Differentiation of Closely Related Carnobacterium Food Isolates Based on 16S-23S Ribosomal DNA Intergenic Spacer Region Polymorphism

A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763^T and C. mobile DSM 4849^T generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766^T and C. piscicola NCDO 2762^T were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNA^Ile and tRNA^Ala genes. The M-ISR included one tRNA^Ala gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with HinfI, TaqI, and HindIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing.     

Identification of Carnobacterium species by restriction fragment length polymorphism of the 16S-23S rRNA gene intergenic spacer region and species-specific PCR

The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNA(Ala) and tRNA(Ile), which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria.     

A rapid PCR procedure for the specific identification of Lactobacillus sanfranciscensis, based on the 16S-23S intergenic spacer regions

AIMS: The organization of ribosomal RNA (rrn) operons in Lactobacillus sanfranciscensis was studied in order to establish an easy-to-perform method for identification of L. sanfranciscensis strains, based on the length and sequence polymorphism of the 16S-23S rDNA intergenic spacer region (ISR). METHODS AND RESULTS: PCR amplification of the 16S-23S rDNA ISRs of L. sanfranciscensis gave three products distinguishing this micro-organism from the remaining Lactobacillus species. Sequence analysis revealed that two of the rrn operons were organized as in previously reported lactobacilli: large spacer (L-ISR), containing tRNA(Ile) and tRNA(Ala) genes; small spacer (S-ISR) without tRNA genes. The third described spacer (medium, M-ISR), original for L. sanfranciscensis, harboured a tRNA-like structure. An oligonucleotide sequence targeting the variable region between tDNA(Ile) and tDNA(Ala) of L. sanfranciscensis L-ISR was approved to be suitable in species-specific identification procedure. Analysis by pulse-field gel electrophoresis of the chromosomal digest with the enzyme I-CeuI showed the presence of seven rrn clusters. Lactobacillus sanfranciscensis genome size was estimated at c. 1.3 Mb. CONCLUSIONS: Direct amplification of 16S-23S ISRs or PCR with specific primer derived from L-ISR showed to be useful for specific typing of L. sanfranciscensis. This was due to the specific rrn operon organization of L. sanfranciscensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have reported a rapid procedure for L. sanfranciscensis identification based on specific structures found in its rrn operon.     

Identification of Carnobacterium Species by Restriction Fragment Length Polymorphism of the 16S-23S rRNA Gene Intergenic Spacer Region and Species-Specific PCR

The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNA^Ala and tRNA^Ile, which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria.     

Polyphasic taxonomic studies of lactic acid bacteria associated with Tunisian fermented meat based on the heterogeneity of the 16S–23S rRNA gene intergenic spacer region

The objective of this work was to investigate the structure and diversity of lactic acid bacteria (LAB) communities in traditionally fermented meat collected from different areas of Tunisia. A polyphasic study, which involves phenotypic tests and ribosomal DNA-based techniques, was used to identify Gram-positive and catalase-negative isolates. PCR amplification of the 16S–23S rDNA ISR of 102 isolates and other reference LAB strains gave (1) one type of rrn operon (M-ISR) for lactococci, (2) two types of rrn operon (S-ISR and M-ISR) for enterococci, (3) two types of rrn operon (S-ISR and L-ISR) for Lactobacilli, and (4) three PCR amplicons (S-ISR, M-ISR, and L-ISR) obtained for Pediococcus spp. and Weissella genus. The clustering and comparison of ISR–RFLP profiles given by the isolates with those given by reference LAB strains, allowed their identification as Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Enterococcus sanguinicola, Enterococcus hawaiiensis, Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus alimentarius, Pediococcus pentosaceus, and Weissella confusa. Combined 16S–23S rDNA ISR and RFLP patterns can be considered as a good potential target for a rapid and reliable differentiation between isolates of LAB and provided further information on the organization of their rrn operons.     

Molecular analysis of the 16S-23S rDNA internal spacer region (ISR) and truncated tRNAAla gene segments in Campylobacter lari

Following PCR amplification and sequencing, nucleotide sequence alignment analyses demonstrated the presence of two kinds of 16S-23S rDNA internal spacer regions (ISRs), namely, long length ISRs of 837-844 base pair (bp) [n = six for urease-negative (UN) Campylobacter lari isolates, UN C. lari JCM2530(T), RM2100, 176, 293, 299 and 448] and short length ISRs of 679-725 bp [n = six for UN C. lari: n = 14 for urease-positive thermophilic Campylobacter (UPTC) isolates]. The analyses also indicated that the short length ISRs mainly lacked the 156 bp sequence from the nucleotide positions 122-277 bp in long length ISRs for UN C. lari JCM2530(T). The 156 bp sequences shared 94.9-96.8 % sequence similarity among six isolates. Surprisingly, atypical tRNA(Ala) gene segment (5' end 35 bp), which was extremely truncated, occurred within the 156 bp sequences in the long length ISRs, as an unexpected tRNA(Ala) pseudogene. An order of the intercistronic tRNA genes within the short nucleotide spacer of 5'-16S rDNA-tRNA(Ala)-tRNA(Ile)-23S rDNA-3' occurred in all the C. lari isolates examined.     

A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences

Aims:  Species-specific primers targeting the 16S–23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis , Lactobacillus panis , Lactobacillus paralimentarius , Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough.
Methods and Results:  The 16S–23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388–1406 of the 16S rRNA gene and to positions 207–189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331 ). Clone libraries of the resulting amplicons were constructed using a pCR2·1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S–23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA^Ile and tRNA^Ala genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested.
Conclusions:  Designed species-specific primers enable a rapid and accurate identification of L. mindensis , L. paralimentarius , L. panis , L. pontis and L. frumenti species among other lactobacilli.
Significance and Impact of the Study:  The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.     

Analysis of 16S-23S intergenic spacer regions of the rRNA operons in Edwardsiella ictaluri and Edwardsiella tarda isolates from fish

AIMS: To analyse interspecies and intraspecies differences based on the 16S-23S rRNA intergenic spacer region (ISR) sequences of the fish pathogens Edwardsiella ictaluri and Edwardsiella tarda. METHODS AND RESULTS: The 16S-23S rRNA spacer regions of 19 Edw. ictaluri and four Edw. tarda isolates from four geographical regions were amplified by PCR with primers complementary to conserved sequences within the flanking 16S-23S rRNA coding sequences. Two products were generated from all isolates, without interspecies or intraspecific size polymorphisms. Sequence analysis of the amplified fragments revealed a smaller ISR of 350 bp, which contained a gene for tRNA(Glu), and a larger ISR of 441 bp, which contained genes for tRNA(Ile) and tRNA(Ala). The sequences of the smaller ISR of different Edw. ictaluri isolates were essentially identical to each other. Partial sequences of larger ISR from several Edw. ictaluri isolates also revealed no differences from the one complete Edw. ictaluri large ISR sequence obtained. The sequences of the smaller ISR of Edw. tarda were 97% identical to the Edw. ictaluri smaller ISR and the larger ISR were 96-98% identical to the Edw. ictaluri larger ISR sequence. The Edw. tarda isolates displayed limited ISR sequence heterogeneity, with > or =97% sequence identity among isolates for both small and large ISR. CONCLUSIONS: There is a high degree of size and sequence similarity of 16S-23S ISR both among isolates within Edw. ictaluri and Edw. tarda species and between the two species. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results confirm a close genetic relationship between Edw. ictaluri and Edw. tarda and the relative homogeneity of Edw. ictaluri isolates compared with Edw. tarda isolates. Because no differences were found in ISR sequences among Edw. ictaluri isolates, sequence analysis of the ISR will not be useful to distinguish isolates of Edw. ictaluri. However, we identified restriction sites that differ between ISR sequences of Edw. ictaluri and Edw. tarda, which will be useful in distinguishing the two species.     

Differentiation of petroleum hydrocarbon-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. based on PCR-RFLP of the 16S-23S rDNA intergenic spacer region

Genetic diversity among 43 petroleum hydrocarbon-degrading Pseudomonas belonging to four different species and the type strain Pseudomonas aeruginosa MTCC1034 was assessed by using restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified 16S–23S rDNA intergenic spacer regions (ISRs) polymorphism. PCR amplification from all Pseudomonas species yielded almost identical ISR amplicons of “?” 800 bp and in nested PCR of “?” 550 bp. The RFLP analysis with MboI and AluI revealed considerable intraspecific variations within the Pseudomonas species. The dendrogram constructed on the basis of the PCR-RFLP patterns of 16S–23S rDNA intergenic spacer regions differentiated all the species into seven different clusters.     

Recombination Drives Evolution of the Clostridium difficile 16S-23S rRNA Intergenic Spacer Region

PCR-ribotyping, a typing method based on size variation in 16S-23S rRNA intergenic spacer region (ISR), has been used widely for molecular epidemiological investigations of C. difficile infections. In the present study, we describe the sequence diversity of ISRs from 43 C. difficile strains, representing different PCR-ribotypes and suggest homologous recombination as a possible mechanism driving the evolution of 16S-23S rRNA ISRs. ISRs of 45 different lengths (ranging from 185 bp to 564 bp) were found among 458 ISRs. All ISRs could be described with one of the 22 different structural groups defined by the presence or absence of different sequence modules; tRNA^Ala genes and different combinations of spacers of different lengths (33 bp, 53 bp or 20 bp) and 9 bp direct repeats separating the spacers. The ISR structural group, in most cases, coincided with the sequence length. ISRs that were of the same lengths had also very similar nucleotide sequence, suggesting that ISRs were not suitable for discriminating between different strains based only on the ISR sequence. Despite large variations in the length, the alignment of ISR sequences, based on the primary sequence and secondary structure information, revealed many conserved regions which were mainly involved in maturation of pre-rRNA. Phylogenetic analysis of the ISR alignment yielded strong evidence for intra- and inter-homologous recombination which could be one of the mechanisms driving the evolution of C. difficile 16S-23S ISRs. The modular structure of the ISR, the high sequence similarities of ISRs of the same sizes and the presence of homologous recombination also suggest that different copies of C. difficile 16S-23S rRNA ISR are evolving in concert.     

Restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer of Aeromonas spp

We analyzed restriction fragment length polymorphism (RFLP) of 16S-23S rDNA intergenic spacer region (ISR) of Aeromonas species. A total of 69 isolates belonging to 18 DNA hybridization groups (HG; equivalent of genomic species) were used in this study. ISRs were amplified by PCR and the products were digested with four restriction endonucleases: Hin6I, Csp6I, TaqI, and TasI. The RFLP patterns obtained after digesting by particular enzymes revealed ISR polymorphism of isolates allocated to individual genomic species. The combined Hin6I, Csp6I, TaqI, and TasI restriction profiles were examined by Dice coefficient (SD) and unweighted pair group method of clustering (UPGMA). The isolates were allocated into 15 groups, three strains were unclustered. The strains belonging to the following genomic species: A. hydrophila, A. bestiarum, A. salmonicida, A. caviae, A. media, A. schubertii, A. allosaccharophila, A. popoffii, and A. culicicola formed distinct clusters. Strains belonging to HG 6, HG 7, HG 11, and HG 16 revealed similar combined RFLP patterns and constituted one group. Similarly, the strains of A. jandaei (HG 9) and the type strain of A. trota were allocated into one cluster. Two isolates of HG 14 formed distinct cluster. We noticed a genetic diversity among A. veronii isolates, the strains were clustered in two groups. Our study showed that combined ISR-RFLP analysis may be used for identification of some species of Aeromonas.     

16S-23S ribosomal DNA intergenic spacer regions in cellulolytic myxobacteria and differentiation of closely related strains

The diversity of 16S-23S rDNA intergenic spacer regions (ISR) among cellulolytic myxobacterial strains was assayed. Agarose gel electrophoresis of PCR amplification products from ten strains shows that there are at least four copies of rRNA operons in the genus Sorangium, based on their size and restriction enzymatic digest maps. There are two sequence organization patterns: tRNA(Ile)-tRNA(Ala)-containing ISR and tRNA-lacking ISR. The tRNA-containing ISRs are highly similar among strains and within a strain (more than 98% similarity) and contain the essential functional regions, such as a ribonuclease III recognition site and an antiterminator recognition site boxA. The tRNA-lacking ISR has no such functional sites that are important for yielding mature rRNA, which suggests that this type of rRNA operons might be degenerate. The tRNA-lacking ISR is divided into two types based on their sizes and sequences, which exhibits about 90% similarity within each type. Thus, the tRNA-lacking ISR polymorphisms can be used to discriminate among different strains of sorangial species.     

16S～23S RDNA间区在链球菌和流感嗜血杆菌分类中的应用

利用16S~23S rDNA间区（intergenic spacer regions,ISR）在不同细菌中拷贝数、碱基排列、序列长度及所含tRNA基因种类和数目的差异,对15株链球菌和流感嗜血杆菌进行属、种、型和株系的分类鉴定。在16S rDNA的3′端和23S rDNA的5′端的保守区中合成引物,PCR扩增16S~23S rDNA ISR序列,对多态片段切胶纯化直接测序。在GenBank上查找对应细菌的ISR序列。用DNAMAN软件进行系统进化分析。链球菌属为单拷贝16S~23Sr RNA ISR、有一个tRNAAla基因编码区、分子大小在269~446bp之间,序列分成4个保守区和4个可变区,可变区碱基排列方式和数目的不同是种分类的依据。7株链球菌的同源率在78%~88%。同种异株的差异反映在碱基的插入和缺失上。流感嗜血杆菌各生物型均为2个拷贝的ISR,小片段为514~519bp,编码1个tRNAGlu基因,有3个狭窄可变区。大片段富含A T碱基,在I、II和IV型中分别是868、848和856bp,编码一个tRNAIle基因和一个tRNAAla基因。不同生物型小分子ISR与标准菌株比较,同源性在97.3%~99.6 %之间。 ISR作为细菌分类的目的基因具有属、种、型和株特异性与灵敏性。简单的基因分离分析技术为认识病原微生物提供了更多的机会。 Abstract:To facilitate species level identification of bacteria without the requirement of presumptive identification,the paper describes a rapid identification method of bacteria by amplification and direct sequencing 16S~23S rDNA intergenic spacer regions (ISR) of the pathogens which cause the upper respiratory tract infective disease by Streptococcus and Haemophilus.Three pairs of primer targeting conserved sequences flanking the 3′ end of 16S and the 5′end of 23S rRNA were used to amplify 16S~23S rRNA ISR of 7 streptococcus strains and 8 Haemophilus strains.The PCR products were separated by 1% agarose gel electrophoresis and the polymorphisms fragments were purified with the Wizard PCR Min-Prep Kit (Promega) and Protocol-SK131(Sangon).The nucleotide sequences of ISR inserts were determined by using the XEQTM DTCS Kit——Terminator Cycle Sequencing and a CEQTM 2000XL DNA Analysis system (Backman Coulter) automatic DAN sequencer.Then those sequences were compared with known seqnences on the GenBank.The alignment of nucleotide sequence,evolutionary distances and phylogenetic tress were analyzed by software DANMAN version 4.0.The PCR products were showed polymorphism patterns with agarose gel.One band was contained in streptococcus genus.The significant variation was found among the spacer sequences of different species in Streptococcus with the lengths of the spacer varying from 269 to 446bp.All the ISR of the streptococcal species had a tRNA Ala gene in the spacer and the sequence identities varied from 78 to 88% within genera.It was found that some spacer sequence blocks were highly conserved between operons of a genome,whereas the presence of others was variable,three regions showed significant spatial variation.Most of the differences between the sequences came from several bases insertions/deletions and substitutions.There are two major bands in the Haemophilus biotypes(515 and 884bp),the small ISR amplicon contained one tDNA coding for tRNAGlu.In contrast to the large one contained two tRNA genes coding for tRANAla and tRNAIle.Two regions of repeating motifs with only A or T were present in higher copy numbers between tRANAla and tRNAIle.The phylogenetic trees varied from 97.5 to 98.8%.The PCR and direct sequencing of 16S~23S rRAN ISR were successful in the pathogen species identification.     

The mosaic nature of intergenic 16S-23S rRNA spacer regions suggests rRNA operon copy number variation in Clostridium difficile strains

Clostridium difficile is a major spore-forming environmental pathogen that causes serious health problems in patients undergoing antibiotic therapy. Consequently, reliable and sensitive methods for typing individual strains are required for epidemiological and environmental studies. Ribotyping is generally considered the best method, but it fails to account for sequence diversity which might exist in intergenic 16S-23S rRNA spacer regions (ISRs) within and among strains of this organism. Therefore, this study was undertaken to compare the sequence of each individual ISR in five strains of C. difficile to explore the extent of this diversity and see whether such information might provide the basis for more sensitive and discriminatory strain typing methods. After targeted PCR amplification, cloning, and sequencing, the diversity of the ISRs was used as a measure of rRNA operon copy number. In C. difficile strains 630, ATCC 43593, A, and B, 11, 11, 7, and 8 ISR length variants, respectively, were found (containing different combinations of sequence groups [i to xiii]), suggesting 11, 11, 7, and 8 rrn copies in the respective strains. Many ISRs of the same length differed markedly in their sequences, and some of these were restricted in occurrence to a single strain. Most of these ISRs did not contain any tRNA genes, and only single copies of the tRNA(Ala) gene were found in those that did. The presence of ISR sequence groups (i to xiii) varied between strains, with some found in one, two, three, four, or all five strains. We conclude that the intergenic 16S-23S rRNA spacer regions showed a high degree of diversity, not only among the rrn operons in different strains and different rrn copies in a single strain but also among ISRs of the same length. It appears that C. difficile ISRs vary more at the inter- and intragenic levels than those of other species as determined by empirical comparison of sequences. The precise characterization of these sequences has demonstrated a high level of mosaic sequence block rearrangements that are present or absent in multiple strain-variable rrn copies within and between five different strains of C. difficile.     

Variation of 16S-23S rRNA intergenic spacer regions (ISRs) in Acinetobacter baylyi (strain B2) isolated from activated sludge

To determine the variability of the 16S-23S rRNA intergenic spacer region (ISR) of the newly described Acinetobacter baylyi, 88 clones containing ISR amplicons were screened and 14 chosen for further analysis. Two different sized 16S-23S rRNA ISRs were distinguished comprising five variable and four conserved nucleotide blocks. The major regions of heterogeneity between the different sized ISRs were due to blocks of substitutions with unique secondary structures interspersed with nucleotide substitutions, rather than differences caused by presence or absence of tRNA genes, which is often the case. Recombination events causing shuffling of nucleotide blocks are considered the most likely explanation for the mosaic structure observed between the different copies of the ISR. Single base differences present in the long ISR (LISR) were then exploited in attempts to detect possible heterogeneity between rrn copies in Acinetobacter baylyi but variability was not detected by RFLP analysis of LISR-specific PCR products. These primers were shown to be highly specific for 3 Acinetobacter baylyi strains based on LISR sequence homogeneity.     

Genetic similarity among Cercospora apii-group species and their detection in host plant tissue by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS)

The objective of this study was to determine the genetic relatedness among the Cercospora and Pseudocercospora species closely related to Cercospora apii by using a polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the internal transcribed spacer (ITS) region. A single PCR fragment (about 550 bp) was obtained from all Cercospora species categorized as the C. apii-group, Pseudocercospora purpurea, Pseudocercospora conyzae, and Pseudocercospora cavarae. Cercospora caricis yielded a 680 bp PCR fragment. The similarity in the PCR fragment size and RFLP profiles among the C. apii-group isolates, including Pseudocercospora purpurea, and Pseudocercospora conyzae strongly suggests that these species are conspecific. Synonymy with C. apii (lectotype) at a subspecific rank has been proposed. Amplified ITS regions of genomic DNA extracted from spinach leaves showing 12 and 233% leaf spot disease symptoms caused by Cercospora beticola yielded two PCR fragments (i.e., one from the fungus and one from the host plant) and were resolved by electrophoresis of the PCR product in 3% LMP agarose. Digestion of the total PCR product with HinfI restriction enzyme yielded RFLP profiles similar to those obtained from amplified DNA from the causative agent, C. beticola. The method described in this preliminary study offers rapid detection and diagnosis of fungal infections in plants for disease prediction and management and screening of plant materials for quarantine purposes.     

Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains

AIMS: To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. METHODS AND RESULTS: The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. CONCLUSIONS: Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. SIGNIFICANCE AND IMPACT OF THE STUDY: A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.     

Use of molecular methods in identification of Candida species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.     

Identification of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus by rDNA-based techniques

Ribosomal DNA-based techniques including the analysis of profiles generated by ISR amplification, ISR restriction and ARDRA have been evaluated as molecular tools for identifying Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. They have been applied for the molecular characterization of 91 strains with the following identities: eight Carnobacterium including the eight type species of the genus; 61 Lactobacillus including 40 type strains out of 45 species, 13 Leuconostoc, out of them 11 are type strains and three are subspecies of Lc. mesenteroides; and nine strains representing the six species of genus Pediococcus. The genetic relationship displayed between these species by rrn-based profiles is sustained by their phylogenetic relationships and can therefore be considered useful for taxonomic purposes. Profiles obtained by ISR amplification allowed identification at genus level of Carnobacterium and Leuconostoc, and even at species level in genus Carnobacterium. Genera Lactobacillus and Pediococcus could not be distinguished from each other by applying this technique. The Lactobacillus species analysed here (45) were differentiated using ARDRA-DdeI and ISR-DdeI profiles, sequentially, and Pediococcus species by ISR-DdeI profiles. It was necessary to combine profiles generated by restriction of ISR-DdeI, ARDRA-DdeI and ARDRA-HaeIII in order to complete the identification of Leuconostoc species.     

DNA array with the groESL intergenic sequence to detect Vibrio parahaemolyticus and Vibrio vulnificus

The untranscribed DNA sequences of the intergenic spacer regions (ISRs) in the groESL gene of 23 Vibrio species were determined and compared. ISR sequence length (41-85 bp) was variable. Vibrio species could be divided into three groups according to the length and homology of their ISR sequences. DNA array hybridization using ISR-specific probes accurately distinguished Vibrio parahaemolyticus and Vibrio vulnificus from other species.