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PubMedDate not supplied

Genotyping of Streptococcus thermophilus evidenced by restriction analysis of ribosomal DNA.

Twenty strains of Streptococcus thermophilus were classified into five different types characterized by ribotyping after DNA digestion with HindIII and hybridization with cDNA from 16S-23S rDNA of Escherichia coli. Ribotyping after digestion with HaeIII and XhoI enabled the same strains to be gathered into three groups as a consequence of the reduced influence of the flanking sequences within the analysis of rRNA operons. Amplified rDNA restriction analysis clearly confirmed the discrimination of these three different ribotypes, producing evidence to suggest that subtaxa are to be recognized within this species.

Bacterial Typing Techniques↗

PubMed2005

Molecular diagnosis of Pneumocystis pneumonia.

The detection of Pneumocystis DNA in clinical specimens by using PCR assays is leading to important advances in Pneumocystis pneumonia (PcP) clinical diagnosis, therapy and epidemiology. Highly sensitive and specific PCR tools improved the clinical diagnosis of PcP allowing an accurate, early diagnosis of Pneumocystis infection, which should lead to a decreased duration from onset of symptoms to treatment, a period with recognized impact on prognosis. This aspect has marked importance in HIV-negative immunocompromised patients, who develop often PcP with lower parasite rates than AIDS patients. The specific amplification of selected polymorphous sequences of Pneumocystis jirovecii genome, especially of internal transcribed spacer regions of the nuclear rRNA operon, has led to the identification of specific parasite genotypes which might be associated with PcP severity. Moreover, multi-locus genotyping revealed to be a useful tool to explore person-to-person transmission. Furthermore, PCR was recently used for detecting P. jirovecii dihydropteroate synthase gene mutations, which are apparently associated with sulfa drug resistance. PCR assays detected Pneumocystis-DNA in bronchoalveolar lavage fluid or biopsy specimens, but also in oropharyngeal washings obtained by rinsing of the mouth. This non-invasive procedure may reach 90%-sensitivity and has been used for monitoring the response to treatment in AIDS patients and for typing Pneumocystis isolates.

DNA, Fungal↗

PubMed2006

Assessment of biases associated with profiling simple, model communities using terminal-restriction fragment length polymorphism-based analyses.

Community profiles based on terminal-restriction fragment length polymorphism (T-RFLP) analyses of amplified ribosomal RNA genes are used to monitor changes in microbial community structure and are sometimes employed for semi-quantitative estimates of species richness and abundance in environmental samples. To assess the accuracy of T-RFLP community profiles representing the relative abundance of bacteria in a sample, five species of ruminal bacteria were used to construct simple "communities". Template DNA for PCR amplification was generated either by mixing equal quantities of genomic DNA from pure cultures or by mixing equal numbers of cells prior to DNA extraction. Pairwise mixtures of Fibrobacter succinogenes S85 with Ruminococcus albus 8, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49 and Streptococcus bovis JB1 were created and a 5-member community was constructed. With genomic DNA mixes, relative abundance calculations based on T-RFLP patterns did not reflect input ratios. These discrepancies could not be accounted for by differences in genome size and rRNA operon copy number. In cell mixing experiments, easily lysed cells were overrepresented. To determine if a numerical correction factor could be used to compensate for observed discrepancies, we attempted to quantify biases attributed to DNA extraction and PCR amplification. Biases attributable to these factors led to deviations from expected PCR product ratios by 6% to 38%. We found that interactions were so complex that a suitable factor could not be derived. The unsystematic dependence of T-RFLP peak ratios on variability of DNA extraction and PCR amplification prevents accurate quantification of the relative abundance of microorganisms designed to represent simplified natural populations.

Bacteria↗

PubMed1990

Antitermination of characterized transcriptional terminators by the Escherichia coli rrnG leader region.

We have used a plasmid antitermination test system to examine the response of an Escherichia coli rRNA operon antiterminator to transcription through Rho-dependent and Rho-independent terminator-containing fragments. We also monitored transcription through multiple copies of a terminator to explore the mechanism of rrn antitermination. Four principal observations were made about antitermination and transcriptional terminators. (1) The rrn antiterminator mediated efficient transcription through Rho-dependent terminators. (2) Under the influence of the rrn antiterminator, RNA polymerase transcribed through two and three copies of the Rho-dependent 16 S----terminator with nearly the same efficiency as through one. (3) The antiterminator had less effect on fragments containing Rho-independent terminators; the rpoC t fragment and three fragments derived from the rrnB terminator region stopped antiterminated transcription. Four other Rho-independent terminator fragments were weakly antiterminated in our test system. (4) Surprisingly, the strength of these terminator fragments was not strongly related to properties such as the -delta G or number of trailing uridine residues of their canonical Rho-independent structures, but appears to be related to additional downstream terminators. We have drawn the following conclusions from these experiments. First, that ribosomal antitermination primarily reverses Rho-dependent termination by modifying the RNA polymerase elongation complex. Transcription through a 1700 nucleotide, multiple terminator sequence showed that the antiterminator caused persistent changes in the transcription process. Second, that fragments derived from the Rho-independent rrnB and rpoBC terminator regions can effectively stop antiterminated transcription. Third, that efficient in vivo termination may often involve regions with complex multiple terminators.

Cloning, Molecular↗

PubMed2003

A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies.

Real-time TaqMan-PCR assays were developed for detection, differentiation and absolute quantification of the pathogenic subspecies of Clavibacter michiganensis (Cm) in one single PCR run. The designed primer pair, targeting intergenic sequences of the rRNA operon (ITS) common in all subspecies, was suitable for the amplification of the expected 223-nt DNA fragments of all subspecies. Closely related bacteria were completely discriminated, except of Rathayibacter iranicus, from which weak PCR product bands appeared on agarose gel after 35 PCR cycles. Sufficient specificity of PCR detection was reached by introduction of the additional subspecies specific probes used in TaqMan-PCR. Only Cm species were detected and there was clear differentiation among the subspecies C. michiganensis sepedonicus (Cms), C. michiganensis michiganensis (Cmm), C. michiganensis nebraskensis (Cmn), C. michiganensis insidiosus (Cmi) and C. michiganensis tessellarius (Cmt). The TaqMan assays were optimized to enable a simultaneous quantification of each subspecies. Validity is shown by comparison with cell counts.

Actinomycetales↗

PubMedDate not supplied

A bioinformatics based approach to discover small RNA genes in the Escherichia coli genome.

The recent explosion in available bacterial genome sequences has initiated the need to improve an ability to annotate important sequence and structural elements in a fast, efficient and accurate manner. In particular, small non-coding RNAs (sRNAs) have been difficult to predict. The sRNAs play an important number of structural, catalytic and regulatory roles in the cell. Although a few groups have recently published prediction methods for annotating sRNAs in bacterial genome, much remains to be done in this field. Toward the goal of developing an efficient method for predicting unknown sRNA genes in the completed Escherichia coli genome, we adopted a bioinformatics approach to search for DNA regions that contain a sigma70 promoter within a short distance of a rho-independent terminator. Among a total of 227 candidate sRNA genes initially identified, 32 were previously described sRNAs, orphan tRNAs, and partial tRNA and rRNA operons. Fifty-one are mRNAs genes encoding annotated extremely small open reading frames (ORFs) following an acceptable ribosome binding site. One hundred forty-four are potentially novel non-translatable sRNA genes. Using total RNA isolated from E. coli MG1655 cells grown under four different conditions, we verified transcripts of some of the genes by Northern hybridization. Here we summarize our data and discuss the rules and advantages/disadvantages of using this approach in annotating sRNA genes on bacterial genomes.

Base Sequence↗

PubMed1997

Physical and genetic map of the chromosome of Dichelobacter nodosus strain A198.

A physical map of the chromosome of Dichelobacter nodosus strain A198 was constructed using the restriction endonucleases EagI and StuI. Mapping data indicated the presence of a single, circular chromosome of 1.54 Mb. The three rRNA operons and the virulence related locus (vrl) were precisely positioned at the junctions of EagI and StuI fragments, and their transcriptional orientations were also determined. Other D. nodosus genes were assigned to specific EagI and StuI fragments. Analysis of the resultant map revealed that the putative virulence genes were not clustered on the chromosome which suggests that the D. nodosus virulence determinants have been acquired gradually and that virulence in D. nodosus is an evolving trait.

Chromosome Mapping↗

PubMed1999

The role of recombination and mutation in 16S-23S rDNA spacer rearrangements.

The intragenomic heterogeneity of the bacterial intergenic (16S-23S rDNA) spacer region (ISR) was analysed from the following species in which sequences for the complete rRNA operon (rrn) set have been determined (rrn number): Enterococcus faecalis (6) and E. faecium (6), Bacillus subtilis (10), Staphylococcus aureus (9), Vibrio cholerae (4), Haemophilus influenzae (6) and Escherichia coli (7). It was found that some spacer sequence blocks were highly conserved between operons of a genome, whereas the presence of others was variable. When these variations were analysed using the program PLATO and partial likelihood phylogenies determined by DNAml for each operon set, three regions showed significant (Z>3.3) spatial variation [Region I was 78-184 nt long (2.1 4.4) possibly due to recombination or selection. Within Region I, there was sequence block variation in all operon sets [some operons contained tRNA genes (tRNAala, tRNAile or tRNAglu), whereas others had sequence blocks such as VS2 (S. aureus) or rsl (E. coli)]. Q Analysis of the ISR sequence from E. faecalis and E. faecium showed that there was more interspecies than intraspecies variation (both in DNA sequence and in the presence or absence of blocks). Dot matrix analysis of the sequence blocks in the nine rrn ISRs from S. aureus showed that there was significant homology between VS2 and VS5/VS6. Furthermore, repeat motifs with only A or T were present in higher copy numbers in VS5/VS6 than in VS2. Since these sequence blocks (VS2 and VS5-VS6) are related, intragenic evolution resulting in AT expansion may have occurred between these two regions. A model is proposed that postulates a role for recombination and AT-expansion in intra-genomic ISR variations. This process may represent a general mechanism of concerted evolution for bacterial ISR rearrangements.

Bacteria↗

PubMed2002

PCR as a diagnostic tool for brucellosis.

Numerous PCR-based assays have been developed for the identification of Brucella to improve diagnostic capabilities. Collectively, the repertoire of assays addresses several aspects of the diagnostic process. For some purposes, the simple identification of Brucella is adequate (e.g. diagnosis of human brucellosis or contamination of food products). In these cases, a genus-specific PCR assay is sufficient. Genus-specific assays tend to be simple, robust, and somewhat permissive of environmental influences. The main genetic targets utilized for these applications are the Brucella BCSP31 gene and the 16S-23S rRNA operon. Other instances require identification of the Brucella species involved. For example, most government-sponsored brucellosis eradication programs include regulations that stipulate a species-specific response. For epidemiological trace back, strain-specific identification is helpful. Typically, differential PCR-based assays tend to be more complex and consequently more difficult to perform. Several strategies have been explored to differentiate among Brucella species and strains, including locus specific multiplexing (e.g. AMOS-PCR based on IS711), PCR-RFLP (e.g. the omp2 locus), arbitrary-primed PCR, and ERIC-PCR to name a few. This paper reviews some of the major advancements in molecular diagnostics for Brucella including the development of procedures designed for the direct analysis of a variety of clinical samples. While the progress to date is impressive, there is still room for improvement.

Animals↗

PubMed2003

Identifying and subtyping species of dangerous pathogens by automated ribotyping.

An investigation of dangerous bacterial pathogens was conducted to determine the usefulness of automated rRNA operon ribotyping (RiboPrinter system) to identify species. A total of 26 isolates comprising Bacillus anthracis, Brucella spp., Burkholderia mallei, Francisella tularensis, and Yersinia pestis were tested using restriction endonucleases EcoRI, PstI, PvuII and AseI. The main problem was that the system's database-relying on EcoRI as restriction enzyme-does not contain the essential dangerous pathogens. B. anthracis was misidentified as B. cereus and Y. pestis as Y. pseudotuberculosis. Two isolates of F. tularensis ssp. holarctica were falsely identified as Vibrio cholerae. This study underscores that riboprint patterns generated with a single restriction enzyme are not always unique for each of the species tested. Using more than one enzyme, the RiboPrinter proved to be a valuable primary typing method. Databases of commercially available systems for the identification of bacteria should include the most important dangerous pathogens.

Austria↗

PubMed1994

rRNA gene restriction patterns as possible taxonomic tools for the genus actinomyces.

Species delineation in the genus Actinomyces remains unclear, particularly regarding the two taxa, A. naeslundii and A. viscosus. The ribotyping patterns of 64 strains of Actinomyces, representing 8 species and comprising different serotypes, were studied as possible taxonomic tools, using an acetyl-aminofluorene (AAF)-labelled E. coli 16S + 23S rRNA probe. Similarities between patterns were assessed using Jaccard's coefficient and clustering achieved using the unweighted pair-group method with average linkage (UPGMA) on a Macintosh II (Apple, Cupertino, USA) computer. The dendrogram obtained from the ribotypes gave results which were in reasonable agreement with many previous reports: A. bovis, A. gerensceriae, A. israelii, A. meyerii, A. odontolyticus and A. pyogenes were found to be distinct species but the two taxa A. naeslundii and A. viscosus remained unclear. Further investigations, using a larger number of A. naeslundii and A. viscosus strains and other endonucleases, need to be carried out to provide more information concerning the relatedness of these two taxa. Nevertheless, these preliminary results suggested that the Actinomyces chromosome contains multiple rRNA operons which may be used as an epidemiological and taxonomical tool.

Actinomyces↗

PubMed2003

Pneumocystis jiroveci in Portuguese immunocompromised patients: association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood.

We analyzed the genetic variation among isolates of Pneumocystis jiroveci from Portuguese immunocompromised patients with PCP at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon and at the dihydropteroate synthase (DHPS) gene. Pulmonary secretions from 42 patients with PCP corresponding to 43 episodes were studied. Demographic, immunological, and clinical data were obtained from all patients. By combining the two regions ITS1 and ITS2, we found 17 different ITS types of P. jiroveci, two of them were new types (Pb and Pe). The four most prevalent ITS types were Eg (23.3%), Eb and Ne (11.6% each), and Bi (9.3%). A single type was detected in 95.3% of the samples and 4.7% had mixed infections with three different ITS types. DHPS mutants were present in 17 (46%), and the wildtype was present in 20 (54%) of 37 isolates. No association was found between ITS and DHPS types and between DHPS types and therapy or response to anti-PCP treatment. Type Ne presented an association with negative response to anti-PCP treatment (P<0.001) and with death before 120 days after PCP diagnosis (P=0.025). Type Eb was significantly more common in children than in adults (P=0.001). Our data suggest an association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood.

Adult↗

PubMed2005

Phenotypic and DNA sequence data comparisons reveal three discrete species in the Ceratocystis polonica species complex.

Ceratocystis polonica and C. laricicola are two morphologically similar species that occur on conifers and reside in the Ceratocystis coerulescens species complex. They, however, represent two ecologically distinct entities. C. polonica causes blue stain on Norway spruce (Picea abies) and other spruce species (Picea spp.) in Eurasia and is associated with the bark beetles Ips typographus, I. typographus japonicus, I. amitinus and I. duplicatus. In contrast, C. laricicola lives in a symbiotic relationship with the bark beetles Ips cembrae and I. subelongatus that infest various larch species (Larix spp.). The objective of this study was to consider the phylogenetic relationships of C. polonica and C. laricicola and more specifically to determine the identity of Japanese isolates from both spruce and larch, based on sequences derived from the ITS regions of the rRNA operon, the beta-tubulin gene and the HMG box of the MAT-2 gene. Isolates were also compared based on morphology and cultural characteristics. Comparisons of anamorph and teleomorph structures confirmed that C. polonica and C. laricicola are indistinguishable based on morphology. Both species had an optimal growth temperature of 25 degrees C. However, at temperatures between 31-33 degrees, C. polonica isolates grew slowly or not at all, while C. laricicola isolates grew more actively at these temperatures. Thus, a growth test at 32 degrees can differentiate these species. Phylograms generated using parsimony for the three gene regions were strongly congruent. These showed three distinct clades supported by high bootstrap values. Two of the clades clearly separate C. laricicola from Europe and C. polonica, supporting the view that they represent two discrete taxa. A third clade included isolates obtained from galleries of Ips subelongatus on Larix kaempferi in Japan. This fungus clearly represents a discrete taxon that is closely related to, but distinct from C. laricicola, which is described here as C. fujiensis sp. nov.

Animals↗

PubMed1998

Heterogeneity in Bacillus cereus PCR products detected by ESI-FTICR mass spectrometry.

PCR amplification of a segment of the 16/23S rDNA interspace region (ISR) from Bacillus cereus 6464 produced a mixture of products. An 89-bp product was predicted on the basis of the reported sequence. The ESI-FTICR analysis revealed three double-stranded products, differing in size by a single nucleotide corresponding to two homoduplexes of 89 and 88 base pairs and a heteroduplex of 89 and 88 nucleotide strands. These were produced from a single preparation of genomic DNA and a single primer pair. ESI-FTICR analysis of the single strands identified a deletion of a T in the coding strand and a corresponding loss of an A in the noncoding strand of this product. The ESI-FTICR analysis indicated the presence of an unreported sequence variation between rRNA operons in this organism. This report illustrates that PCR products amplified from templates differing by a single nucleotide can be resolved and identified using ESI-FTICR at the 89-bp level. Furthermore, the ESI-FTICR mass measurements provided the identity of the deletion, which is indicative of interoperon variability.

Bacillus cereus↗

PubMed2001

The root rot fungus Armillaria mellea introduced into South Africa by early Dutch settlers.

Dead and dying oak (Quercus) and numerous other woody ornamental trees and shrubs showing signs and symptoms of Armillaria root rot were identified in the Company Gardens, Cape Town, South Africa, which were established in the mid-1600s by the Dutch East Indies Trading Company. Nineteen isolates from dying trees or from mushrooms were collected and analysed to identify and characterize the Armillaria sp. responsible for the disease. The AluI digestion of the amplified product of the first intergenic spacer region (IGS-1) of the rRNA operon of 19 isolates from the Company Gardens was identical to that of some of the European isolates of A. mellea s. s. The IGS-1 region and the internal transcribed spacers (ITS) were sequenced for some of the Cape Town isolates. Phylogenetic analyses placed the Cape Town isolates in the European clade of A. mellea, which is distinct from the Asian and North American clades of this species. Identification based on sexual compatibility was conducted using A. mellea tester strains in diploid-haploid pairings, which showed some compatibility between the Cape Town isolates and testers from Europe. Somatic compatibility tests (diploid-diploid pairings) and DNA fingerprinting with multilocus, microsatellite probes indicated that the Cape Town isolates were genetically identical and may have resulted from vegetative (clonal) spread from a single focus in the centre of the original Company Gardens (c. 1652). The colonized area is at least 345 m in diameter. Assuming a linear spread rate underground of 0.3 m/year to 1.6 m/year, the genet (clone) was estimated to be between 108 and 575 years old. These data suggest that A. mellea was introduced into Cape Town from Europe, perhaps on potted plants, such as grapes or citrus, planted in the Company Gardens more than 300 years ago.

Agaricales↗

PubMed2003

Identification of functional lox sites in the plastid genome.

Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.

3' Untranslated Regions↗

PubMed2004

The louse-borne human pathogen Bartonella quintana is a genomic derivative of the zoonotic agent Bartonella henselae.

We present the complete genomes of two human pathogens, Bartonella quintana (1,581,384 bp) and Bartonella henselae (1,931,047 bp). The two pathogens maintain several similarities in being transmitted by insect vectors, using mammalian reservoirs, infecting similar cell types (endothelial cells and erythrocytes) and causing vasculoproliferative changes in immunocompromised hosts. A primary difference between the two pathogens is their reservoir ecology. Whereas B. quintana is a specialist, using only the human as a reservoir, B. henselae is more promiscuous and is frequently isolated from both cats and humans. Genome comparison elucidated a high degree of overall similarity with major differences being B. henselae specific genomic islands coding for filamentous hemagglutinin, and evidence of extensive genome reduction in B. quintana, reminiscent of that found in Rickettsia prowazekii. Both genomes are reduced versions of chromosome I from the highly related pathogen Brucella melitensis. Flanked by two rRNA operons is a segment with similarity to genes located on chromosome II of B. melitensis, suggesting that it was acquired by integration of megareplicon DNA in a common ancestor of the two Bartonella species. Comparisons of the vector-host ecology of these organisms suggest that the utilization of host-restricted vectors is associated with accelerated rates of genome degradation and may explain why human pathogens transmitted by specialist vectors are outnumbered by zoonotic agents, which use vectors of broad host ranges.

Animals↗

PubMed2004

Genome sequence of the deep-sea gamma-proteobacterium Idiomarina loihiensis reveals amino acid fermentation as a source of carbon and energy.

We report the complete genome sequence of the deep-sea gamma-proteobacterium, Idiomarina loihiensis, isolated recently from a hydrothermal vent at 1,300-m depth on the Loihi submarine volcano, Hawaii. The I. loihiensis genome comprises a single chromosome of 2,839,318 base pairs, encoding 2,640 proteins, four rRNA operons, and 56 tRNA genes. A comparison of I. loihiensis to the genomes of other gamma-proteobacteria reveals abundance of amino acid transport and degradation enzymes, but a loss of sugar transport systems and certain enzymes of sugar metabolism. This finding suggests that I. loihiensis relies primarily on amino acid catabolism, rather than on sugar fermentation, for carbon and energy. Enzymes for biosynthesis of purines, pyrimidines, the majority of amino acids, and coenzymes are encoded in the genome, but biosynthetic pathways for Leu, Ile, Val, Thr, and Met are incomplete. Auxotrophy for Val and Thr was confirmed by in vivo experiments. The I. loihiensis genome contains a cluster of 32 genes encoding enzymes for exopolysaccharide and capsular polysaccharide synthesis. It also encodes diverse peptidases, a variety of peptide and amino acid uptake systems, and versatile signal transduction machinery. We propose that the source of amino acids for I. loihiensis growth are the proteinaceous particles present in the deep sea hydrothermal vent waters. I. loihiensis would colonize these particles by using the secreted exopolysaccharide, digest these proteins, and metabolize the resulting peptides and amino acids. In summary, the I. loihiensis genome reveals an integrated mechanism of metabolic adaptation to the constantly changing deep-sea hydrothermal ecosystem.

Amino Acids↗