Variation in the 16S-23S rRNA intergenic spacer regions in Vibrio parahaemolyticus strains are due to indels nearby their tRNAGlu.
TL;DR: Comparison of the nucleotide sequence between spacer classes suggests that intragenomic nonreciprocal recombination causes the size variations observed in the spacer regions of V. parahaemolyticus strains.
Abstract: Vibrio parahaemolyticus contains 11 rRNA operons each including one of six 16S–23S rRNA gene intergenic spacer classes differing in size and nucleotide sequence. Some of the spacer classes may differ between isolates. We observed that the differences in the spacers between isolates are generally in two spacer classes present in single copies in the genome, one class containing tRNAAla and tRNAGlu and the other tRNAGlu exclusively. Moreover, these differences are due to indels located nearby their tRNAGlu. Comparison of the nucleotide sequence between spacer classes suggests that intragenomic nonreciprocal recombination causes the size variations observed in the spacer regions of V. parahaemolyticus strains.
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TL;DR: This study underscores the potential contribution of interoperon ITS variation to bacterial microdiversity studies, as well as unequivocally demonstrates the pervasiveness of concerted evolution in the rrn gene family.
Abstract: Variation in the internal transcribed spacer (ITS) of the rRNA (rrn) operon is increasingly used to infer population-level diversity in bacterial communities. However, intragenomic ITS variation may skew diversity estimates that do not correct for multiple rrn operons within a genome. This study characterizes variation in ITS length, tRNA composition, and intragenomic nucleotide divergence across 155 Bacteria genomes. On average, these genomes encode 4.8 rrn operons (range: 2–15) and contain 2.4 unique ITS length variants (range: 1–12) and 2.8 unique sequence variants (range: 1–12). ITS variation stems primarily from differences in tRNA gene composition, with ITS regions containing tRNA-Ala + tRNA-Ile (48% of sequences), tRNA-Ala or tRNA-Ile (10%), tRNA-Glu (11%), other tRNAs (3%), or no tRNA genes (27%). Intragenomic divergence among paralogous ITS sequences grouped by tRNA composition ranges from 0% to 12.11% (mean: 0.94%). Low divergence values indicate extensive homogenization among ITS copies. In 78% of alignments, divergence is <1%, with 54% showing zero variation and 81% containing at least two identical sequences. ITS homogenization occurs over relatively long sequence tracts, frequently spanning the entire ITS, and is largely independent of the distance (basepairs) between operons. This study underscores the potential contribution of interoperon ITS variation to bacterial microdiversity studies, as well as unequivocally demonstrates the pervasiveness of concerted evolution in the rrn gene family.
67 citations
Cites background from "Variation in the 16S-23S rRNA inter..."
...…variation at the interstrain and interspecies levels, particularly with respect to medically relevant organisms (Chun et al. 1999; Osorio et al. 2005; González-Escalona et al. 2006), for which knowledge of ITS composition is valuable for differentiating and tracking pathogenic genetic variants....
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TL;DR: This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well.
Abstract: The genus Vibrio is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous Vibrio species. In this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal Vibrio strains, representing 48 Vibrio species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among Vibrio. Furthermore, testing 36 strains each of V. parahaemolyticus and V. vulnificus, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well. This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations providing information more quickly than other time-honoured methods traditionally used in these types of analyses.
42 citations
Cites result from "Variation in the 16S-23S rRNA inter..."
...This particular observation deviated significantly from an earlier study [26] proposing that V....
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...Furthermore, the previous study [26], suggests that mismatches in the L1 (Jensen) primer [21] gave rise to a different and, presumably, an incorrect banding pattern from that generated when using their own primer set, although the L1/G1 pattern they present in their representation is clearly in agreement with the pattern that would be theoretically obtained from the NCBI genomic sequence of the V....
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TL;DR: Despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.
Abstract: Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that “enhanced infection” is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.
26 citations
Cites methods from "Variation in the 16S-23S rRNA inter..."
...The bands on the gel (only fragments between sizes of 500 bp and 2500, the size range well resolved in this gel) were visualized by silver staining, as described previously [29]....
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TL;DR: Although the pathology of this disease appears similar in the various different hosts, sequencing and examination of the phylogenetic relationships reveal 4 distinct RLB involved in the infection process.
Abstract: Black tiger shrimp Penaeus monodon, European shore crab Carcinus maenas and spiny lobster Panulirus spp. can be affected by milky hemolymph syndrome (MHS). Four rickettsia-like bacteria (RLB) isolates of MHS originating from 5 geographical areas have been identified to date. The histopathology of the disease was characterized and a multiplex PCR assay was developed for detection of the 4 bacterial isolates. The 16S rRNA gene and 16-23S rRNA intergenic spacer region (ISR) were used to examine the phylogeny of the MHS isolates. Although the pathology of this disease appears similar in the various different hosts, sequencing and examination of the phylogenetic relationships reveal 4 distinct RLB involved in the infection process.
25 citations
TL;DR: DGREA established clearer relationships among V. vulnificus strains and was more consistent with MLST than with PFGE, and is a very promising tool for epidemiological and ecological studies of Vibrio vulnIFICus.
Abstract: We compared the potential of direct genome restriction enzyme analysis (DGREA) and pulsed-field gel electrophoresis (PFGE) for discriminating Vibrio vulnificus isolates from clinical (23) and environmental (17) sources. The genotypes generated by both methodologies were compared to previous multilocus sequence typing (MLST) data. DGREA established clearer relationships among V. vulnificus strains and was more consistent with MLST than with PFGE. DGREA is a very promising tool for epidemiological and ecological studies of V. vulnificus.
20 citations
Cites methods from "Variation in the 16S-23S rRNA inter..."
...Bands were visualized by silver staining as described previously (8) and photographed using a Canon PowerShot A620 digital camera....
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References
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
35,309 citations
Book•
01 Jan 2001
TL;DR: The content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
25,596 citations
TL;DR: A novel fluorimetric method to estimate the G+C mol% content in microorganisms using double-stranded DNA and its thermal denaturalization was followed by measuring a decrease in fluorescence using a real-time PCR thermocycler.
Abstract: G+C mol% content in microorganisms is one of the recommended characteristics for the standard description of bacterial species. In this study we present a novel fluorimetric method to estimate the G+C mol% content in microorganisms. Double-stranded DNA was specifically stained with SYBR Green I, and its thermal denaturalization was followed by measuring a decrease in fluorescence using a real-time PCR thermocycler. Unlike most previous determinations of G+C mol%, in this study only DNA from microorganisms with an available completely sequenced genome were used to prepare the calibration curves. Calibration curves showed a linear relationship between G+C mol% content and melting temperature and they were performed both in the absence and presence of 30% formamide. This protocol proves to be a rapid and inexpensive method to estimate DNA base ratios of novel microorganisms.
1,650 citations
TL;DR: This finding explains clinical features of V parahaemolyticus infections, which commonly include inflammatory diarrhoea and in some cases systemic manifestations such as septicaemia, distinct from those of V cholerae infections,Which are generally associated with non-inflammatory diarrhoeA.
933 citations