Structure and rearrangements of rRNA genes in chloroplast DNA in two strains of Euglena gracilis

Summary The organisation of the rRNA genes in the chloroplast genomes of two strains of Euglena gracilis were analyzed and compared. It was previously shown that the bacillaris strain contains three complete rrn (rRNA) operons (7) and that the Z-S strain contains one operon (21). Using heteroduplex analysis it was found that the bacillaris strain contains, apart from the three complete rrn operons, an extra 16S rRNA gene, an extra partial 23S rRNA gene sequence and an inverted duplication of a stretch within the 5S–16S spacer. In addition a short (<100 bp) inverted repeat sequence (13) which forms a stem/loop structure in single-stranded cpDNA was located between the 3-end of the extra 16S rRNA gene and the partial 23 S rRNA sequence.The Z-S strain differs from the bacillaris strain by a deletion of two units of the complete rrn operons. The region upstream of the single complete rrn operon, including the inverted repeats, the partial 23S and the extra 16S rRNA sequences is identical with the bacillaris strain.The only non-homology found in heteroduplexes between the SalI fragments of B of the two strains is the deletion-insertion loop which represents the two rrn operons. A small deletion loop was found occasionally in hetero-and in homoduplexes of both strands in the region of variable size. Apart from the deletion/insertion of two rrn operons the two genomes appear to be colinear as can be seen from partial denaturation mapping. The organisation of the rRNA genes of the two strains is compared with those of the Z strain and the bacillaris-ATCC strain.     

Life history implications of rRNA gene copy number in Escherichia coli

The role of the rRNA gene copy number as a central component of bacterial life histories was studied by using strains of Escherichia coli in which one or two of the seven rRNA operons (rrnA and/or rrnB) were deleted. The relative fitness of these strains was determined in competition experiments in both batch and chemostat cultures. In batch cultures, the decrease in relative fitness corresponded to the number of rRNA operons deleted, which could be accounted for completely by increased lag times and decreased growth rates. The magnitude of the deleterious effect varied with the environment in which fitness was measured: the negative consequences of rRNA operon deletions increased under culture conditions permitting more-rapid growth. The rRNA operon deletion strains were not more effective competitors under the regimen of constant, limited resources provided in chemostat cultures. Enhanced fitness in chemostat cultures would have suggested a simple tradeoff in which deletion strains grew faster (due to more efficient resource utilization) under resource limitation. The contributions of growth rate, lag time, Ks, and death rate to the fitness of each strain were verified through mathematical simulation of competition experiments. These data support the hypothesis that multiple rRNA operons are a component of bacterial life history and that they confer a selective advantage permitting microbes to respond quickly and grow rapidly in environments characterized by fluctuations in resource availability.     

Comparative analysis of the whole set of rRNA operons between an enterohemorrhagic Escherichia coli O157:H7 Sakai strain and an Escherichia coli K-12 strain MG1655

Two primer sets for direct sequence determination of all seven rRNA operons (rrn) of Escherichia coli have been developed; one is for specific-amplification of each rrn operon and the other is for direct sequencing of the amplified operons. Using these primer sets, we determined the nucleotide sequences of seven rrn operons, including promoter and terminator regions, of an enterohemorrhagic E. coli (EHEC) O157:H7 Sakai strain. To elucidate the intercistronic or intraspecific variation of rrn operons, their sequences were compared with those for the K-12 rrn operons. The rrn genes and the internal transcribed spacer regions showed a higher similarity to each other in each strain than between the corresponding operons of the two strains. However, the degree of intercistronic homogeneity was much higher in the EHEC strain than in K-12. In contrast, promoter and terminator regions in each operons were conserved between the corresponding operons of the two strains, which exceeded intercistronic similarity.     

Evaluation of the rrn operon copy number in Bifidobacterium using real-time PCR

AIMS: A real-time PCR-based method was developed to evaluate the Bifidobacterium rRNA operon copy number. As a result of their repetitive nature, rRNA operons are very suitable targets for chromosomal integration of heterologous genes. METHODS AND RESULTS: The rrn operon multiplicity per chromosome was determined by real-time PCR quantification of the 16S rRNA amplicons obtained from genomic DNA. The values obtained in several bifidobacterial strains of human origin ranged from 1 to 5. The reliability of the method developed was confirmed by Southern hybridization technique. CONCLUSIONS: In the Bifidobacterium genus the rrn operon copies showed variability at species and strain level. The identification of Bifidobacterium strains with high rRNA multiplicity allowed the selection of potential hosts for chromosomal integration. SIGNIFICANCE AND IMPACT OF THE STUDY: The methodology here proposed represents a rapid, reliable and sensitive new tool for the quantification of rrn operon copy number in bacteria.     

Isolation of spectinomycin resistance mutations in the 16S rRNA of Salmonella enterica serovar Typhimurium and expression in Escherichia coli and Salmonella

Two single-base mutations in 16S rRNA conferring high-level resistance to spectinomycin were isolated on a plasmid-borne copy of the rrnD operon from Salmonella enterica serovar Typhimurium. Neither of the mutations (C1066U and C1192U) had appreciable effects on cell growth, but each had differential effects on resistance to spectinomycin and fusidic acid. Both mutations also conferred resistance to spectinomycin in Escherichia coli strains containing deletions of all seven chromosomal rrn operons and expressing plasmid-encoded Salmonella rRNA exclusively. In contrast, when expressed in E. coli strains containing intact chromosomal rrn operons, the strains were sensitive to spectinomycin. However, chromosomal mutations arose that allowed expression of the rRNA-dependent spectinomycin resistance phenotype. It is proposed that in heterogeneous rRNA populations, the native E. coli rRNA out-competes the heterologous Salmonella rRNA for binding to ribosomal proteins, translation factors, or ribosome assembly, thus limiting entry of the antibiotic-resistant 30S subunits into the functioning ribosome pool.     

Instability of rRNA operons in Bacillus subtilis.

Many laboratory strains of Bacillus subtilis contain 9 rather than 10 rRNA operons due to deletions occurring within the rrnJ-rrnW or rrnI-rrnH-rrnG gene cluster. These operons are members of two sets of closely spaced clusters located in the cysA-aroI region. Analysis of rescued DNA from integrants with insertions into rrnG and rrnH indicated that these tandemly arranged operons allowed frequent deletions of an rrn operon equivalent. These events may arise spontaneously by intrachromosomal recombination or by simultaneous double crossovers with a multimeric integrative plasmid.     

rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation.

Here we present evidence that only five of the seven rRNA operons present in Escherichia coli are necessary to support near-optimal growth on complex media. Seven rrn operons are necessary, however, for rapid adaptation to nutrient and temperature changes, suggesting it is the ability to adapt quickly to changing environmental conditions that has provided the selective pressure for the persistence of seven rrn operons in E. coli. We have also found that one consequence of rrn operon inactivation is a miscoordination of the concentrations of initiation factor IF3 and ribosomes.     

The microbiology of butyrate formation in the human colon

A physical map of the Enterococcus faecium ATCC19434 chromosome was constructed by NotI, I-CeuI and Sse8387I. The chromosome was a circular DNA of 2600 kb in size, and contained six rRNA operons (rrn). The locations and orientations of the six rrn operons and 24 different determinants were mapped. Genomes of three additional E. faecium strains were also analyzed by I-CeuI digestion, and the genome sizes were found to vary from 2550 to 2995 kb. We further investigated the genome sizes and number of rrn operons in four E. faecalis, one E. avium, and one E. durans strains. The genome sizes were larger than E. faecium: 3000-3250 kb in E. faecalis, 3445 kb in E. avium, and 3070 kb in E. durans. E. avium and E. durans contained six rrn operons as in E. faecium, but all the E. faecalis strains possessed four rrn operons.     

Variable numbers of rRNA gene operons in Bacillus cereus strains

Abstract Ribosornal RNA operon organisation was analysed in two Bacillus cereus strains of different chromosome size, ATCC 10987 (5.4 Mb) and F0837/76 (2.4 Mb). We estimated that there were twelve and nine copies of the rRNA operons in these two strains, respectively. In B. cereus ATCC 10987 six rRNA operons were less than 10 kb apart, while in B. cereus F0837/76 four rRNA operons were similarly clustered. The origin of replication was located in the vicinity of a rRNA operon in both strains.     

Modulation of 16S rRNA function by ribosomal protein S12

Ribosomal protein S12 is a critical component of the decoding center of the 30S ribosomal subunit and is involved in both tRNA selection and the response to streptomycin. We have investigated the interplay between S12 and some of the surrounding 16S rRNA residues by examining the phenotypes of double-mutant ribosomes in strains of Escherichia coli carrying deletions in all chromosomal rrn operons and expressing total rRNA from a single plasmid-borne rrn operon. We show that the combination of S12 and otherwise benign mutations at positions C1409-G1491 in 16S rRNA severely compromises cell growth while the level and range of aminoglycoside resistances conferred by the G1491U/C substitutions is markedly increased by a mutant S12 protein. The G1491U/C mutations in addition confer resistance to the unrelated antibiotic, capreomycin. S12 also interacts with the 912 region of 16S rRNA. Genetic selection of suppressors of streptomycin dependence caused by mutations at proline 90 in S12 yielded a C912U substitution in 16S rRNA. The C912U mutation on its own confers resistance to streptomycin and restricts miscoding, properties that distinguish it from a majority of the previously described error-promoting ram mutants that also reverse streptomycin dependence.     

Tn10-mediated inversions fuse uridine phosphorylase (udp) and rRNA genes of Escherichia coli.

Two strains carrying metE::Tn10 insertions (upstream of the udp gene) were used to isolate mutants of Escherichia coli overexpressing udp. These strains differ in their gene order; one contains an inversion between the rrnD and rrnE rRNA operons. Selection was based on the ability of overexpressed Udp to complement thymine auxotrophy. Chromosomal rearrangements that connect the udp gene and promoters of different rrn operons were obtained by this selection. Seven of 14 independent mutants selected in one of the initial strains contained similar inversions of the metE-rrnD segment of the chromosome (about 12% of its length). Another mutant contained traces of a more complicated event, inversion between rrnB and rrnG operons, which was followed by reinversion of the segment between metE and the hybrid rrnG/B operon. Similar inversions (udp-rrn) in a strain already carrying an rrnE-rrnD inversion flip the chromosomal segment between metE and rrnD/E in the opposite direction. In this case, inversions are also accompanied by duplications of the chromosomal region between the rrnA and hybrid udp-rrnD/E operons. PCR amplification with a set of oligonucleotides from the rrn, Tn5, and met genes was used for more detailed mapping. Amplified fragments of the rearranged chromosomes connecting rrnD sequences and insertion elements were sequenced, and inversion endpoints were established.     

Variation in 16S-23S rRNA intergenic spacer regions among Bacillus subtilis 168 isolates

The genome of the Bacillus subtilis 168-type strain contains 10 ribosomal RNA (rRNA) operons. In the intergenic spacer region (ISR) between the 16S and 23S rRNA genes, five rRNA operons, rrnI-H-G and rrnJ-W, lack a trinucleotide signature region. Precise determination of molecular weight (MW), using electrospray mass spectrometry (MS), of the polymerase chain reaction (PCR) products from a segment of the ISR from the 168-type strain and B. subtilis 168-like strain 23071 demonstrated 114 and 111 basepair (bp) PCR products (due to the presence or absence of the insert in the operons) as predicted from sequence. However, PCR of the ISR segment for five other B. subtilis 168 isolates generated only a 114 bp PCR product, suggesting the presence of the trinucleotide signature region in all rRNA operons for these strains. Additional genetic variability between the seven B. subtilis 168 isolates was demonstrated by restriction fragment length polymorphism (RFLP) of the rRNA operons, with three distinct patterns found upon Southern blot analysis. The 168-type strain and three others (23066, 23067, and 23071) exhibited the same Southern pattern. Thus, operon deletion is not responsible for the absence of a 111 bp product on MS analysis for strains 23066 and 23067. Restriction analysis confirmed the presence of the trinucleotide signature region in the ISR of all rRNA operons for five B. subtilis 168 isolates; sequencing of rrnW/H from a representative strain also upheld this finding. These results help provide a better understanding of variations in sequence, operon number and chromosomal organization, both within a genome and among isolates of B. subtilis subgroup 168. It is also hypothesized that the presence of the trinucleotide insert in certain rRNA operons may play a role in rRNA maturation and protein synthesis.     

Rates and consequences of recombination between rRNA operons

A mutant strain of Escherichia coli was created by inserting a cassette encoding sucrose sensitivity and neomycin resistance (sacB-neo) into the small-subunit rRNA-encoding gene rrs in the rrnB operon. During growth in a complex medium, the cassette was lost from the population, and a complete rrs gene was restored at a rate of 5 x 10(-9) per cell division. Repair of this lesion required flanking regions of DNA that were similar to the six remaining intact rRNA operons and reestablished the full complement of seven rRNA operons. The relative fitness of strains with restored rrnB operons was 1 to 2% higher than that of the mutant strain. The rrnB operon normally contains a spacer region between the 16S and 23S rRNA-encoding genes that is similar in length and tRNA gene content to the spacer in rrnC, -E, and -G. In 2 of the 14 strains in which rrnB was restored, the spacer region had the same length as the spacer region in rrnA, -D, and -H. The requirement for flanking regions of nearly identical DNA and the replication of the spacer region from other rRNA operons during the repair of rrnB suggest that the restoration was accomplished via gene conversion. The rate of gene conversion was 10-fold less than the fixation of point mutations in the same region of the chromosome but was apparently sufficient to homogenize the sequences of rRNA genes in E. coli. These findings are discussed in the context of a conceptual model describing the presence of sequence heterogeneity in coevolving rRNA genes.     

Polymorphism and gene conversion of the 16S rRNA genes in the multiple rRNA operons of Vibrio parahaemolyticus

The genome sequence of a strain of Vibrio parahaemolyticus holds 11 copies of rRNA operons (rrn) with identical 16S rRNA genes (rrs). Conversely, the species type strain contains two rrs classes differing in 10 nucleotide sites within a short segment of 25 bp. Furthermore, we show here that the sequence of this particular segment largely differs between some strains of this species. We also show that of the eleven rrn operons in the species type strain, seven contain one rrs class and four the other, indicating gene conversion. Our results support the hypothesis that the rrs differences observed between strains of this species were caused by lateral transfer of an rrs segment and subsequent conversion.     

Genetic, physiological and biochemical characterization of multiple methanol methyltransferase isozymes in Methanosarcina acetivorans C2A

Biochemical evidence suggests that methanol catabolism in Methanosarcina species requires the concerted effort of methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB), a corrinoid-containing methyl-accepting protein (MtaC) and Co-methyl-5-hydroxybenzimidazolylcobamide:2-mercapto-ethanesulphonic acid methyltransferase (MtaA). Here we show that Methanosarcina acetivorans possesses three operons encoding putative methanol-specific MtaB and corrinoid proteins: mtaCB1, mtaCB2 and mtaCB3. Deletion mutants lacking the three operons, in all possible combinations, were constructed and characterized. Strains deleted for any two of the operons grew on methanol, whereas strains lacking all three did not. Therefore, each operon encodes a bona fide methanol-utilizing MtaB/corrinoid protein pair. Most of the mutants were similar to the wild-type strain, with the exception of the DeltamtaCB1 DeltamtaCB2 double mutant, which grew more slowly and had reduced cell yields on methanol medium. However, all mutants displayed significantly longer lag times when switching from growth on trimethylamine to growth on methanol. This indicates that all three operons are required for wild-type growth on methanol and suggests that each operon has a distinct role in the metabolism of this substrate. The combined methanol:CoM methyltransferase activity of strains carrying only mtaCB1 was twofold higher than strains carrying only mtaCB2 and fourfold higher than strains carrying only mtaCB3. Interestingly, the presence of the mtaCB2 and mtaCB3 operons, in addition to the mtaCB1 operon, did not increase the overall methyltransferase activity, suggesting that these strains may be limited by MtaA availability. All deletion mutants were unaffected with respect to growth on trimethylamine and acetate corroborating biochemical evidence indicating that each methanogenic substrate has specific methyltransfer enzymes.