Genome plasticity among related ++Lactococcus strains: identification of genetic events associated with macrorestriction polymorphisms

The genomic diversity of nine strains of the Lactococcus lactis subsp. cremoris (NCDO712, NCDO505, NCDO2031, NCDO763, MMS36, C2, LM0230, LM2301, and MG1363) was studied by macrorestriction enzyme analysis using pulsed-field gel electrophoresis. These strains were considered adequate for the investigation of genomic plasticity because they have been described as belonging to the same genetic lineage. Comparison of ApaI and SmaI genome fingerprints of each strain revealed the presence of several macrorestriction fragment length polymorphisms (RFLPs), despite a high degree of similarity of the generated restriction patterns. The physical map of the MG1363 chromosome was used to establish a genome map of the other strains and allocate the RFLPs to five regions. Southern hybridization analysis correlated the polymorphic regions with genetic events such as chromosomal inversion, integration of prophage DNA, and location of the transposon-like structures carrying conjugative factor or oligopeptide transport system.     

DNA polymorphisms in strains of Legionella pneumophila serogroups 3 and 4 detected by macrorestriction analysis and their use for epidemiological investigation of nosocomial legionellosis.

Genomic DNAs of clinical and environmental isolates of Legionella pneumophila belonging to serogroups 3 and 4 were analyzed by macrorestriction analysis by pulsed-field gel electrophoresis. The restriction enzymes SfiI and NotI allowed easy visual separation of epidemiologically unrelated serogroup 3 strains. Three unrelated serogroup 3 strains that were isolated from different locations were identical by this genome mapping technique. Five unrelated serogroup 4 strains were separable by this technique. The electrophoretic patterns obtained after SfiI or NotI cleavage of the DNA of strains isolated from four patients with hospital-acquired legionellosis were identical to the patterns of strains isolated from the hot water supply systems of the buildings in which the patients were hospitalized. In conclusion, macrorestriction analysis is a valuable tool for epidemiological studies of infections caused by L. pneumophila serogroups 3 and 4.     

Genes but not genomes reveal bacterial domestication of Lactococcus lactis

Background

The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST) scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE).

Methodology/Principal Findings

The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content) did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST) differing by up to 230 kb in genome size.

Conclusion/Significance

The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between “environmental” strains, the main contributors to the genetic diversity within the subspecies, and “domesticated” strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the “domesticated” strains essentially arose through substantial genomic flux within the dispensable genome.     

Genomic Relatedness of Chlamydia Isolates Determined by Amplified Fragment Length Polymorphism Analysis

The genomic relatedness of 19 Chlamydia pneumoniae isolates (17 from respiratory origin and 2 from atherosclerotic origin), 21 Chlamydia trachomatis isolates (all serovars from the human biovar, an isolate from the mouse biovar, and a porcine isolate), 6 Chlamydia psittaci isolates (5 avian isolates and 1 feline isolate), and 1 Chlamydia pecorum isolate was studied by analyzing genomic amplified fragment length polymorphism (AFLP) fingerprints. The AFLP procedure was adapted from a previously developed method for characterization of clinical C. trachomatis isolates. The fingerprints of all C. pneumoniae isolates were nearly identical, clustering together at a Dice similarity of 92.6% (+/- 1.6% standard deviation). The fingerprints of the C. trachomatis isolates of human, mouse, and swine origin were clearly distinct from each other. The fingerprints of the isolates from the human biovar could be divided into at least 12 different types when the presence or absence of specific bands was taken into account. The C. psittaci fingerprints could be divided into a parakeet, a pigeon, and a feline type. The fingerprint of C. pecorum was clearly distinct from all others. Cluster analysis of selected isolates from all species revealed groups other than those based on sequence data from single genes (in particular, omp1 and rRNA genes) but was in agreement with available DNA-DNA hybridization data. In conclusion, cluster analysis of AFLP fingerprints of representatives of all species provided suggestions for a grouping of chlamydiae based on the analysis of the whole genome. Furthermore, genomic AFLP analysis showed that the genome of C. pneumoniae is highly conserved and that no differences exist between isolates of respiratory and atherosclerotic origins.     

Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains

Background  

Bacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome.     

Suitability of genomic DNA synthesized by strand displacement amplification (SDA) for AFLP analysis: genotyping single spores of arbuscular mycorrhizal (AM) fungi

Limited biological samples of microbial origin often yield insufficient amounts of genomic DNA, making application of standard techniques of genetic analysis, like amplified fragment length polymorphism (AFLP), virtually impossible. The Phi29 DNA polymerase based whole genome amplification (WGA) method has the potential to alleviate this technical bottleneck. In the present work, we have sought to investigate the suitability of genomic DNA synthesized using Phi29 based WGA for AFLP analysis. We first used genomic DNA from Saccharomyces cerevisiae to optimize the protocol for the use of SDA-amplified DNA for AFLP analysis. Based on the optimized protocol we obtained AFLP fingerprints which were indistinguishable from the non-amplified genomic DNA. Finally, AFLP analysis was performed using SDA synthesized genomic DNA from single spores of various species of arbuscular mycorrhizal (AM) fungi. Unique and highly reproducible fingerprints for each species were obtained. The present study introduces the application of WGA-mediated AFLP to AM fungal biology; similarly, our protocol could be useful for other microbial genomes currently not amenable to genetic analysis owing to the paucity of starting template.     

Phylogeography of sulfate-reducing bacteria among disturbed sediments, disclosed by analysis of the dissimilatory sulfite reductase genes (dsrAB)

Sediment samples were collected worldwide from 16 locations on four continents (in New York, California, New Jersey, Virginia, Puerto Rico, Venezuela, Italy, Latvia, and South Korea) to assess the extent of the diversity and the distribution patterns of sulfate-reducing bacteria (SRB) in contaminated sediments. The SRB communities were examined by terminal restriction fragment (TRF) length polymorphism (TRFLP) analysis of the dissimilatory sulfite reductase genes (dsrAB) with NdeII digests. The fingerprints of dsrAB genes contained a total of 369 fluorescent TRFs, of which <20% were present in the GenBank database. The global sulfidogenic communities appeared to be significantly different among the anthropogenically impacted (petroleum-contaminated) sites, but nearly all were less diverse than pristine habitats, such as mangroves. A global SRB indicator species of petroleum pollution was not identified. However, several dsrAB gene sequences corresponding to hydrocarbon-degrading isolates or consortium members were detected in geographically widely separated polluted sites. Finally, a cluster analysis of the TRFLP fingerprints indicated that many SRB microbial communities were most similar on the basis of close geographic proximity (tens of kilometers). Yet, on larger scales (hundreds to thousands of kilometers) SRB communities could cluster with geographically widely separated sites and not necessarily with the site with the closest proximity. These data demonstrate that SRB populations do not adhere to a biogeographic distribution pattern similar to that of larger eukaryotic organisms, with the greatest species diversity radiating from the Indo-Pacific region. Rather, a patchy SRB distribution is encountered, implying an initially uniform SRB community that has differentiated over time.     

High density whole genome fingerprinting of methicillin-resistant and -susceptible strains of Staphylococcus aureus in search of phenotype-specific molecular determinants

AFLP is a selective restriction fragment amplification method generating DNA fingerprints for microbial isolates. We present high-throughput AFLP (htAFLP) to characterize molecular markers associated with bacterial phenotypes. Methicillin-resistant and -susceptible isolates of Staphylococcus aureus have been used for this model study in conjunction with the available S. aureus genome sequences. This facilitates the calculation of theoretical AFLP fingerprints, comparison of these fingerprints with genuine experimental fingerprints, and the subsequent identification of polymorphic AFLP markers without sequence analysis. Analysis of 46 MRSA and 46 MSSA strains by 39 different AFLP reactions generated more than 2500 fragments per strain and an overall number of 6180 scorable markers within all strains. We successfully identify MRSA specific markers and elaborate on the general applicability of the htAFLP approach. This method can be applied to any microbial species for which at least one full-genome sequence is available.     

Rep-PCR mediated genomic fingerprinting of rhizobia and computer-assisted phylogenetic pattern analysis

A rapid and reproducible method has been developed for genomic fingerprinting of rhizobia and other soil microbes interacting with plants. The method is based on the use of oligonucleotide primers, corresponding to conserved motifs in naturally occurring interspersed repetitive DNA elements in bacteria (rep-elements), and the polymerase chain reaction (rep-PCR). Rep-PCR results in the amplification of inter-element genomic DNA fragments of characteristic lengths and thereby generates a genomic fingerprint. These fingerprints resemble UPC bar code patterns, and can be used to identify bacteria at the sub-species and strain level, as well as for phylogenetic analyses. Here we show that highly characteristic and very reproducible rep-PCR generated genomic fingerprints can be obtained not only from purified genomic DNA, but also directly from rhizobial cells derived from liquid cultures or from colonies on plates, as well as from nodule tissue. We examine the effect of growth phase of the bacterial cells, serial subculturing and other parameters on the reproducibility of the rep-PCR fingerprinting protocol. Moreover, we describe the results of mixing experiments designed to determine if individual genomic fingerprints can be recognized in mixtures of strains. Lastly, we review the use of computer-based fragment detection and phylogentic analysis packages to analyse rep-PCR generated genomic fingerprints of a collection of Rhizobium loti and Bradyrhizobium strains nodulating different Lotus spp.The authors are with the NSF Center for Microbial Ecology and the MSU-DOE Plant Research Laboratory. F. J. de Bruijn is also with the Microbiology Department and Genetics Program of Michigan State University, E. Lansing, MI 48824 USA.     

Comparative genomic analysis of isolates belonging to the six species of the genus Thermus using pulsed-field gel electrophoresis and ribotyping

Fifty isolates belonging to the six validly described species of the genus Thermus (T. aquaticus, T. filiformis, T. thermophilus, T. scotoductus, T. brockianus, and T. oshimai) isolated from hot springs of different geographical areas were compared using macrorestriction analysis of genomic DNA and ribotyping. With the exception of presumed clones, the macrorestriction patterns of isolates obtained with EcoRI or NdeI were distinct. However, isolates belonging to the same species exhibited similar profiles particularly when they were isolated from the same hot spring. The estimated genomic size of strains of the Thermus spp. varied between approximately 1.8 and 2.5 Mbp. Ribotyping with BamHI and HindIII produced 30 and 35 distinct ribotypes, respectively. In spite of the variability of the hybridization patterns produced, the ribotypes obtained for isolates belonging to the same species also shared, in general, several fragments of identical size, and these fragments were similar when isolates originated from the same spring. Received: 7 October 1996 / Accepted: 10 March 1997     

Intraspecies variation in bacterial genomes: the need for a species genome concept

Bacterial populations are clonal. Their evolution involves not only divergence between orthologous genes but also gain of genes from other clones or species, which has only recently been widely appreciated through macrorestriction mapping, genomic subtraction and complete genome sequencing. Genes can also be lost in response to selection or by random mutation after becoming redundant. The bacterial genome is a dynamic structure and intraspecies variation needs to be included in genome analysis if we are to gain insight into the full species genome.     

Endophytic and ectophytic potato-associated bacterial communities differ in structure and antagonistic function against plant pathogenic fungi

Differences between endophytic and ectophytic bacterial communities with stress on antagonistic bacteria, were studied by comparing the composition of communities isolated from the rhizosphere, phyllosphere, endorhiza and endosphere of field-grown potato plants using a multiphasic approach. Terminal restriction fragment length polymorphism analysis of 16S rDNA of the bacterial communities revealed discrete microenvironment-specific patterns. To measure the antagonistic potential of potato-associated bacteria, a total of 2648 bacteria were screened by dual testing of antagonism to the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. Composition and diversity of bacterial antagonists were mainly specific for each microenvironment. The rhizosphere and endorhiza were the main reservoirs for antagonistic bacteria and showed the highest similarity in their colonisation by antagonists. The most prominent species of all microenvironments was Pseudomonas putida, and rep-PCR with BOX primers showed that these isolates showed microenvironment-specific DNA fingerprints. P. putida isolates from the rhizosphere and endorhiza gave nearly identical fingerprints confirming the high similarity of bacterial populations. The phlD gene, involved in the production of the antibiotic 2,4-diacetyl-phloroglucinol, was found only among Pseudomonas isolates from the rhizosphere and endorhiza. Evaluation of the bacterial isolates for biocontrol potential based on fungal antagonism and physiological characteristics resulted in the selection of five promising isolates from each microenvironment. The most effective isolate was Serratia plymuthica 3Re4-18 isolated from the endorhiza.     

Oligonucleotide fingerprint identification for microarray-based pathogen diagnostic assays

MOTIVATION: Advances in DNA microarray technology and computational methods have unlocked new opportunities to identify 'DNA fingerprints', i.e. oligonucleotide sequences that uniquely identify a specific genome. We present an integrated approach for the computational identification of DNA fingerprints for design of microarray-based pathogen diagnostic assays. We provide a quantifiable definition of a DNA fingerprint stated both from a computational as well as an experimental point of view, and the analytical proof that all in silico fingerprints satisfying the stated definition are found using our approach. RESULTS: The presented computational approach is implemented in an integrated high-performance computing (HPC) software tool for oligonucleotide fingerprint identification termed TOFI. We employed TOFI to identify in silico DNA fingerprints for several bacteria and plasmid sequences, which were then experimentally evaluated as potential probes for microarray-based diagnostic assays. Results and analysis of approximately 150 in silico DNA fingerprints for Yersinia pestis and 250 fingerprints for Francisella tularensis are presented. AVAILABILITY: The implemented algorithm is available upon request.     

Phylogenetic relationships in the stone fruit group of Prunus as revealed by restriction fragment analysis of chloroplast DNA.

In order to clarify the genetic relationships among stone fruits, a restriction fragment analysis of chloroplast DNAs (cpDNAs) was applied to cultivated Prunus species, whose genetic diagnoses are difficult because of their heterogeneity and long life span. Chloroplast DNAs (cpDNAs) were extracted from leaves of nine stone fruit accessions covering six species of Prunus. A restriction fragment analysis was conducted by gel electrophoresis after digestion with these endonucleases. The genome sizes of the cpDNAs were about 135-139 kbp, and the fruits were classified into seven chloroplast genome types according to their restriction fragment patterns. Two peach cultivars and nectarine were found to harbor identical plastomes, differing from those of two wild peaches and the European plum. This suggests that two cultivated peaches (P. persica) did not receive the cytoplasm from the wild peaches, P. mira and P. davidiana. Phylogenetic relationships among these types were then estimated based on the shared common fragments among the species.     

Genome analysis of Clostridium botulinum type A by pulsed-field gel electrophoresis.

Genomic DNA from type A Clostridium botulinum was digested with restriction endonucleases that cut at rare sites, and the large fragments were separated by pulsed-field gel electrophoresis. Of 15 restriction enzymes tested, MluI, RsrII, SmaI, NruI, KspI, NaeI, and XhoI generated satisfactory digestion patterns of genomic DNA of various C. botulinum strains, enabling the use of the method for genomic fingerprinting. The genomes of four group I (type A) C. botulinum strains examined had similar restriction patterns. However, each strain had unique digestion patterns, reflecting genotypic differences. The genome size of C. botulinum strain 62A was estimated to be 4,039 +/- 40 kbp from the summation of restriction fragments from MluI, RsrII, and SmaI digestions. Genes encoding proteins involved in the toxinogenicity of C. botulinum, including neurotoxin, hemagglutinin A, and genes for a temperate phage, as well as various transposon Tn916 insertion sites in C. botulinum 62A, were mapped by pulsed-field gel electrophoresis. The genes encoding neurotoxin and hemagglutinin A-1, were located on the same fragment in several cases, indicating their probable physical linkage. The macrorestriction analysis established here should be useful for genetic and epidemiological studies of C. botulinum.     

Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.     

Analysis of the genome of the gram-negative moderate halophiles Halomonas and Chromohalobacter by using pulsed-field gel electrophoresis

The genomes of 11 moderately halophilic bacteria belonging to the genera Halomonas and Chromohalobacter have been analyzed by restriction endonuclease digestion and pulsed-field gel electrophoresis (PFGE). By using the infrequently cutting restriction endonucleases SpeI and SwaI, highly characteristic fingerprintings were obtained for each of the isolates studied. On the basis of the lengths of the SpeI and SwaI fragments, separated by PFGE, the genome size of the 11 strains studied was estimated. The genome size for 8 Halomonas strains ranged from 1450 to 2830 kb, whereas for the 3 Chromohalobacter strains studied it ranged from 1770 to 2295 kb. Finally, we show that macrorestriction fingerprints could be a useful tool to elucidate the taxonomic position of bacteria belonging to the Halomonas–Deleya complex. Received: October 13, 1997 / Accepted: May 12, 1998     

Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence

Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identified distinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans and M. marinum, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS2404 and IS2606, have recently been reported in M. ulcerans, and significantly, these elements are not present in M. marinum. Hybridization of the AseI restriction fragments from M. ulcerans with IS2404 and IS2606 indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently diverged from M. marinum by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of M. tuberculosis, where species diversity is being driven mainly by the activity of mobile DNA elements.     

Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.