Strain-specific differentiation of environmental Escherichia coli isolates via denaturing gradient gel electrophoresis (DGGE) analysis of the 16S-23S intergenic spacer region

Denaturing gradient gel electrophoresis (DGGE) was applied to the 16S-23S rRNA intergenic spacer region (ISR) as a means to evaluate strain level differences in Escherichia coli. The ISRs of 81 environmental E. coli isolates obtained from bovine, poultry, and human sources yielded a total of 41 unique DGGE banding patterns, with identical patterns and common bands within each source and no overlapping patterns among sources. An additional 51 isolates from two nearby streams yielded 45 unique banding patterns with no overlap between sites. However, two of the isolates from the streams had identical banding patterns to those from two of the source isolates, resulting in a total of 84 unique DGGE banding patterns out of 132 isolates identified in this study. These results revealed high diversity among environmental E. coli isolates, which made it difficult to unambiguously ascribe strains found in water samples to specific host organisms.     

Physical mapping of the ribosomal RNA genes of Mycoplasma capricolum.

Physical mapping of the rRNA genes of Mycoplasma capricolum was done by digestion of the mycoplasmal DNA with EcoRI, PstI and BglII and hybridization with nick-translated probes consisting of defined portions of the rrnB ribosomal RNA operon of Escherichia coli. The results indicate that the rRNA genes in the chromosome of M. capricolum are arranged in two clusters, each organized in the order 5'-16S-23S-5S-3', resembling the order of the genes in the rrnB operon, with no large spacer regions separating the genes in each cluster.     

Use of 16S-23S rRNA intergenic spacer region PCR and repetitive extragenic palindromic PCR analyses of Escherichia coli isolates to identify nonpoint fecal sources

Despite efforts to minimize fecal input into waterways, this kind of pollution continues to be a problem due to an inability to reliably identify nonpoint sources. Our objective was to find candidate source-specific Escherichia coli fingerprints as potential genotypic markers for raw sewage, horses, dogs, gulls, and cows. We evaluated 16S-23S rRNA intergenic spacer region (ISR)-PCR and repetitive extragenic palindromic (rep)-PCR analyses of E. coli isolates as tools to identify nonpoint fecal sources. The BOXA1R primer was used for rep-PCR analysis. A total of 267 E. coli isolates from different fecal sources were typed with both techniques. E. coli was found to be highly diverse. Only two candidate source-specific E. coli fingerprints, one for cow and one for raw sewage, were identified out of 87 ISR fingerprints. Similarly, there was only one candidate source-specific E. coli fingerprint for horse out of 59 BOX fingerprints. Jackknife analysis resulted in an average rate of correct classification (ARCC) of 83% for BOX-PCR analysis and 67% for ISR-PCR analysis for the five source categories of this study. When nonhuman sources were pooled so that each isolate was classified as animal or human derived (raw sewage), ARCCs of 82% for BOX-PCR analysis and 72% for ISR-PCR analysis were obtained. Critical factors affecting the utility of these methods, namely sample size and fingerprint stability, were also assessed. Chao1 estimation showed that generally 32 isolates per fecal source individual were sufficient to characterize the richness of the E. coli population of that source. The results of a fingerprint stability experiment indicated that BOX and ISR fingerprints were stable in natural waters at 4, 12, and 28 degrees C for 150 days. In conclusion, 16S-23S rRNA ISR-PCR and rep-PCR analyses of E. coli isolates have the potential to identify nonpoint fecal sources. A fairly small number of isolates was needed to find candidate source-specific E. coli fingerprints that were stable under the simulated environmental conditions.     

Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution

A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E. coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E. coli was very great and is most likely accounting for our inability to correctly classify many environmental E. coli isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.     

Mycoplasmas (Mollicutes) have a low number of rRNA genes.

DNA from Mycoplasma, Ureaplasma, Acholeplasma, and Spiroplasma species digested by restriction endonucleases was hybridized with probes consisting of portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of Mycoplasma capricolum. The results indicate the presence of only one or two sets of rRNA genes in the genome of Mollicutes linked in the procaryotic fashion, 16S-23S-5S.     

Transmission and detection of Flavobacterium columnare in channel catfish Ictalurus punctatus

A specific and rapid PCR detection method for Flavobacterium columnare based on the 16S-23S rDNA intergenic spacer region (ISR) of the ribosomal RNA operon has been developed. The ISR of 30 F. columnare strains and other Flavobacterium species was amplified using universal primers and sequenced. Once F. columnare specific sequences within the ISR were recognized, specific PCR primers were designed against them (FCISRFL and FCISRR1). The primers were sensitive and able to detect as low as 7 colony forming units from pure culture by PCR. The new PCR detection method was applied to experimentally infected channel catfish. Two different experiments in which channel catfish fingerlings were infected by intramuscular injection or by immersion bath showed the advantage of the PCR method over standard culture techniques. F. columnare was detected by PCR in both tank water and catfish tissue samples with a higher frequency and in less time than standard microbiological methods. Furthermore, PCR detection confirmed that F. columnare can be transmitted horizontally indirectly through the water column without fish-to-fish contact. The newly developed PCR detection method for F. columnare was more sensitive and rapid than standard culture on bacteriological media for detection of F. columnare in channel catfish tissues and in tank water.     

Evaluation of denaturing gradient gel electrophoresis to differentiate Escherichia coli populations in secondary environments

The development of methodology to differentiate mixed populations of Escherichia coli in the secondary habitat might improve monitoring of fecal pollution indicators and facilitate the development of strategies to mitigate bacterial pollution. The objective of this study was to determine the ability of denaturing gradient gel electrophoresis (DGGE) to differentiate mixed assemblages of E. coli in the natural environment. After confirming the identity of 184 environmental bacterial isolates as E. coli, each was subjected to polymerase chain reaction (PCR) of the beta-glucuronidase gene (uidA) followed by DGGE fingerprinting. The ability of DGGE to discriminate individual isolates at the strain level was determined by comparing fingerprints to those resulting from a standard, library-dependent fingerprinting method, BOX-PCR. Computerized analysis of fingerprints indicated that DGGE and BOX-PCR identified 15 and 21 unique phylotypes respectively. Rank-abundance plots comparing the numerical distribution of unique E. coli phylotypes detected by both methods revealed no difference in resolution at the population level. In water and sediment samples from two beaches, DGGE effectively distinguished indigenous E. coli populations with an average rate of correct classification (site-based) of 83%. Denaturing gradient gel electrophoresis of uidA genes isolated and PCR-amplified from environmental samples appears to be an effective tool to differentiate unique E. coli populations and should be useful to characterize E. coli dynamics in the secondary environment.     

Loss of the spacer loop sequence from the rrnB operon in the Escherichia coli K-12 subline that bears the relA1 mutation.

A polymorphism affecting the spacer region of the rrnB rRNA operon is described. Strains from a major Escherichia coli K-12 subbranch are missing a 106-nucleotide portion of the rrnB 16S-to-23S spacer, and a 20-nucleotide sequence is found in its place. We have called this mutant operon rrnB2. The rrnB2 spacer was most probably derived from either rrnC or rrnE. This alteration of rrnB may have occurred by a recombinational exchange or by gene conversion. In the genealogy of E. coli K-12 strains, the appearance of rrnB2 is associated with the spontaneous occurrence of the first relaxed mutation, but attempts to show a selective relationship between the two mutational events have had negative results. The sequences of the rrnG and rrnC 16S-to-23S spacers have also been determined and their comparisons to the other rrn operons encoding tRNAGlu2 are presented.     

A single base mutation at position 2661 in E. coli 23S ribosomal RNA affects the binding of ternary complex to the ribosome.

A single base substitution mutation from guanine to cytosine was constructed at position 2661 of Escherichia coli 23S rRNA and cloned into the rrnB operon of the multi-copy plasmid pKK3535. The mutant plasmid was transformed into E.coli to determine the effect of the mutation on cell growth as well as the structural and functional properties of the mutant ribosomes in vivo and in vitro. The results show that the mutant ribosomes have a slower elongation rate and an altered affinity for EF-Tu-tRNA-GTP ternary complex. This supports previous findings which indicated that position 2661 is part of a region of 23S rRNA that forms a recognition site for binding of the ternary complex in the ribosomal A site. Combinations of the 2661 mutation with various mutations in ribosomal protein S12 also demonstrate that elements of both ribosomal subunits work in concert to form this binding site.     

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.     

Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.     

Fusion of the promoter region of rRNA operon rrnB to lac Z gene

A Lambda phage was constructed in which the structural gene for beta galactosidase is fused to a DNA segment carrying the ribosomal promoter rrnB of E. coli. In this hybrid operon beta galactosidase synthesis in vitro is repressed by ppGpp. Repression of beta galactosidase synthesis by cAMP is reported.     

Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients.

A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes in the environment with PCR and temperature or denaturing gradient gel electrophoresis (TGGE or DGGE, respectively). The specificity of the forward primer in combination with different reverse ones was tested with genomic DNA from a variety of bacterial strains. Most actinomycetes investigated could be separated by TGGE and DGGE, with both techniques giving similar results. Two strategies were employed to study natural microbial communities. First, we used the selective amplification of actinomycete sequences (E. coli positions 226 to 528) for direct analysis of the products in denaturing gradients. Second, a nested PCR providing actinomycete-specific fragments (E. coli positions 226 to 1401) was used which served as template for a PCR when conserved primers were used. The products (E. coli positions 968 to 1401) of this indirect approach were then separated by use of gradient gels. Both approaches allowed detection of actinomycete communities in soil. The second strategy allowed the estimation of the relative abundance of actinomycetes within the bacterial community. Mixtures of PCR-derived 16S rDNA fragments were used as model communities consisting of five actinomycetes and five other bacterial species. Actinomycete products were obtained over a 100-fold dilution range of the actinomycete DNA in the model community by specific PCR; detection of the diluted actinomycete DNA was not possible when conserved primers were used. The methods tested for detection were applied to monitor actinomycete community changes in potato rhizosphere and to investigate actinomycete diversity in different soils.     

Development of primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR.

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.     

Identification of highly conserved hydrophobic amino acid motif in deduced amino acid sequence of Elymus sibiricus L. mitochondrial S13 ribosomal protein

The nucleotide sequence of mitochondrial ribosomal protein rps13 gene from wild perennial grass Elymus sibiricus is presented. It was determined by the method of PCR amplification with specific oligonucleotide primers and the direct sequencing of the amplification product. The sequence of E. sibiricus mitochondrial gene for S13 predicts a hydrophobic ribosomal protein of 116 amino acids that shows strong similarity to those of wheat (99.7% identity) and maize (98%). The deduced amino acid sequence of S13 protein from E. sibiricus and homologous plant's (Zea mays, Daucus carota, Nicotiana tabacum, Marchantia polymorpha) and nonplant's (Escherichia coli) proteins shows the presence of hydrophobic amino acids' motif -L-X10-L-X10-M-X10-L-X10-L-. Slightly modified it can be found in many other ribosomal proteins. This conserved motif is presumed to be particularly important for association of the ribosomal S13 protein with other proteins in the small subunit of the mitochondrial ribosome.     

Evaluation of virulence factor profiling in the characterization of veterinary Escherichia coli isolates

Escherichia coli has been used as an indicator organism for fecal contamination of water and other environments and is often a commensal organism in healthy animals, yet a number of strains can cause disease in young or immunocompromised animals. In this study, 281 E. coli isolates from bovine, porcine, chicken, canine, equine, feline, and other veterinary sources were analyzed by BOXA1R PCR and by virulence factor profiling of 35 factors to determine whether they had utility in identifying the animal source of the isolates. The results of BOXA1R PCR analysis demonstrated a high degree of diversity; less than half of the isolates fell into one of 27 clusters with at least three isolates (based on 90% similarity). Nearly 60% of these clusters contained isolates from more than one animal source. Conversely, the results of virulence factor profiling demonstrated clustering by animal source. Three clusters, named Bovine, Chicken, and Porcine, based on discriminant components analysis, were represented by 90% or more of the respective isolates. A fourth group, termed Companion, was the most diverse, containing at least 84% of isolates from canine, feline, equine, and other animal sources. Based on these results, it appears that virulence factor profiling may have utility, helping identify the likely animal host species sources of certain E. coli isolates.     

Molecular tools for isolate and community studies of Pyrenomycete fungi

The Pyrenomycetes, defined physiologically by the formation of a flask-shaped fruiting body present in the sexual form, are a monophyletic group of fungi that consist of a wide diversity of populations including human and plant pathogens. Based on sequence analysis of 18S ribosomal DNA (rDNA), rDNA regions conserved among the Pyrenomycetes but divergent among other organisms were identified and used to develop selective PCR primers and a highly specific primer set. The primers presented here were used to amplify large portions of the 18S rDNA as well as the entire internal transcribed spacer (ITS) region (ITS 1, 5.8S rDNA, and ITS 2). In addition to database searches, the specificity of the primers was verified by PCR amplification of DNA extracted from pure culture isolates and by sequence analysis of fungal rDNA PCR-amplified from environmental samples. In addition, denaturing gradient gel electrophoresis (DGGE) analyses were performed on closely related Colletotrichum isolates serving as a model pathogenic genus of the Pyrenomycetes. Although both ITS and 18S rDNA DGGE analyses of Colletotrichum were consistent with a phylogeny established from sequence analysis of the ITS region, DGGE analysis of the ITS region was found to be more sensitive than DGGE analysis of the 18S rDNA. This study introduces molecular tools for the study of Pyrenomycete fungi by the development of two specific primers, demonstration of the enhanced sensitivity of ITS-DGGE for typing of closely related isolates and application of these tools to environmental samples.