Some rRNA operons in E. coli have tRNA genes at their distal ends.

We have previously isolated seven rRNA operons on plasmids or lambda transducing phages and identified various tRNAs encoded by these operons. Each of the seven operons has one of two different spacer tRNA gene arrangements between the genes for 16S and 23S rRNA: either tRNAGlu2 or both tRNAIle1 and tRNAAla1B genes. In addition, various tRNA genes are located at or near the distal ends of rRNA operons. In particular, genes for tRNATrp and tRNAAsp1 are located at the distal end of rrnC at 83 min on the E. coli chromosome. Experiments with various hybrid plasmids, some of which lack the rRNA promoter, have now demonstrated that this promoter is necessary for expression of the distal tRNA genes. Rifampicin run-out experiments have also provided evidence that the tRNATrp gene is located farther from its promoter than the spacer tRNA gene or the 5S RNA gene. These results confirm the localization of genes for tRNATrp and tRNAAsp1 at the distal end of rrnC and strongly suggest that they are co-transcribed with the genes for 16S, tRNAGlu2, 23S and 5S RNA. Other such distal tRNAs have been identified, and it is suggested that they too are part of rRNA operons.     

Loss of the spacer loop sequence from the rrnB operon in the Escherichia coli K-12 subline that bears the relA1 mutation.

A polymorphism affecting the spacer region of the rrnB rRNA operon is described. Strains from a major Escherichia coli K-12 subbranch are missing a 106-nucleotide portion of the rrnB 16S-to-23S spacer, and a 20-nucleotide sequence is found in its place. We have called this mutant operon rrnB2. The rrnB2 spacer was most probably derived from either rrnC or rrnE. This alteration of rrnB may have occurred by a recombinational exchange or by gene conversion. In the genealogy of E. coli K-12 strains, the appearance of rrnB2 is associated with the spontaneous occurrence of the first relaxed mutation, but attempts to show a selective relationship between the two mutational events have had negative results. The sequences of the rrnG and rrnC 16S-to-23S spacers have also been determined and their comparisons to the other rrn operons encoding tRNAGlu2 are presented.     

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.     

Involvement of ribosomal ribonucleic acid operons in Salmonella typhimurium chromosomal rearrangements.

As part of our efforts to understand factors influencing chromosomal organization and rearrangements, we studied a family of Salmonella typhimurium tandum duplication mutants. We found that the duplications were originally generated by unequal recombination between pairs of similarly oriented ribosomal ribonucleic acid operons (rrn). This demonstration involved the physical isolation of the duplicated material as circular deoxyribonucleic acid excised from the duplication. The four rrn operons involved embraced the ilv pur D segment of the chromosome and occurred at positions closely analogous to those previously observed for Escherichia coli. The interval between rrnC and rrnA of S. typhimurium was similar in size to that of E. coli (43 versus 39 kilobases), as was the interval between rrnB and rrnE (94 versus 91 kilobases). The rrnA-to-rrnB interval of S. typhimurium, however, was 155 kilobases, substantially greater than the 126 kilobases observed for E. coli.     

Ribosomal RNA operon anti-termination. Function of leader and spacer region box B-box A sequences and their conservation in diverse micro-organisms

All Escherichia coli rrn operons show a common motif in which anti-terminator box B-box A sequences occur twice, first in the leader and again in the 16 S-23 S spacer. In this study we have analyzed several aspects of rrn anti-termination by leader and spacer anti-terminator sequences. Using DNA synthesis and a plasmid test system, we incorporated random changes into the leader anti-terminator region and examined these mutations for their ability to read through a strong terminator. We also examined anti-termination by synthetic box A and by rrn spacer region sequences. Information derived from these experiments was used to search the rrn sequences of other micro-organisms for possible anti-termination features. Our principal conclusions were that: (1) box A was sufficient for terminator readthrough; (2) we could show no positive requirement for box B in our test system; (3) many of the negative anti-terminator mutations caused a promoter up-effect in the absence of a terminator; (4) the search of rrn operons from other micro-organisms revealed that anti-terminator-like box B-box A sequences exist in leader and spacer regions of both eubacteria and archaebacteria. The frequent occurrence of this pattern suggested that the E. coli rrn anti-termination motif is widespread in nature and has been conserved in microbial evolution.     

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.     

Sequences of the envM gene and of two mutated alleles in Escherichia coli.

The nucleotide sequence of the Escherichia coli envM gene was determined. It codes for a protein of 262 amino acids. The sequences of the E. coli and Salmonella typhimurium EnvM proteins are 98% identical. Gene envM is preceded in E. coli by a 43-nucleotide-long structural element, termed 'box c', which occurs in several E. coli operons between structural genes. This sequence element is totally absent in S. typhimurium. Gene envM was mapped at coordinate position 1366.8 kb of the physical map of Kohara et al. (Cell, 1987, 50, 495-508). As in S. typhimurium, a Gly for Ser exchange at position 93 of the amino acid sequence leads to a diazaborine-resistant E. coli phenotype. A Ser for Phe exchange at position 241 of the EnvM protein results in a temperature-sensitive growth phenotype. Comparison of the EnvM amino acid sequence with sequences available in databases showed significant homology with the family of short-chain alcohol dehydrogenases.     

Exchange of Spacer Regions between Rrna Operons in Escherichia Coli

The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions. The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala1B). We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type. Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala1B) spacer (at rrnA). While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected. At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer. In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal. In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons. Two examples of inversion of one-half of the E. coli chromosome between rrnG and rrnH were observed. The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids.     

Merodiploidy in ESCHERICHIA COLI-SALMONELLA TYPHIMURIUM Crosses: The Role of Unequal Recombination between Ribosomal RNA Genes

Previous workers have shown that intergeneric crosses between Salmonella typhimurium and Escherichia coli produce a high proportion of merodiploid recombinants among the viable progeny. We have examined the unequal cross-over event that was responsible for a number of intergeneric merodiploids. The merodiploids that we studied were all heterozygous for the metB-argH interval and were the products of intergeneric conjugal crosses. We found that when the S. typhimurium donor had its transfer origin closely linked to metB and argH, all recombinants examined were merodiploid, and they generally arose as F-prime factors. Many of these F-prime factors had been created by recombination between flanking rrn genes in the donor. When the S. typhimurium Hfr transfer origin was more distant from the selected markers, quite different results were obtained. Depending on the donor, 19-47% of the recombinants that acquired the donor argH+ or metB+ genes were merodiploid for these loci, but none of the recombinants were F-prime. A majority of the merodiploids had a novel (nonparental) rrn gene, indicating that unequal recombination between nonidentical rrn genes was a prevalent mechanism for establishing the merodiploidy. Both tandem and nontandem duplications were found. Some of the merodiploids duplicated E. coli genes in addition to acquiring S. typhimurium genes. Some merodiploids contained the oriC region from each parent. Of a total of 118 intergeneric merodiploids characterized from all donors, 48 different genotypes were observed, and 38 of the 48 had one or more nonparental rrn operons.     

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.     

DNA sequence of the 16S rRNA/23S rRNA intercistronic spacer of two rDNA operons of the archaebacterium Methanococcus vannielii

The DNA sequence of the spacer (plus flanking) regions separating the 16S rRNA and 23S rRNA genes of two presumptive rDNA operons of the archaebacterium Methanococcus vannielii was determined. The spacers are 156 and 242 base pairs in size and they share a sequence homology of 49 base pairs following the 3' terminus of the 16S rRNA gene and of about 60 base pairs preceding the 5' end of the 23S rRNA gene. The 242 base pair spacer, in addition contains a sequence which can be transcribed into tRNAAla, whereas no tRNA-like secondary structure can be delineated from the 156 base pair spacer region. Almost complete sequence homology was detected between the end of the 16S rRNA gene and the 3' termini of either Escherichia coli or Halobacterium halobium 16S rRNA, whereas the putative 5' terminal 23S rRNA sequence shared partial homology with E. coli 23S rRNA and eukaryotic 5.8S rRNA.     

Structure of Recombinants from Conjugational Crosses between Escherichia Coli Donor and Mismatch-Repair Deficient Salmonella Typhimurium Recipients

To get more insight into the control of homologous recombination between diverged DNA by the Mut proteins of the long-patch mismatch repair system, we have studied interspecies Escherichia coli/Salmonella typhimurium recombination. Knowing that the same recombination pathway (RecABCD) is responsible for intraspecies and interspecies recombination, we have now studied the structure (replacement vs. addition-type or other rearrangement-type recombinants) of 81 interspecies recombinants obtained in conjugational crosses between E. coli donor and mutL, mutS, mutH, mutU or mut(+) S. typhimurium recipients. Taking advantage of high interspecies sequence divergence, a physical analysis was performed on one third of the E. coli Hfr genome, which was expected to be transferred to S. typhimurium F(-) recipients during 40 min before interruption of the mating. Probes specific for each species were hybridized on dot blots of genomic DNA, or on colonies, and the composition of the rrn operons was determined from purified genomic DNA. With very few exceptions, the structure of these interspecies recombinants corresponds to replacements of one continuous block of the recipient genome by the corresponding region of the donor genome.     

A rapid PCR procedure for the specific identification of Lactobacillus sanfranciscensis, based on the 16S-23S intergenic spacer regions

AIMS: The organization of ribosomal RNA (rrn) operons in Lactobacillus sanfranciscensis was studied in order to establish an easy-to-perform method for identification of L. sanfranciscensis strains, based on the length and sequence polymorphism of the 16S-23S rDNA intergenic spacer region (ISR). METHODS AND RESULTS: PCR amplification of the 16S-23S rDNA ISRs of L. sanfranciscensis gave three products distinguishing this micro-organism from the remaining Lactobacillus species. Sequence analysis revealed that two of the rrn operons were organized as in previously reported lactobacilli: large spacer (L-ISR), containing tRNA(Ile) and tRNA(Ala) genes; small spacer (S-ISR) without tRNA genes. The third described spacer (medium, M-ISR), original for L. sanfranciscensis, harboured a tRNA-like structure. An oligonucleotide sequence targeting the variable region between tDNA(Ile) and tDNA(Ala) of L. sanfranciscensis L-ISR was approved to be suitable in species-specific identification procedure. Analysis by pulse-field gel electrophoresis of the chromosomal digest with the enzyme I-CeuI showed the presence of seven rrn clusters. Lactobacillus sanfranciscensis genome size was estimated at c. 1.3 Mb. CONCLUSIONS: Direct amplification of 16S-23S ISRs or PCR with specific primer derived from L-ISR showed to be useful for specific typing of L. sanfranciscensis. This was due to the specific rrn operon organization of L. sanfranciscensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have reported a rapid procedure for L. sanfranciscensis identification based on specific structures found in its rrn operon.     

Molecular cloning and physical and functional characterization of the Salmonella typhimurium and Salmonella typhi galactose utilization operons.

The chromosomally encoded galactose utilization (gal) operons of Salmonella typhimurium and S. typhi were each cloned on similar 5.5-kilobase HindIII fragments into pBR322 and were identified by complementation of Gal- Escherichia coli strains. Restriction endonuclease analyses indicated that these Salmonellae operons share considerable homology, but some heterogeneities in restriction sites were observed. Subcloning and exonuclease mapping experiments showed that both operons have the same genetic organization as that established for the E. coli gal operon (i.e., 5' end, promoter, epimerase, transferase, kinase, and 3' end). Two gal operator regions (oE and oI) of S. typhimurium, identified by repressor titration in an E. coli superrepressor [galR(Sup)] mutant, were sequenced and found to flank the promoter region. This promoter region is identical to the -10 and -35 regions of the E. coli gal operon. Minicell studies demonstrated that the three gal structural genes of S. typhimurium encode separate polypeptides of 39 kilodaltons (kDa) (epimerase, 337 amino acids [aa's]), 41 kDa (transferase, 348 aa's), and 43 kDa (kinase, 380 aa's). Despite functional and organizational similarities, DNA sequence analysis revealed that the S. typhimurium gal genes show less than 70% homology to the E. coli gal operon. Because of codon degeneracy, the deduced amino acid sequences of these polypeptides are highly conserved (greater than 90% homology) as compared with those of the E. coli gal enzymes. These studies have defined basic genetic parameters of the gal genes of two medically important Salmonella species, and our findings support the hypothesized divergent evolution of E. coli and Salmonella spp. from a common ancestral parent bacterium.     

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.     

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 (2.14.4) 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.     

Comparison of the overlapping frd and ampC operons of Escherichia coli with the corresponding DNA sequences in other gram-negative bacteria.

Specific DNA probes from Escherichia coli K-12 were used to analyze the sequence divergence of the frd and ampC operons in various species of gram-negative bacteria. These operons code for the fumarate reductase complex and the chromosomal beta-lactamase, respectively. We demonstrate that the two operons show the same general pattern of divergence, although the frd operon is considerably more conserved than is the ampC operon. The major exception is Salmonella typhimurium LT2, which shows a strong homology to the E. coli frd probe but none to the E. coli ampC probe. The operons from Citrobacter freundii and Shigella sonnei were cloned and characterized by physical mapping, Southern hybridization, and protein synthesis in minicells. In S. sonnei, as in E. coli K-12, the frd and ampC operons overlap (T. Grundstr?m and B. Jaurin, Proc. Natl. Acad. Sci. U.S.A. 79:1111-1115, 1982). Only minor discrepancies between the two operons were found over the entire frd-ampC region. In C. freundii, the ampC and frd operons do not overlap, being separated by about 1,100 base pairs. Presumably the inducible property of the C. freundii chromosomal beta-lactamase is encoded by this 1,100-base-pair DNA segment.