonov sta po rezultatih CCA red vodotoka in vrednost saprobneega indeksa, kar izpostavi taksone, ki so bili najbolj zastopani na prvem in zadnjem vzorčnem mestu. References ALLAN J. D. 2004: Stream ecology. Structure and function of running waters. Kluwer Academic Pu- blishers, Dordrecht/Boston/London, 388 pp. BAUERNFEIND E., HUMPESCH U.H. 2001: Die Eintagsfl iegen Zentraleuropas (Insecta: Ephemeroptera): Bestimmung und Ökologie. Verlag des Naturhistorischen Museums, Wien, 237 pp. BRINKHURST R. O. 1971: A guide for the identifi cation of British aquatic Oligochaeta. Freshwater Biological Association, Ambelside, 52 pp. CAO Y., BARK A.W. & WILLIAMS P. 1996: Measuring the responses of macroinvertebrate communities to water pollution: a comparison of multivariate approaches, biotic and diversity indices. Hydro- biologia 341: 1–19. ELLIOT J. M. 1977: A key to the larvae and adults of British freshwater Megaloptera and Neuroptera with notes on their life cycles and ecology. Freshwater biological association, 52 pp. M. Pust, M. Toman: Quantitative analysis of the macroinvertebrate community in the river ... 32 Acta Biologica Slovenica, 49 (1), 2006 GERKEN B., STERNBERG K: 1999: Die Exuvien Europäischer Libellen (Insecta: Odonata). Höxter und Jena, 354 pp. GLOER P. 2002: Mollusca I. Süsswassergastropoden. Nord und Mitteleuropas. Bestimungschlüssel, Lebensweise, Verbreitung. ConchBooks, 327 pp. KARAMAN G. S., PINKSTER S. 1977: Freshwater Gammarus species from Europe, North Africa and adjacent regions of Asia. Part I. Gammarus pulex – group and related species. Bijdr. Dierk. 47(1): 1–97. LOUNACI A., BROSSE S., THOMAS A., LEK S. 2000: Abundance, diversity and community structure of macroinvertebrates in an Algerian stream: the Sébaou wadi. Annls. Limnol. 36 (2): 132–133. MCCUNE B., MEFFORD M. J. 1999. PC-ORD. Multivariate analysis of Ecological Data, Version 4. MjM Software design, Gleneden Beach, Oregon, USA. MOOG O. 1995: Fauna aquatica Austriaca. Katalog zur autökologischen Einstufung aquatischer Orga- nismen Österreichs. Bundesmoinisterium für Land- und Worstwirtschaft, umwelt und Wasserwirtschaft, Wien, 426 pp. NESEMANN H. 1997: Egel und Krebsegel (Clitellata: Hirudinea, Branchiobdellida) Österreichs. Son- derheft der Ersten Vorarlberger Malakologischen Gesellschaft, Rankweil, 104 pp. PUST M. 2005: Kvantitativna analiza združbe makroinvertebratov v reki Temenici. Diplomska naloga, Univerza v Ljubljani, Biotehniška fakulteta, Odd. za biologijo, 72 pp. REYNOLDSON T.B. 1978: A key to the British species of Freshwater Triclads (Turbellaria, Paludicola). Freshwater biological association, 32 pp. ROSSARO B., PIETRANGELO A. 1993: Macroinvertebrate distribution in streams: a comparison of CA ordination with biotic indices. Hydrobiologia 263: 109–118. SCHMEDTJE U., KOHMANN F. 1992: Bestimugschlüssel für die Saprobier-DIN-Arten (Makrooganismen). Bayerisches Landesamt für Wasserwitschaft, 274 pp. TACHET H. 2000: Invertebrés d‘eau douce; systematique, biologie, écologie. CNRS editions, 590 pp. TER BRAAK C. J. F., ŠMILAUER P. 1998: CANOCO release 4 reference manual and user’s guide to Canoco for Windows – Software for canonocal community ordination. Ithaca, New york, Micro- computer Power. TRONTELJ P., SKET B. 2000: Molecular re-assesment of some phylogenetic, taxonomic and biogeo- graphic relationships between the leech genera Dina and Trocheta (Hirudinea: Erpobdellidae). Hydrobiologia 438: 227–235. URBANIČ G., WARINGER J., GRAF W. 2003. The larva and distribution of Psychomyia klapaleki Malicky, 1995 (Trichoptera: Psychomyiidae). Lauterbornia, 46: 135–140. URBANIČ G. 2004: Ekologija in razširjenost mladoletnic (Insecta: Trichoptera) v nekaterih vodotokih v Sloveniji. Dokt. disertacija. Ljubljana, Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za biologijo, 188 pp. URBANIČ G., TOMAN M. J. & KRUŠNIK C. 2004: Microhabitat type selection of caddisfl y larvae (Insecta: Trichoptera) in a shallow lowland stream. Hydrobiologia 00: 1–12. WEGL R. 1983. Index für die Limnosaprobität. Wasser und abwasser 26: 1–175. ŽIŠKO A. 2000. Morfološke prilagoditve bibice (Synurella ambulans, Crustacea: Amphipoda) na po- dzemeljske habitate. Dipl. delo, Ljubljana, Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za biologijo, 46 pp. 33 NAVODILA AVTORJEM 1. Vrste prispevkov a) ZNANSTVENI ČLANEK je celovit opis originalne raziskave in vključuje teoretični pregled tematike, po- drobno predstavljene rezultate z diskusijo in sklepe ter literaturni pregled: shema IMRAD (Introduction, Methods, Results And Discussion). Dolžina članka, vključno s tabelami, grafi in slikami, na sme presegati 15 strani; razmak med vrsticami je dvojen. Recenzirata ga dva recenzenta. b) PREGLEDNI ČLANEK objavi revija po posvetu uredniškega odbora z avtorjem. Število strani je lahko večje od 15. c) KRATKA NOTICA je originalni prispevek z različnih bioloških področij (sistematike, biokemije, genetike, mikrobiologije, ekologije itd.), ki ne vsebuje podrobnega teoretičnega pregleda. Njen namen je seznaniti bralca s preliminarnimi ali delnimi rezultati raziskave. Dolžina na sme presegati 5 strani. Recenzira ga en recenzent. d) KONGRESNA VEST seznanja bralce z vsebinami in sklepi pomembnih kongresov in posvetovanj doma in v tujini. e) DRUŠTVENA VEST poroča o delovanju slovenskih bioloških društev. 2. Originalnost prispevka Članek, objavljen v reviji Acta Biologica Slovenica, ne sme biti predhodno objavljen v drugih revijah ali kongresnih knjigah. 3. Jezik Teksti naj bodo pisani v angleškem jeziku, izjemoma v slovenskem, če je tematika zelo lokalna. Kongresne in društvene vesti so praviloma v slovenskem jeziku. 4. Naslov prispevka Naslov (v slovenskem in angleškem jeziku) mora biti kratek, informativen in razumljiv. Za naslovom sledijo imena avtorjev in njihovi polni naslovi (če je mogoče, tudi štev. faxa in e-mail). 5. Izvleček – Abstract Podati mora jedrnato informacijo o namenu, uporabljenih metodah, dobljenih rezultatih in zaključkih. Primerna dolžina za znanstveni članek naj bo približno 250 besed, za kratko notico pa 100 besed. 6. Ključne besede – Keywords Število naj ne presega 10 besed, predstavljati morajo področje raziskave, predstavljene v članku. Člankom v slovenskem jeziku morajo avtorji dodati ključne besede v angleškem jeziku. 7. Uvod Nanašati se mora le na tematiko, ki je predstavljena v članku ali kratki notici. 8. Slike in tabele Tabele in slike (grafi , dendrogrami, risbe, fotografi je idr.) naj v članku ne presegajo števila 10, v članku naj bo njihovo mesto nedvoumno označeno. Ves slikovni material naj bo oddan kot fi zični original (fotografi ja ali slika). Tabele in legende naj bodo tipkane na posebnih listih (v tabelah naj bodo le vodoravne črte). Naslove tabel pišemo nad njimi, naslove slik in fotografi j pod njimi. Naslovi tabel in slik ter legenda so v slovenskem in angleškem jeziku. Pri citiranju tabel in slik v besedilu uporabljamo okrajšave (npr. Tab. 1 ali Tabs. 1-2, Fig. 1 ali Figs. 1-2; Tab. 1 in Sl. 1). 9. Zakjučki Članek končamo s povzetkom glavnih ugotovitev, ki jih lahko zapišemo tudi po točkah. 34 Acta Biologica Slovenica, 49 (1), 2006 10. Povzetek – Summary Članek, ki je pisan v slovenskem jeziku, mora vsebovati še obširnejši angleški povzetek. Velja tudi obratno. 11. Literatura Uporabljene literaturne vire citiramo med tekstom. Če citiramo enega avtorja, pišemo ALLAN (1995) ali (ALLAN 1995), če sta dva avtorja (TRINAJSTIĆ & FRANJIĆ 1994), če je več avtorjev (PULLIN & al. 1995). Kadar navajamo citat iz večih del hkrati, pišemo (HONSIG-ERLENBURG & al. 1992, WARD 1994a, ALLAN 1995, PULLIN & al. 1995). V primeru, če citiramo več del istega avtorja, objavljenih v enem letu, posamezno delo označimo s črkami a, b, c itd. (WARD 1994a,b). Če navajamo dobesedni citat, označimo dodatno še strani: TOMAN (1992: 5) ali (TOMAN 1992: 5-6). Literaturo uredimo po abecednem redu, začnemo s priimkom prvega avtorja, sledi leto izdaje in naslov članka, mednarodna kratica za revijo (časopis), volumen poudarjeno, številka v oklepaju in strani. Npr.: HONSIG-ERLENBURG W., K. KRAINER, P. MILDNER & C. WIESER 1992: Zur Flora und Fauna des Webersees. Carinthia II 182/102 (1): 159-173. TRINAJSTIĆ & J. FRANJIĆ 1994: Ass. Salicetum elaeagno-daphnoides (BR.-BL. et VOLK, 1940) M. MOOR 1958 (Salicion elaeagni) in the Vegetation in Croatia. Nat. Croat. 3 (2): 253-256. WARD J. V. 1994a: Ecology of Alpine Streams. Freshwater Biology 32 (1): 10-15. WARD J. V. 1994b: Ecology of Prealpine Streams. Freshwater Biology 32 (2): 10-15. Knjige, poglavja iz knjig, poročila, kongresne povzetke citiramo sledeče: ALLAN J. D. 1995: Stream Ecology. Structure and Function of Running Waters, 1st ed. Chapman & Hall, London, 388 pp. PULLIN A. S., I. F. G. MCLEAN & M. R. WEBB 1995: Ecology and Conservation of Lycaena dispar: British and European Perspectives. In: PULLIN A. S. (ed.): Ecology and Conservation of Butterfl ies, 1st ed. Chapman & Hall, London, pp. 150-164. TOMAN M. J. 1992: Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s posvetovanja DZVS, Gozd Martuljek, pp. 1-7. 12. Format in oblika članka Članek naj bo poslan v obliki Word dokumenta (doc) ali kot obogateno besedilo (rtf) v pisavi “Times New Roman CE 12” z dvojnim medvrstnim razmakom in levo poravnavo ter s 3 cm robovi na A4 formatu. Odstavki naj bodo med seboj ločeni s prazno vrstico. Naslov članka in poglavij naj bodo pisani krepko in v velikosti pisave 14. Vsa latinska imena morajo biti napisana ležeče. Uporabljene nomenklaturne vire navedemo v poglavju Metode. Tabele in slike so posebej priložene tekstu. Vse strani (vključno s tabelami in slikami) morajo biti oštevilčene. Glavnemu uredniku je potrebno oddati original, dve kopiji in elektronski zapis na disketi 3,5”, na CD-romu ali kot priponko elektronske pošte (slednjega odda avtor po opravljenih strokovnih in jezikovnih popravkih). 13. Recenzije Vsak znanstveni članek bosta recenzirala dva recenzenta (en domači in en tuji), kratko notico pa domači recenzent. Avtor lahko v spremnem dopisu predlaga tuje recenzente. Recenziran članek, ki bo sprejet v objavo, popravi avtor. Po objavi prejme 30 brezplačnih izvodov. V primeru zavrnitve se originalne materiale vrne avtorju skupaj z negativno odločitvijo glavnega urednika. INSTRUCTIONS FOR AUTHORS 1. Types of Articles a) SCIENTIFIC ARTICLES are comprehensive descriptions of original research and include a theoretical survey of the topic, a detailed presentation of results with discussion and conclusion, and a bibliography according to the IMRAD outline (Introduction, Methods, Results, and Discussion). The length of an article including tables, 35 graphs, and illustrations may not exceed fi fteen (15) pages; lines must be double-spaced. Scientifi c articles shall be subject to peer review by two experts in the fi eld. b) REVIEW ARTICLES will be published in the journal after consultation between the editorial board and the author. Review articles may be longer than fi fteen (15) pages. c) BRIEF NOTES are original articles from various biological fi elds (systematics, biochemistry, genetics, microbiology, ecology, etc.) that do not include a detailed theoretical discussion. Their aim is to acquaint readers with preliminary or partial results of research. They should not be longer than fi ve (5) pages. Brief note articles shall be subject to peer review by one expert in the fi eld. d) CONGRESS NEWS acquaints readers with the content and conclusions of important congresses and seminars at home and abroad. e) ASSOCIATION NEWS reports on the work of Slovene biology associations. 2. Originality of Articles Manuscripts submitted for publication in Acta Biologica Slovenica should not contain previously published material and should not be under consideration for publication elsewhere. 3. Language Articles and notes should be submitted in English, or as an exception in Slovene if the topic is very local. As a rule, congress and association news will appear in Slovene. 4. Titles of Articles Titles (in Slovene and English) must be short, informative, and understandable. The title should be followed by the name and full address of the author (and if possible, fax number and e-mail address). 5. Abstract The abstract must give concise information about the objective, the methods used, the results obtained, and the conclusions. The suitable length for scientifi c articles is approximately 250 words, and for brief note articles, 100 words. 6. Keywords There should be no more than ten (10) keywords; they must refl ect the fi eld of research covered in the article. Authors must add keywords in English to articles written in Slovene. 7. Introduction The introduction must refer only to topics presented in the article or brief note. 8. Illustrations and Tables Articles should not contain more than ten (10) illustrations (graphs, dendrograms, pictures, photos etc.) and tables, and their positions in the article should be clearly indicated. All illustrative material should be provided as physical originals (photographs or illustrations). Tables with their legends should be submitted on separate pages (only horizontal lines should be used in tables). Titles of tables should appear above the tables, and titles of photographs and illustrations below. Titles of tables and illustrations and their legends should be in both Slovene and English. Tables and illustrations should be cited shortly in the text (Tab. 1 or Tabs. 1-2, Fig. 1 or Figs. 1-2; Tab. 1 and Sl. 1). 9. Conclusions Articles shall end with a summary of the main fi ndings which may be written in point form. 36 Acta Biologica Slovenica, 49 (1), 2006 10. Summary Articles written in Slovene must contain a more extensive English summary. The reverse also applies. 11. Literature References shall be cited in the text. If a reference work by one author is cited, we write ALLAN (1995) or (ALLAN 1995); if a work by two authors is cited, (TRINAJSTIĆ & FRANJIĆ 1994); if a work by three or more authors is cited, (PULLIN & al. 1995); and if the reference appears in several works, (HONSIG-ERLENBURG & al. 1992, WARD 1994a, ALLAN 1995, PULLIN & al. 1995). If several works by the same author published in the same year are cited, the individual works are indicated with the added letters a, b, c, etc.: (WARD 1994a,b). If direct quotations are used, the page numbers should be included: TOMAN (1992: 5) or (TOMAN 1992: 5-6). The bibliography shall be arranged in alphabetical order beginning with the surname of the fi rst author fol- lowed by the year of publication, the title of the article, the international abbreviation for the journal (periodical), the vo-lume (in bold print), the number in parenthesis, and the pages. Examples: HONSIG-ERLENBURG W., K. KRAINER, P. MILDNER & C. WIESER 1992: Zur Flora und Fauna des Webersees. Carinthia II 182/102 (1): 159-173. TRINAJSTIĆ & J. FRANJIĆ 1994: Ass. Salicetum elaeagno-daphnoides (BR.-BL. et VOLK, 1940) M. MOOR 1958 (Salicion elaeagni) in the Vegetation in Croatia. Nat. Croat. 3 (2): 253-256. WARD J. V. 1994a: Ecology of Alpine Streams. Freshwater Biology 32 (1): 10-15. WARD J. V. 1994b: Ecology of Prealpine Streams. Freshwater Biology 32 (2): 10-15. Books, chapters from books, reports, and congress anthologies use the following forms: ALLAN J. D. 1995: Stream Ecology. Structure and Function of Running Waters, 1st ed. Chapman & Hall, London, 388 pp. PULLIN A. S., I. F. G. Mclean & M. R. Webb 1995: Ecology and Conservation of Lycaena dispar: British and European Perspectives. In: Pullin A. S. (ed.): Ecology and Conservation of Butterfl ies, 1st ed. Chapman & Hall, London, pp. 150-164. TOMAN M. J. 1992: Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s posvetovanja DZVS, Gozd Martuljek, pp. 1-7. 12. Format and Form of Articles Articles should be send as Word document (doc) or Rich text format (rtf) using “Times New Roman CE 12” font with double spacing, align left and margins of 3 cm on A4 pages. Paragraphs should be separated with an empty line. The title and chapters should be written bold in font size 14. All scientifi c names must be properly italicized. Used nomenclature source should be cited in the Methods section. Tables and illustrations shall accompany the texts separately. All pages including tables and fi gures should be numbered. The original manuscript, two copies, and an electronic copy (after all corrections) on a 3.5” computer diskette, on CD-ROM or by e-mail must be given to the editor-in-chief. All articles must be proofread for professional and language errors before submission. 13. Peer Review All Scientifi c Articles shall be subject to peer review by two experts in the fi eld (one Slovene and one foreign) and Brief Note articles by one Slovene expert in the fi eld. Authors may nominate a foreign reviewer in an accom- panying letter. Reviewed articles accepted for publication shall be corrected by the author. Authors shall receive thirty (30) free copies of the journal upon publication. In the event an article is rejected, the original material shall be returned to the author together with the negative determination of the editor-in-chief.