Intragenomic 16S rDNA Divergence in <Emphasis Type="Italic">Haloarcula marismortui</Emphasis> Is an Adaptation to Different Temperatures

The halophilic archaeon Haloarcula marismortui contains three ribosomal RNA operons, designated rrnA, rrnB, and rrnC. Operons A and C are virtually identical, whereas operon B presents a high divergence in nucleotide sequence, having up to 135 nucleotide polymorphisms among the three 16S, 23S, and 5S ribosomal RNA genes. Quantitative PCR and structural analyses have been performed to elucidate whether the presence of this intragenomic heterogeneity could be an adaptation to the variable environmental conditions in the natural habitat of H. marismortui. Variation in salt concentration did not affect expression but variation in incubation temperature did produce significant changes, with operon B displaying an expression level four times higher than the other two together at 50°C and three times lower at 15°C. We show that the putative promoter region of operon B is also different. In addition, the predicted secondary structure of these genes indicated that they have distinct stabilities at different temperatures and a mutant strain lacking operon B grew slower at high temperatures. This study supports the idea that divergent rRNA genes can be adaptive, with different variants being functional under different environmental conditions (e.g., temperature). The same phenomenon could take place in other halophiles or thermophiles with intragenomic rDNA heterogeneity, where the use of 16S rDNA as a phylogenetic marker and indicator of biodiversity should be used with caution.     

Organization of three rRNA (rrn) operons from Sphingobium chungbukense DJ77

The nucleotide sequences of all three rRNA operons (rrnA, rrnB, and rrnC) of Sphingobium chungbukense DJ77 were determined. The three rrn operons have the same gene order (16S rRNA-tRNA^Ile-tRNA^Ala-23S rRNA-5S rRNA-tRNA^fMet). The nucleotide sequences were identical over a 5,468 bp region spanning the 16S rRNA gene to the 5S rRNA gene. Variability was observed in the 5S rRNA-tRNA^fMet spacer sequence of rrnB. The tRNA^fMet gene sequences were identical except for two bases (T₅₇₉₄ and A₅₈₇₁ in rrnB, T₅₉₄₂ and A₅₉₅₆ in rrnA, but C₅₉₄₂ and G₅₉₅₆ in rrnC). Comparative sequence analyses of ribosomal RNA operons from DJ77 with those of the class Alphaproteobacteria, to which the genus Sphingobium belongs, reveal close evolutionary relationships with other members of the order Sphingomonadales.     

Amino acid sequence of the ribosomal protein HS23 from the halophilicHaloarcula marismortui and homology studies to other ribosomal proteins

The ribosomal protein HS23 from the 30S subunit of the extreme halophilicHaloarcula marismortui, belonging to the group of archaea, was isolated either by RP-HLPLC or two-dimensional polyacrylamide gel electrophoresis. The complete amino acid sequence was determined by automated N-terminal microsequencing. The protein consists of 123 residues with a corresponding molecular mass of 12,552 Da as determined by electrospray mass spectroscopy; the pI is 11.04. Homology studies reveal similarities to the eukaryotic ribosomal protein S8 fromHomo sapiens, Rattus norvegicus, Leishmania major, andSaccharomyces cerevisiae.Abbreviations H. marismortui Haloarcula marismortui - PVDF polyvinylidene difluoride - PTH phenylthiohydantoin - RP-HPLC reversed-phase high-performance liquid chromatography - TFA trifluoro acetic acid - TP30 total protein mixture from the 30S ribosomal subunit ofH. marismortui     

Sequence and characterisation of a ribosomal RNA operon fromAgrobacterium vitis

One of the four ribosomal RNA operons (rrnA) from theAgrobacterium vitis vitopine strain S4 was sequenced.rrnA is most closely related to therrn operons ofBradyrhizobium japonicum andRhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case ofR. sphaeroides. The 16S rRNA sequence of S4 differs from theA. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3 ends of the three otherrrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3 ends of the four repeats and defines two groups:rrnA/rrnB andrrnC/rrnD.     

Sequence and characterisation of a ribosomal RNA operon fromAgrobacterium vitis

One of the four ribosomal RNA operons (rrnA) from theAgrobacterium vitis vitopine strain S4 was sequenced.rrnA is most closely related to therrn operons ofBradyrhizobium japonicum andRhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case ofR. sphaeroides. The 16S rRNA sequence of S4 differs from theA. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3′ ends of the three otherrrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3′ ends of the four repeats and defines two groups:rrnA/rrnB andrrnC/rrnD.     

E. coli ribosomal protein L4 is a feedback regulatory protein

We studied the synthesis of ribosomal proteins encoded by the S10 operon, an eleven gene operon from the str-spc region of the E. coli chromosome, using a λfus3 DNA-directed, in vitro protein synthesizing system. Addition of ribosomal protein L4 (1 μM) to in vitro protein synthesis reactions caused selective inhibition of synthesis of the promoter-proximal proteins of the S10 operon, S10, L3, L4, L23 and possibly L2. Proteins of the S10 operon other than L4 did not cause selective inhibition of protein synthesis. Autoregulatory ribosomal proteins previously identified from other operons, L1, S4 and S8, did not inhibit protein synthesis from the S10 operon; nor did L4 cause significant inhibition of protein synthesis from operons other than the S10 operon. As with L1, S4 and S8, L4 inhibits gene expression at the level of translation.     

Expression of Marker RNAs in Pseudomonas putida

A broad-host-range vector that expresses a unique artificial RNA in Pseudomonas putida has been developed. This vector was derived from the plasmid pBBR1MCS and incorporates regulatory regions from the Escherichia coli ribosomal operon, rrnB. These include the promoters P1 and P2, and the terminators T1 and T2. The gene for the artificial RNA was derived from Vibrio proteolyticus 5S rRNA. The artificial RNA product accumulates to a level that is 10–20% of the total 5S rRNA in P. putida. The RNA product is not incorporated into ribosomes and has a minimal effect on cell growth rate. In contrast, when wild-type V. proteolyticus 5S rRNA was expressed from the vector, it was incorporated into ribosomes. It is expected that this new vector system will allow artificial RNA expression systems to be readily developed for a large variety of species. Received: 16 June 1999 / Accepted: 11 August 1999     

The life-cycle of operons

Operons are a major feature of all prokaryotic genomes, but how and why operon structures vary is not well understood. To elucidate the life-cycle of operons, we compared gene order between Escherichia coli K12 and its relatives and identified the recently formed and destroyed operons in E. coli. This allowed us to determine how operons form, how they become closely spaced, and how they die. Our findings suggest that operon evolution may be driven by selection on gene expression patterns. First, both operon creation and operon destruction lead to large changes in gene expression patterns. For example, the removal of lysA and ruvA from ancestral operons that contained essential genes allowed their expression to respond to lysine levels and DNA damage, respectively. Second, some operons have undergone accelerated evolution, with multiple new genes being added during a brief period. Third, although genes within operons are usually closely spaced because of a neutral bias toward deletion and because of selection against large overlaps, genes in highly expressed operons tend to be widely spaced because of regulatory fine-tuning by intervening sequences. Although operon evolution may be adaptive, it need not be optimal: new operons often comprise functionally unrelated genes that were already in proximity before the operon formed.