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PubMed2004

Physical and genetic map of the Weissella paramesenteroides DSMZ 20288T chromosome and characterization of different rrn operons by ITS analysis.

The Weissella paramesenteroides DSMZ 20288T chromosome was analysed by pulsed-field gel electrophoresis, enabling the construction of a physical and genetic map. A total of 21 recognition sites of the restriction enzymes AscI, I-CeuI, NotI and SfiI were mapped on the chromosome, which was found to be circular with an estimated size of 2026 kb. This is believed to constitute the first study into the genomic organization of a strain of this genus, addressing the localization of important chromosomal regions such as oriC and terC. A total of 23 genetic markers were mapped, including eight rrn operons that were precisely assigned in 37 % of the W. paramesenteroides chromosome. The transcription direction of rrn loci was determined and three different rrn clusters were recognized regarding the presence/absence of tRNA genes in ITS regions.

Chromosomes, Bacterial↗
PubMed1999

Role of genomic rearrangements in producing new ribotypes of Salmonella typhi.

Salmonella typhi is the only species of Salmonella which grows exclusively in humans, in whom it causes enteric typhoid fever. Strains of S. typhi show very little variation in electrophoretic types, restriction fragment length polymorphisms, cell envelope proteins, and intervening sequences, but the same strains are very heterogeneous for ribotypes which are detected with the restriction endonuclease PstI. In addition, the genome of S. typhi has been proven to undergo genomic rearrangement due to homologous recombination between the seven copies of rrn genes. The relationship between ribotype heterogeneity and genomic rearrangement was investigated. Strains of S. typhi which belong to 23 different genome types were analyzed by ribotyping. A limited number of ribotypes were found within the same genome type group; e. g., most strains of genome type 3 belonged to only two different ribotypes, which result from recombination between rrnH and rrnG operons. Different genome type groups normally have different ribotypes. The size and identity of the PstI fragment containing each of the seven different rrn operons from S. typhi Ty2 were determined, and from these data, one can infer how genomic rearrangement forms new ribotypes. It is postulated that genomic rearrangement, rather than mutation, is largely responsible for producing the ribotype heterogeneity in S. typhi.

Bacterial Typing Techniques↗
PubMed1997

Organ-specific transcription of the rrn operon in spinach plastids.

The spinach rrn operon is used as a model system to study transcriptional regulation in higher plant photosynthetic and non-photosynthetic plastids. We performed capping experiments to determine whether P1, PC, or P2 promoters are employed for rrn transcription start sites in cotyledon and root tissues. By using a new method of analysis of capped RNA we demonstrate for the first time that 1) in both organs the rrn operon is expressed in a constitutive manner by cotranscription with the preceding tRNA(GAC)Val gene, and 2) the PC transcription start site is used only in cotyledons and leaves, i.e. we demonstrate the organ-specific usage of a plastid promoter. Both start sites, PC and that of the tRNA(GAC)Val cotranscript, lack Escherichia coli-like consensus sequences. The cotranscript is initiated 457 base pairs upstream of the tRNA(GAC)Val gene. The PC-specific DNA-binding factor, CDF2, is not detectable in root tissues confirming its regulatory role in PC-initiated rrn expression and the organ specificity of PC expression. Furthermore, our results show that rrn operon expression patterns differ in spinach and tobacco indicating species-specific transcriptional regulation of plant plastid gene expression.

Base Sequence↗
PubMed1992

Effects of template topology on RNA polymerase pausing during in vitro transcription of the Escherichia coli rrnB leader region.

Transcription elongation catalysed by DNA-dependent RNA polymerase does not occur at a constant rate. Instead, during the transcription of many genes pausing occurs at defined template positions. Pausing is known to be influenced by the intracellular NTP concentration, the secondary structure of the growing transcript or by transcription factors like NusA. We have investigated the effects of the template topology of transcriptional pauses in the presence and absence on purified NusA protein. Taking advantage of a method for quantifying transcriptional pauses we have studied pausing behaviour during in vitro transcription of the early region of a plasmid-encoded ribosomal RNA operon. Plasmid templates with different superhelical densities (sigma between +0.0017 and -0.055) were employed in transcription elongation assays. If linearized or relaxed templates are used, some of the characteristic pauses can no longer be detected. For the stronger pauses we could demonstrate a direct correlation between pause strength and the negative superhelical densities of the templates used. This correlation is observed regardless of whether or not pauses are dependent upon NusA. Changes in the average transcription elongation rate, caused by variations in the NTP concentration or the temperature, do not appear to have a comparable effect on transcription pausing. The results are consistent with the assumption that the template topology has a regulatory function in transcription elongation of rRNA genes in Escherichia coli.

Bacterial Proteins↗
PubMed1999

Resurgent Vibrio cholerae O139: rearrangement of cholera toxin genetic elements and amplification of rrn operon.

The unprecedented genesis of a novel non-O1 Vibrio cholerae strain belonging to serogroup O139, which caused an epidemic in late 1992 in the Indian subcontinent, and its subsequent displacement by El Tor O1 vibrios after 18 months initiated a renewed investigation of the aspects of the organism that are related to pathogenesis. The reappearance of V. cholerae O139 with altered antibiotic sensitivity compared to O139 Bengal (O139B) in late 1996 has complicated the epidemiological scenario of V. cholerae and has necessitated an examination of possible rearrangements in the genome underlying such rapid changes in the phenotypic traits. With a view to investigating whether the phenotypic changes that have occurred are associated with alteration in the genome, the genome of the resurgent V. cholerae O139 (O139R) strains were examined. Pulsed-field gel electrophoresis analysis of NotI- and SfiI-digested genomic DNA of O139R isolates showed restriction fragment length polymorphism including in the cholera toxin (CTX) genetic element locus and with O139B isolates. Analyses of the organization of the CTX genetic elements in O139R strains showed that in contrast to two copies of the elements connected by two direct-repeat sequences (RS) in most of the genomes of O139B isolates, the genomes of all O139R strains examined, except strain AS192, have three such elements connected by a single RS. While the RS present in the upstream of the CTX genetic elements in the genome of O139R is of O139B origin, the RS connecting the cores of the elements has several new restriction sites and has lost the BglII site which is supposed to be conserved in all O1 strains and O139B. The endonuclease I-CeuI, which has sites only in the rrn operons in the genomes of all organisms examined so far, has 10 sites in the genomes of O139R strains, compared to 9 in the genomes of O139B strains. The recent isolates of V. cholerae O139 have thus gained one rrn operon. This variation in the number of rrn operons within a serogroup has not been reported for any other organism. The results presented in this report suggest that like the pathogenic El Tor O1 strains, the genomes of O139 strains are undergoing rapid alterations.

Cholera Toxin↗
PubMed1997

Nucleotide sequence and analysis of the phoB-rrnE-groESL region of the Bacillus subtilis chromosome.

A 36 kb sequence of the phoB-rrnE-groESL region of the Bacillus subtilis chromosome at around 55 degrees has been determined. The sequenced region contains 36 ORFs including the phoB and groESL genes, and the whole rrnE operon. The phoB gene is transcribed in the direction opposite to that of chromosome replication, while most ORFs, including groESL and the rrnE operon, are transcribed in the same direction. Two newly identified tRNA genes upstream of the rrnE operon were those for Arg-tRNA and Gly-tRNA. The sequenced region contains an operon consisting of genes for degradation and uptake of mannan. The rrnE operon and its downstream ORFs are well conserved among Mycoplasma genitalium, Haemophilus influenzae, Synechocystis sp. and Methanococcus jannaschii. delta H consensus sequences are present in the promoter regions of three ORFs, including groESL.

Amino Acid Sequence↗
PubMed1999

Construction of a genomic map of Moraxella (Branhamella) catarrhalis ATCC 25238 and physical mapping of virulence-associated genes.

A physical genome map of the Moraxella catarrhalis type strain (ATCC 25238) has been constructed using pulsed field gel electrophoresis. Macrorestriction analyses of the genome of M. catarrhalis were performed by digestion with the restriction enzymes SmaI, NotI, and RsrII, which cleave the single circular chromosome into 9, 10, and 6 fragments, respectively. The chromosomal fragments generated by pulsed field gel electrophoresis were converted to a linkage map utilizing a combination of partial digestions, and cross-hybridizations. Moraxella catarrhalis, like a number of other respiratory pathogens, has a relatively small genome estimated at 1750 kilobase pairs or about 40% of the size of the Escherichia coli genome. The locations of the four ribosomal RNA operons (rrnLS) were determined by Southern hybridization and by digestion with I-CeuI endonuclease. A number of genes involved in virulence have been placed onto the physical map by Southern hybridization including those encoding the predominant outer-membrane proteins and the chromosomal gene encoding beta-lactamase.

Blotting, Southern↗
PubMed2000

Pathovars of Pseudomonas syringae causing bacterial brown spot and halo blight in phaseolus vulgaris L. are distinguishable by ribotyping.

Ribotyping was evaluated as a method to differentiate between Pseudomonas syringae pv. phaseolicola and pv. syringae strains causing bacterial brown spot and halo blight diseases in Phaseolus vulgaris L. Ribotyping, with restriction enzymes BglI and SalI and using the Escherichia coli rrnB operon as the probe, differentiated 11 and 14 ribotypes, respectively, and a combination of data from both procedures yielded 19 combined ribotypes. Cluster analysis of the combined ribotypes differentiated the pathovars phaseolicola and syringae, as well as different clonal lineages within these pathovars. The potential of ribotyping to screen for correlations between lineages and factors such as geographical region and/or bean varieties is also reported.

Cluster Analysis↗
PubMed1999

Simkania negevensis strain ZT: growth, antigenic and genome characteristics.

Simkania negevensis is the type species of Simkaniaceae, a recently proposed family in the order Chlamydiales. In the current study, growth, antigenic and genomic characteristics of this intracellular bacterium were investigated and compared to those of members of the family Chlamydiaceae. Growth of the organism, as assessed by infectivity assays, reached a plateau in 2-3 d although by light microscopy the cytopathic effect on the host cells increased for 12 or more days after infection. S. negevensis growth was unaffected by sulfadiazine. Cells infected by S. negevensis strain ZT were not recognized by either of two monoclonal antibodies specific for Chlamydiaceae LPS and several specific Chlamydiaceae ompA primers were unable to PCR amplify a S. negevensis gene. The S. negevensis genome contained one copy of the ribosomal operon. The genome size of S. negevensis strain ZT was determined by PFGE to be 1.7 Mbp, and the G + C content was 42.5 mol%. These data, taken together with other published data, are consistent with the proposal that S. negevensis belongs to a distinct family in the order Chlamydiales.

Animals↗
PubMed2001

General method of rapid Smith/Birnstiel mapping adds for gap closure in shotgun microbial genome sequencing projects: application to Pseudomonas putida KT2440.

A physical mapping strategy has been developed to verify and accelerate the assembly and gap closure phase of a microbial genome shotgun-sequencing project. The protocol was worked out during the ongoing Pseudomonas putida KT2440 genome project. A macro-restriction map was constructed by linking probe hybridisation of SwaI- or I-CeuI-restricted chromosomes to serve as a backbone for the quick quality control of sequence and contig assemblies. The library of PCR-generated SwaI linking probes was derived from the sequence assembly after 3- and 6-fold genome coverage. In order to support gap closure in regions with ambiguous assemblies such as the repetitive sequence of the seven ribosomal operons, high-resolution Smith/Birnstiel maps were generated by Southern hybridisation of pulsed-field gel electrophoresis-separated rare-cutter complete/frequent-cutter partial digestions with rare-cutter fragment end probes. Overall 1.5 Mb of the 6.1 Mb P.putida KT2440 genome has been subjected to high-resolution physical mapping in order to align assemblies generated from shotgun sequencing.

DNA, Bacterial↗
PubMed1979

Transcription termination in the Escherichia coli ribosomal RNA operon rrnC.

The distal portion of the Escherichia coli ribosomal RNA operon rrnC has been analyzed to determine (a) the transcription boundaries of an operon containing rRNA genes distal to rDNA, and (b) the sequence elements which dictate efficient termination of RNA synthesis. I have sequenced a 505-base pair DNA restriction fragment containing the genes which specify 5 S rRNA, TRNA1Asp, and tRNATrp, in that order. Segments of a spacer DNA separate these genes: the 5 S-tRNA1Asp and tRNA1Asp-tRNATrp spacers are 52 and 8 base pairs long, respectively. In vitro transcription of restriction endonuclease fragments containing the distal portion of rrnC indicates that termination of transcription occurs at a locus beginning 28 base pairs after the tRNATrp gene. The rrnC terminator shares several features with many other known sites of transcription termination in E. coli.

Base Sequence↗
PubMed1994

Characterization of Serpulina hyodysenteriae isolates of serotypes 8 and 9 from Quebec by restriction endonuclease fingerprinting and ribotyping.

This study was undertaken to assess the discriminatory value of restriction endonuclease fingerprinting (REF) analysis and ribotyping of 21 Serpulina hyodysenteriae isolates of serotypes 8 and 9. For REF analysis, DNAs were digested with the BglII restriction enzyme and the resultant fragments were separated by polyacrylamide gel electrophoresis. For ribotyping, hybridization of BglII genomic fragments with a probe of rrnB operon using an Escherichia coli rDNA probe was performed on all isolates. Although many isolates shared a common pattern by BglII REF and BglII ribotyping analysis, differences among some S. hyodysenteriae isolates were observed. REF and ribotyping using BglII restriction enzyme, were not specific for serotypes. The predominance of an REF and a ribotype pattern among S. hyodysenteriae isolates from Quebec suggested that epidemiologically important S. hyodysenteriae types occur in different swine herds.

Bacterial Typing Techniques↗
PubMed1999

Molecular identification of Lactobacillus hilgardii and genetic relatedness with Lactobacillus brevis.

Conventional phenotypic methods lead to misidentification of the lactic acid bacteria Lactobacillus hilgardii and Lactobacillus brevis. Random amplified polymorphic DNA (RAPD) and repetitive element PCR (REP-PCR) techniques were developed for a molecular study of these two species. The taxonomic relationships were confirmed by analysis of the ribosomal operon. Amplified DNA fragments were chosen to isolate L. hilgardii-specific probes. In addition to rapid molecular methods for identification of L. hilgardii, these results convincingly proved that some strains first identified as L. brevis must be reclassified as L. hilgardii. The data clearly showed that these molecular methods are more efficient than phenotypic or biochemical studies for bacterial identification at the species level.

Amino Acid Sequence↗
PubMed1998

Rapid isolation of chromosomal DNA from oral streptococci and polymerase chain reaction-oriented restriction fragment-length polymorphism analysis for genetic heterogeneity.

We have extensively modified the published method for the lysis of gram-positive bacteria to isolate chromosomal DNA from only 1 ml of oral streptococcal overnight culture. Cells were incubated with lysozyme and R Nase A in the presence of polyethylene glycol. After centrifugation, cells were lysed with sodium dodecyl sulfate and proteinase K. Following ethanol precipitation, sodium dodecyl sulfate solution was added to the residue, and the pellet was completely dispersed by incubating at 65 degrees C. The chromosome was purified by extraction over phenol and chloroform. Two regions corresponding to the ribosomal RNA (rrn) operon and the glucosyltransferase gene were amplified using the chromosome from Streptococcus mutans and Streptococcus sobrinus by polymerase chain reaction (PCR). Genetic heterogeneity was assessed by restriction fragment-length polymorphism (PCR-RFLP). The PCR-RFLP analysis readily allowed us to subtype each strain, suggesting that the strategy presented here will provide a useful tool to verify epidemiological studies at the molecular level.

Base Sequence↗
PubMed2000

Riboprints as a tool for rapid preliminary identification of sphingomonads.

Fourtythree strains of the genus Sphingomonas and close relatives were subjected to riboprint analyses generated after digestion of genomic DNA with the restriction enzyme EcoRI and hybridization with E. coli rrnB operon. The majority of strains were characterized by a complex banding pattern in the riboprints. High degrees of similarities in the riboprints were only observed among strains of the same species such as S. yanoikuyae, S. aromaticivorans, S. subarctica and S. chlorophenolica. Strains of different species including close phylogenetic relatives such as S. asaccharolytica, S. mali and S. pruni were easily distinguished by the differences in the riboprints even after visual evaluation. Thus, our data demonstrate that riboprint analysis is useful for preliminary identification of new sphingomonad isolates at the species level.

Alphaproteobacteria↗
PubMed1994

Pseudomonas pseudomallei isolates collected over 25 years from a non-tropical endemic focus show clonality on the basis of ribotyping.

Between 1966 and 1991, melioidosis, a disease caused by Pseudomonas pseudomallei that is mostly confined to tropical regions, occurred in farm animals and a farmer in temperate south-west Western Australia. Using an Escherichia coli probe containing a ribosomal RNA operon, P. pseudomallei DNA from isolates from 8 animals, a soil sample and the human case showed an identical ribotype on Southern blotting. The ribotype was different from the 3 commonest ribotypes seen in tropical Australia. This molecular typing supports the theory of clonal introduction of P. pseudomallei into a non-endemic region, with environmental contamination, local dissemination and persistence over 25 years. As melioidosis is often fatal in humans, such persistence in a temperate region is cause for concern.

Animals↗
PubMed2005

Diversity of genome structure in Salmonella enterica serovar Typhi populations.

The genomes of most strains of Salmonella and Escherichia coli are highly conserved. In contrast, all 136 wild-type strains of Salmonella enterica serovar Typhi analyzed by partial digestion with I-CeuI (an endonuclease which cuts within the rrn operons) and pulsed-field gel electrophoresis and by PCR have rearrangements due to homologous recombination between the rrn operons leading to inversions and translocations. Recombination between rrn operons in culture is known to be equally frequent in S. enterica serovar Typhi and S. enterica serovar Typhimurium; thus, the recombinants in S. enterica serovar Typhi, but not those in S. enterica serovar Typhimurium, are able to survive in nature. However, even in S. enterica serovar Typhi the need for genome balance and the need for gene dosage impose limits on rearrangements. Of 100 strains of genome types 1 to 6, 72 were only 25.5 kb off genome balance (the relative lengths of the replichores during bidirectional replication from oriC to the termination of replication [Ter]), while 28 strains were less balanced (41 kb off balance), indicating that the survival of the best-balanced strains was greater. In addition, the need for appropriate gene dosage apparently selected against rearrangements which moved genes from their accustomed distance from oriC. Although rearrangements involving the seven rrn operons are very common in S. enterica serovar Typhi, other duplicated regions, such as the 25 IS200 elements, are very rarely involved in rearrangements. Large deletions and insertions in the genome are uncommon, except for deletions of Salmonella pathogenicity island 7 (usually 134 kb) from fragment I-CeuI-G and 40-kb insertions, possibly a prophage, in fragment I-CeuI-E. The phage types were determined, and the origins of the phage types appeared to be independent of the origins of the genome types.

Chromosomes, Bacterial↗
PubMed2002

The genome structure of Pseudomonas putida: high-resolution mapping and microarray analysis.

As part of a collaborative project aimed at sequencing and functionally analysing the entire genome of Pseudomonas putida strain KT2440, a physical clone map was produced as an initial resource. To this end, a high-coverage cosmid library was arrayed and ordered by clone hybridizations. Restriction fragments generated by rare-cutting enzymes and plasmids containing the rrn operon and 23S rDNA of Pseudomonas aeruginosa were used as probes and, parts of the cosmids were end-sequenced. This provided the information necessary for merging and comparing the macro-restriction map, cosmid clone order and sequence information, thereby assuring co-linearity of the eventual sequence assembly with the actual genome. A tiling path of clones was selected, from the shotgun clones used for sequencing, for the production of DNA microarrays that represent the entire genome including its non-coding portions.

Cloning, Molecular↗