Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro-intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low-resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum-marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism.

The genus Xanthomonas contains a large number of strains, which have been characterized by a variety of phenotypic and genotypic classification methods. The Xanthomonas collection constitutes one of the largest groups of bacteria that have been characterized phylogenetically by DNA-DNA homology studies and genomic fingerprinting. Presently, a total genomic DNA-DNA homology value of 70 % represents an internationally accepted criterion to define bacterial species levels. However, the complexity of DNA-DNA reassociation kinetics methods precludes the rapid analysis of large numbers of bacterial isolates, which is imperative for molecular microbial diversity studies. Therefore, the aim of this study was to compare more facile PCR-based genomic fingerprinting techniques, such as repetitive-sequence-based (rep)-PCR and AFLP genomic fingerprinting, to DNA-DNA hybridization studies. Using three different primer sets, rep-PCR genomic fingerprint patterns were generated for 178 Xanthomonas strains, belonging to all 20 previously defined DNA-DNA homology groups, and one Stenotrophomonas maltophilia strain. In addition, AFLP genomic fingerprints were produced for a subset of 80 Xanthomonas strains belonging to the 20 DNA-DNA homology groups and for the S. maltophilia strain. Similarity values derived from rep-PCR-and AFLPgenerated fingerprinting analyses were calculated and used to determine the correlation between rep-PCR-or AFLP-derived relationships and DNA-DNA homology values. A high correlation was observed, suggesting that genomic fingerprinting techniques truly reveal genotypic and phylogenetic relationships of organisms. On the basis of these studies, we propose that genomic fingerprinting techniques such as rep-PCR and AFLP can be used as rapid, highly discriminatory screening techniques to determine the taxonomic diversity and phylogenetic structure of bacterial populations.

The advent of molecular biology in general and the polymerase chain reaction in particular have greatly facilitated genomic analyses of microorganisms, provide enhanced capability to characterize and classify strains, and facilitate research to assess the genetic diversity of populations. The diversity of large populations can be assessed in a relatively efficient manner using rep-PCR-, AFLP-, and AP-PCR/RAPD-based genomic fingerprinting methods, especially when combined with computer-assisted pattern analysis. Genetic diversity maps provide a framework to understand the taxonomy, population structure, and dynamics of phytobacteria and provide a high-resolution framework to devise sensitive, specific, and rapid methods for pathogen detection, plant disease diagnosis, as well as management of disease risk. A variety of PCR-based fingerprinting protocols such as rDNA-based PCR, ITS-PCR, ARDRA, T-RFLPs, and tRNA-PCR have been devised, and numerous innovative approaches using specific primers have been adopted to enhance both the detection and identification of phytobacteria. PCR-based protocols, combined with computer-based analysis, have provided novel fundamental knowledge of the ecology and population dynamics of bacterial pathogens, and present exciting new opportunities for basic and applied studies in plant pathology.

In the past decade, various methods have been developed for the identification and typing of prokaryotic and eukaryotic organisms at the DNA level. These methods differ in their taxonomic range, discriminatory power, reproducibility, and ease of interpretation and standardization (62,67,86,87,101,106,110,116). The ideal genotyping method produces results that are invariable from laboratory to laboratory and allows unambiguous comparative analyses and the establishment of reliable databases.One of the newest and most promising methods is amplifiedfragment length polymorphism (AFLP) analysis (11,118,122), developed by Keygene BV, Wageningen, The Netherlands. This method combines universal applicability with high powers of discrimination and reproducibility (45). An increasing number of reports describe the use of AFLP analysis for plant and animal genetic mapping, medical diagnostics, phylogenetic studies, and microbial typing. This minireview describes the principles, advantages, and disadvantages of AFLP analysis and summarizes its applications in different fields. PRINCIPLE OF AFLPIn the nomenclature of Vaneechoutte (110), AFLP analysis belongs to the category of selective restriction fragment amplification techniques, which are based on the ligation of adapters (i.e., linkers and indexers) to genomic restriction fragments followed by a PCR-based amplification with adapterspecific primers. For AFLP analysis ( Fig. 1), only a small amount of purified genomic DNA is needed; this is digested with two restriction enzymes, one with an average cutting frequency (like EcoRI) and a second one with a higher cutting frequency (like MseI or TaqI). Double-stranded oligonucleotide adapters are designed in such a way that the initial restriction site is not restored after ligation, which allows simultaneous restriction and ligation, while religated fragments are cleaved again. An aliquot is then subjected to two subsequent PCR amplifications under highly stringent conditions with adapter-specific primers that have at their 3Ј ends an extension of one to three nucleotides running into the unknown chromosomal restriction fragment. An extension of one selective nucleotide amplifies 1 of 4 of the ligated fragments, whereas three selective nucleotides in both primers amplify 1 of 4,096 of the fragments. The PCR primer which spans the average-frequency restriction site is labeled. After polyacrylamide gel electrophoresis a highly informative pattern of 40 to 200 bands is obtained. The patterns obtained from different strains are polymorphic due to (i) mutations in the restriction sites, (ii) mutations in the sequences adjacent to the restriction sites and complementary to the selective primer extensions, and (iii) insertions or deletions within the amplified fragments.Since the original publication by Vos et al. in 1995 (118) several enzyme combinations have been used, such as EcoRI, PstI, HindIII, or ApaI combined with MseI or TaqI. For animal genomes EcoRI and TaqI appear to be the most suitable (2). Alternative AFLP typing proce...