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PubMed1992

The Fis protein: it's not just for DNA inversion anymore.

Higher-order nucleoprotein complexes are associated with many biological processes. In bacteria the formation of these macromolecular structures for DNA recombination, replication, and transcription often requires not only the participation of specific enzymes and co-factors, but also a class of DNA-binding proteins collectively known as 'nucleoid-associated' or 'histone-like' proteins. Examples of this class of proteins are HU, Integration Host Factor, H-NS, and Fis. Fis was originally identified as the factor for inversion stimulation of the homologous Hin and Gin site-specific DNA recombinases of Salmonella and phage Mu, respectively. This small, basic, DNA-bending protein has recently been shown to function in many other reactions including phage lambda site-specific recombination, transcriptional activation of rRNA and tRNA operons, repression of its own synthesis, and oriC-directed DNA replication. Cellular concentrations of Fis vary tremendously under different growth conditions which may have important regulatory implications for the physiological role of Fis in these different reactions. The X-ray crystal structure of Fis has been determined and insights into its mode of DNA binding and mechanisms of action in these disparate systems are being made.

Bacterial Proteins↗
PubMed1995

High-resolution alignment of a 1-megabase-long genome region of three strains of Rhodobacter capsulatus.

A detailed restriction map of the genome of Rhodobacter capsulatus SB1003 was constructed recently by using an ordered set of overlapping cosmids. Pulsed-field gel electrophoresis-generated restriction patterns of the chromosomes of 14 other R. capsulatus strains were compared. Two of them, St. Louis and 2.3.1, were chosen for high-resolution alignment of their genomes with that of SB1003. A 1-Mb segment of the R. capsulatus SB1003 cosmid set was used as a source of ordered probes to group cosmids from the other strains. Selected cosmids were linked into one 800-kb contig and two smaller contigs of 100 kb each. EcoRV and BamHI restriction maps of the newly ordered cosmids were constructed by using lambda terminase. Long-range gene order in the new strains was mainly conserved for the regions studied. However, one large genome rearrangement inverted a 470-kb DNA fragment of the St. Louis strain between the rrnA and rrnB operons. A 50-kb deletion covering three SB1003 probes was found in strain 2.3.1 near rrnB. Conservation of about 50% of the positions of restriction sites in all these strains and nearly 80% for the pair 2.3.1- St. Louis made it possible to produce high-resolution alignment of the contiguous 800-kb genome segment. Ten deletions of 2 to 27 kb, one 30-kb inversion, and three translocations were found in this region. Strong clustering of the positions of polymorphic restriction sites was observed. For a 50-kb size interval, two patterns of the distribution of restriction sites were found, one with about 90% and the other with 5 to 30% conservation of sites. This structure may be explained by independent acquisition of these divergent regions from other Rhodobacter strains.

Chromosome Inversion↗
PubMed1997

Genomic organization of the acidophilic chemolithoautotrophic bacterium Thiobacillus ferrooxidans ATCC 21834.

The genomic organization of the acidophilic chemolithoautotrophic bacterium Thiobacillus ferrooxidans ATCC 21834 has been studied by pulsed-field gel electrophoresis (PFGE). Analysis of its intact DNA, as well as the restriction patterns obtained with several endonucleases, allowed the characterization of one circular chromosome of 2.9 Mb and one plasmid of 8.6 kb. The first complete and highly resolved physical map (86 restriction sites) of the chromosome of an acidophilic obligate chemolithoautotrophic bacterium has been constructed by using endonucleases PmeI, SwaI, XbaI, and SpeI. The rRNA and str operons have been located on the chromosomal physical map.

Acidithiobacillus thiooxidans↗
PubMed1980

The distal end of the ribosomal RNA operon rrnD of Escherichia coli contains a tRNA1thr gene, two 5s rRNA genes and a transcription terminator.

The distal region of the Escherichia coli ribosomal RNA operon rrnD has been sequenced to identify transfer RNA genes which might be contained within this transcriptional unit, and to define a possible transcription termination site. A nucleotide sequence corresponding to the gene for tRNA1thr is located between two genes for 5S rRNA. The spacer region between the proximal 5S rRNA gene and the tRNA1thr gene is 12 base pairs long, whereas the spacer region between the distal 5S rRNA gene and the tRNA1thr gene is 37 base pairs. Possible sites for RNA processing are discussed, and the sequence of the tRNA1thr gene is found to possess the same anticodon as the tRNA3thr gene. A putative transcription termination site for rrnD was located about 25 base pairs distal to the 3' end of the second 5S rRNA gene. The rrnD and rrnC terminators are compared with each other and with other prokaryotic terminators. Located at exactly the same site on the complementary strand is another putative transcription termination site for a gene being transcribed towards rrnD. The possibility of overlapping terminators is discussed.

Base Composition↗
PubMed1992

The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris.

The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.

Animals↗
PubMed1992

rRNA gene restriction patterns as taxonomic tools for the genus Aeromonas.

In the genus Aeromonas there are at least 13 DNA hybridization groups, which are difficult to differentiate biochemically. We investigated the usefulness of rRNA gene restriction patterns for characterization and identification of the various groups. Genomic DNA was digested with restriction endonuclease SmaI, transferred to a nylon membrane, and hybridized with biotinylated plasmid pKK3535 containing the rrnB operon of Escherichia coli. The SmaI bands at 0.8 to 4 kb but not those at positions corresponding to sizes larger than 4 kb showed a good correlation with hybridization groups, allowing identification of strains to the level of genetic species. We demonstrated that the 567-bp fragment localized between positions 80 and 647 of the 16S ribosomal gene of E. coli was essential for hybridization to the low-molecular-weight fragments, whereas the remainder of the operon did not hybridize to these fragments. On the basis of these results, we concluded that the Aeromonas chromosome contains multiple rRNA operons which may be used for species identification.

Aeromonas↗
PubMedDate not supplied

Antisignature oligonucleotides and their analogs as inhibitors of mollicutes-cofactors of HIV.

Inhibition of mollicutes by synthetic oligonucleotides and their analogs complementary to specific "signature" regions of 16S rRNA and corresponding sequences of ribosomal operon DNA was studied. It was shown that antisignature oligonucleotides inhibited transcription in vitro for above 79% interacting specifically with ribosomal operon and non-specific with DNA-dependent RNA-polymerase. The inhibition efficiency depended on oligonucleotide sequence and type of modification. Translation in vitro was suppressed most efficiently (up to 60%) by oligonucleotides complementary to 3'-end region of 16S rRNA, also depending on their modification. Translation in vivo was inhibited most efficiently (up to 73%) by thiophosphate analogs of oligonucleotides complementary to sequences 499-507 and 523-532 of 16S rRNA responsible for binding of ribosomal "core" protein S4 starting the assembly of 30S ribosome subunit. With the simultaneous use of the last two oligonucleotides, the growth of mollicutes in SM IMV-72 medium rich in exogenous sources of nucleosides was suppressed for over 90%. It is supposed that under conditions where mollicutes have no free access to starting materials for their own synthesis of nucleic acid these nucleotides could suppress microorganisms completely. Antisignature oligonucleotides are considered as superspecific agents not leading to the development of resistance of mollicutes and believed to be the main future remedy against diseased caused by microorganisms lacking the system of nucleoside synthesis.

Acholeplasma laidlawii↗
PubMed2004

Evolution of the RNA polymerase B' subunit gene (rpoB') in Halobacteriales: a complementary molecular marker to the SSU rRNA gene.

Many prokaryotes have multiple ribosomal RNA operons. Generally, sequence differences between small subunit (SSU) rRNA genes are minor (<1%) and cause little concern for phylogenetic inference or environmental diversity studies. For Halobacteriales, an order of extremely halophilic, aerobic Archaea, within-genome SSU rRNA sequence divergence can exceed 5%, rendering phylogenetic assignment problematic. The RNA polymerase B' subunit gene (rpoB') is a single-copy conserved gene that may be an appropriate alternative phylogenetic marker for Halobacteriales. We sequenced a fragment of the rpoB' gene from 21 species, encompassing 15 genera of Halobacteriales. To examine the utility of rpoB' as a phylogenetic marker in Halobacteriales, we investigated three properties of rpoB' trees: the variation in resolution between trees inferred from the rpoB' DNA and RpoB' protein alignment, the degree of mutational saturation between taxa, and congruence with the SSU rRNA tree. The rpoB' DNA and protein trees were for the most part congruent and consistently recovered two well-supported monophyletic groups, the clade I and clade II haloarchaea, within a collection of less well resolved Halobacteriales lineages. A comparison of observed versus inferred numbers of substitution revealed mutational saturation in the rpoB' DNA data set, particularly between more distant species. Thus, the RpoB' protein sequence may be more reliable than the rpoB' DNA sequence for inferring Halobacteriales phylogeny. AU tests of tree selection indicated the trees inferred from rpoB' DNA and protein alignments were significantly incongruent with the SSU rRNA tree. We discuss possible explanations for this incongruence, including tree reconstruction artifact, differential paralog sampling, and lateral gene transfer. This is the first study of Halobacteriales evolution based on a marker other than the SSU rRNA gene. In addition, we present a valuable phylogenetic framework encompassing a broad diversity of Halobacteriales, in which novel sequences can be inserted for evolutionary, ecological, or taxonomic investigations.

Base Sequence↗
PubMed1997

Depletion of pre-16S rRNA in starved Escherichia coli cells.

Specific hybridization assays for intermediates in rRNA synthesis (pre-rRNA) may become useful for monitoring the growth activity of individual microbial species in complex natural systems. This possibility depends upon the assumption that rRNA processing in microbial cells continues after growth and pre-rRNA synthesis cease, resulting in drainage of the pre-rRNA pool. This is not the case in many eukaryotic cells, but less is known about the situation in bacteria. Therefore, we used DNA probes to measure steady-state cellular pre-16S rRNA pools during growth state transitions in Escherichia coli. Pre-16S rRNA became undetectable when cells entered the stationary phase on rich medium and was replenished upon restoration of favorable growth conditions. These fluctuations were of much greater magnitude than concurrent fluctuations in the mature 16S rRNA pool. The extent of pre-16S rRNA depletion depended upon the circumstances limiting growth. It was significantly more pronounced in carbon-energy-starved cells than in nitrogen-starved cells or in cells treated with energy uncouplers. In the presence of the transcriptional inhibitor rifampin, rates of pre-16S rRNA depletion in carbon-energy-starved cells and nitrogen-starved cells were similar, suggesting that the difference between these conditions resides primarily at the level of pre-rRNA synthesis. Chloramphenicol, which inhibits the final steps in rRNA maturation, halted pre-16S rRNA depletion under all conditions. The data show that E. coli cells continue to process pre-rRNA after growth and rrn operon transcription cease, leading to drainage of the pre-rRNA pool. This supports the feasibility of using pre-rRNA-targeted probes to monitor bacterial growth in natural systems, with the caveat that patterns of pre-rRNA depletion vary with the conditions limiting growth.

Anti-Bacterial Agents↗
PubMed2001

[Transcription regulation in the ribosomal RNA operon of mycobacteria].

Tuberculosis is a extremely important infectious disease, caused by the bacilli Mycobacterium tuberculosis. One of the characteristic of this bacteria is its very slow rate of growth, that allows it to survive for long periods of time inside the host cells. Among the genetic elements involved in growth regulation the operon rrn is of extreme importance. This operon contains the genes that code the three rRNA molecules, essential components of the bacterial ribosome. The tuberculosis bacilli, differently from most of the microorganisms, has a single copy of the rrn operon per genome, meaning that it must be submitted to very strict control mechanisms. Another important conclusion is that the sequences of the rrn operon constitute ideal targets for anti-mycobacterial drugs. In this work we have studied some of the elements involved in transcription control in M. tuberculosis, particularly those present in the leader region of the operon. By using basic molecular biology techniques we have identified sequence elements in the leader region that seem to be involved in the control of transcription elongation, by a mechanism related to anti-termination.

Base Sequence↗
PubMed1980

Euglena gracilis chloroplast ribosomal RNA transcription units. II. Nucleotide sequence homology between the 16 S--23 S ribosomal RNA spacer and the 16 S ribosomal RNA leader regions.

The DNA sequences of two segments of the ribosomal RNA transcription units of Euglena gracilis Pringsheim strain Z chloroplast DNA have been determined. The first is from the 16 S to 23 S rRNA spacer region. The nucleotide sequence determined includes 64 bp from the 3'-end of the 16 S rRNA gene, the adjacent 87-bp spacer containing 68A-T base pairs, a tRNAIle gene, a 9-bp spacer, a tRNAAla gene, a spacer of approximately 15 bp, and the first 120 bp from the 5'-end of the 23 S rRNA gene. The gene organization of the 16 S to 23 S rRNA spacer, the identity of the tRNA genes, and the tRNA anticodons for the E. gracilis rRNA transcription units are identical with that of the rrnA, D, and X operons of Escherichia coli. The second DNA segment which was sequenced is from a region preceding the 5'-end of the 16 S rRNA gene. Within a continuous region of 189 bp in this 16 S rRNA leader sequence, 68% of the bases are homologous to the 16 S rRNA to 23 S rRNA spacer region. This homology includes the 3'-end of the 16 S rRNA gene, the adjacent spacer, and a complete "pseudo" tRNAIle gene. This leader sequence which has the same polarity as the rRNA transcripts, is flanked by nucleotide sequences resembling partial tRNA genes.

Base Sequence↗
PubMed1988

Mutational analysis of the L1 binding site of 23S rRNA in Escherichia coli.

The L11 ribosomal protein operon of Escherichia coli contains the genes for L11 and L1 and is feedback regulated by the translational repressor L1. Both the L1 binding site on 23S rRNA and the L1 repressor target site on L11 operon mRNA share similar proposed secondary structures and contain some primary sequence identity. Several site-directed mutations in the binding region of 23S rRNA were constructed and their effects on binding were examined. For in vitro analysis, a filter binding method was used. For in vivo analysis, a conditional expression system was used to overproduce a 23S rRNA fragment containing the L1 binding region, which leads to specific derepression of the synthesis of L11 and L1. Changes in the shared region of the 23S rRNA L1 binding site produced effects on L1 binding similar to those found previously in analysis of corresponding changes in the L11 operon mRNA target site. The results support the hypothesis that r-protein L1 interacts with both 23S rRNA and L11 operon mRNA by recognizing similar features on both RNAs.

Bacterial Proteins↗
PubMed1984

Evidence for antitermination in Escherichia coli RRNA transcription.

The stable RNA operons of Escherichia coli do not exhibit polarity, even though they make an RNA product that is not translated. By contrast, most E. coli operons that specify proteins exhibit polarity if their translation is interrupted. The transcriptional component of this polarity depends on the action of Rho protein on the exposed mRNA, which results in premature transcription termination. Here we examine how a stable RNA operon (rrnG) transcript is protected from the Rho protein-mediated polarity response. We compared transcription from the ara and the rrnG promoters through a 16S DNA segment. In each case, the promoter-16S sequences were joined to a trp-lac fusion, and lacZ mRNA was examined in rho+ and rho-115 strains. We found significant Rho protein-dependent termination of transcripts from the ara promoter but little or no Rho protein effect on transcription from the rrnG promoter. We concluded that the transcript of the 16S ribosomal DNA segment does contain Rho protein-dependent transcription terminators, but there is an antitermination system in the rrnG control region that allows it to transcribe through those terminators.

Base Sequence↗
PubMed1997

Ribosomal RNA (rrn) operons in Streptococcus mutans and nucleotide sequence of tRNA(Pro) gene associated with rrnB.

Using a cloned 0.5-kb probe containing an internal fragment of 23S ribosomal RNA from the rrnB operon of Streptococcus mutans, we analyzed various endonuclease digests of the chromosomal DNA isolated from human-derived strains of mutans streptococci. Thus far, the examined S. mutans strains exhibited five ribosomal operons. Here, we describe a ribotyping technique for S. mutans based on restriction and Southern blot analyses with the biotin-labeled homologous probe and chemiluminescence detection. We cloned and sequenced a unique gene encoding tRNA(Pro) downstream from 23S rRNA gene at the 3' end of the operon. Primers designed to the 3' end of the rrnB operon PCR-amplified a 2.3-kb DNA fragment in all tested strains. Restriction fragment length polymorphism analysis of the amplicon revealed a diversity of the single locus among S. mutans isolates, thus establishing a potential use of the technique for the molecular epidemiology of mutans streptococci.

Base Sequence↗
PubMed2001

Extensive ribosomal DNA genic variation in the columnar cactus Lophocereus.

Sequence analysis of the hypervariable internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) is commonly used to gain insights into plant and animal population structure and phylogeny. We characterized ITS1, ITS2, and the 5.8S coding region of 18 senita (Lophocereus) individuals from 12 different populations in Baja as well as from closely related cactus species. Analyses of multiple clones demonstrated extensive paralogy in the senita rDNA gene family. We identified at least two putatively non-recombining rDNA operons in senita as well as multiple paralogous sequences within each operon. Usage of PCR, reverse transcriptase (RT)-PCR, Southern blot, primary sequence analyses of the 18S rDNA gene, and secondary structure analyses of the 5.8S rRNA showed that one of the operons encodes rDNA pseudogenes in a low copy-number (Truncated), whereas the second operon encodes an expressed rRNA (Functional). Surprisingly, we found extensive paralogy not only in the ITS regions but also in the 5.8S coding regions in senita both within and between operons. Phylogenetic analyses suggest that the second rDNA operon originated prior to the divergence of Lophocereus. A significant (p < 0.05) divergence-rate acceleration was found in the Lophocereus 5.8S rDNA coding region in the Functional operon in comparison to Pereskiopsis porteri (Cactaceae) and Portulaca molokiniensis (Portulacaceae) with Silene dioica and Spinacia oleracea as the outgroups.

Base Sequence↗
PubMed1999

Molecular characterization of a new ribotype of Vibrio cholerae O139 Bengal associated with an outbreak of cholera in Bangladesh.

Vibrio cholerae O139 Bengal initially appeared in the southern coastal region of Bangladesh and spread northward, causing explosive epidemics during 1992 and 1993. The resurgence of V. cholerae O139 during 1995 after its transient displacement by a new clone of El Tor vibrios demonstrated rapid changes in the epidemiology of cholera in Bangladesh. A recent outbreak of cholera in two north-central districts of Bangladesh caused by V. cholerae O139 led us to analyze strains collected from the outbreak and compare them with V. cholerae O139 strains isolated from other regions of Bangladesh and neighboring India to investigate their origins. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA (ribotype) revealed that the recently isolated V. cholerae O139 strains belonged to a new ribotype which was distinct from previously described ribotypes of toxigenic V. cholerae O139. All strains carried the genes for toxin-coregulated pili (tcpA and tcpI) and accessory colonization factor (acfB), the regulatory gene toxR, and multiple copies of the lysogenic phage genome encoding cholera toxin (CTXPhi) and belonged to a previously described ctxA genotype. Comparative analysis of the rfb gene cluster by PCR revealed the absence of a large region of the O1-specific rfb operon downstream of the rfaD gene and the presence of an O139-specific genomic region in all O139 strains. Southern hybridization analysis of the O139-specific genomic region also produced identical restriction patterns in strains belonging to the new ribotype and those of previously described ribotypes. These results suggested that the new ribotype of Bengal vibrios possibly originated from an existing strain of V. cholerae O139 by genetic changes in the rRNA operons. In contrast to previously isolated O139 strains which mostly had resistance to trimethoprim, sulfamethoxazole, and streptomycin encoded by a transposon (SXT element), 68.6% of the toxigenic strains analyzed in the present study, including all strains belonging to the new ribotype, were susceptible to these antibiotics. Molecular analysis of the SXT element revealed possible deletion of a 3.6-kb region of the SXT element in strains which were susceptible to the antibiotics. Thus, V. cholerae O139 strains in Bangladesh are also undergoing considerable reassortments in genetic elements encoding antimicrobial resistance.

Bangladesh↗
PubMed1989

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia coli rrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affinity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis.

Escherichia coli↗