Search for Alternative RNA Secondary Structures Regulating Expression of Bacterial Genes

Expression of many bacterial genes is regulated by formation of alternative secondary RNA structure within the leader mRNA sequence. Our algorithm designed to search for these structures (basing on analysis of one nucleotide sequence) was applied to analyze operons of amino acid biosynthesis in alpha- and gamma-proteobacteria. The attenuators of these operons are predicted for genomes of some poorly known gamma-proteobacteria including Shewanella putrefaciens, attenuators of the tryptophan operon in some alpha-proteobacteria are also predicted.     

A theoretical study of the attenuation control mechanism

The attenuator control mechanism, used in a number of amino acid biosynthetic operons, is considered from a theoretical point of view. The physics of RNA hairpin-loop formation is discussed, and rules for predicting which codons in the leader peptide that will affect operon expression are suggested. Manabe's (1981) stochastic model for the attenuator mechanism is used to analyse a number of known attenuators, showing a need for a “polymerase pause-site” in most of the attenuators, and providing some quantitative arguments in favour of the use of unusual codons in the control region.     

Conservation of ribosomal protein gene ordering in 16 complete genomes

The organization of ribosomal proteins in 16 prokaryotic genomes was studied as an example of comparative genome analyses of gene systems. Hypothetical ribosomal protein-containing operons were constructed. These operons also contained putative genes and other non-ribosomal genes. The correspondences among these genes across different organisms were clarified by sequence homology computations. In this way a cross tabulation of 70 ribosomal proteins genes was constructed. On average, these were organized into 9-14 operons in each genome. There were also 25 non-ribosomal or putative genes in these mainly ribosomal protein operons. Hence the table contains 95 genes in total. It was found that: (i) the conservation of the block of about 20 r-proteins in the L3 and L4 operons across almost the entire eubacteria and ar-chaebacteria is remarkable; (ii) some operons only belong to eubacteria or archaebacte-ria; (iii) although the ribosomal protein operons are highly conserved within domain, there are fine variat     

Conservation of ribosomal protein gene ordering in 16 complete genomes

The organization of ribosomal proteins in 16 prokaryotic genomes was studied as an example of comparative genome analyses of gene systems. Hypothetical ribosomal protein-containing operons were constructed. These operons also contained putative genes and other non-ribosomal genes. The correspondences among these genes across different organisms were clarified by sequence homology computations. In this way a cross tabulation of 70 ribosomal proteins genes was constructed. On average, these were organized into 9-14 operons in each genome. There were also 25 non-ribosomal or putative genes in these mainly ribosomal protein operons. Hence the table contains 95 genes in total. It was found that: (i) the conservation of the block of about 20 r-proteins in the L3 and L4 operons across almost the entire eubacteria and archaebacteria is remarkable; (ii) some operons only belong to eubacteria or archaebacteria; (iii) although the ribosomal protein operons are highly conserved within domain, there are fine variations in some operons across different organisms within each domain, and these variations are informative on the evolutionary relations among the organisms. This method provides a new potential for studying the origin and evolution of old species.     

Phylogenetic analysis of gamma-proteobacteria inferred from nucleotide sequence comparisons of the house-keeping genes adk, aroE and gdh: comparisons with phylogeny inferred from 16S rRNA gene sequences

Nucleotide sequence comparisons of three house-keeping genes, adenylate kinase (adk), shikimate dehydrogenase (aroE), and glucose-6-phosphate dehydrogenase (gdh), were used to infer the phylogeny of 33 gamma-proteobacteria. Phylogenetic trees inferred from each gene, and from the concatenated sequences of all three genes, are, in general, similar to a 16S rRNA gene-inferred tree. Similar grouping of bacteria are revealed at the family, genus, species and strain levels in all five trees. The house-keeping genes, however, show a higher rate of nucleotide sequence substitutions. Consequently, they can possibly probe deeper branches of a phylogenetic tree than the 16S rRNA gene. However, because their nucleotide sequences are not as highly conserved among gamma-proteobacteria, family- or genus-specific primers would need to be designed for the amplification of any of these three house-keeping genes. Since these genes are used in multilocus sequence typing, it is expected that the number of sequences publicly available for many taxa will increase over time proving them very useful either at complementing 16S rRNA-inferred phylogenies or for specific, targeted, phylogenetic analysis.     

Computer analysis of regulatory signals in complete bacterial genomes. Translation initiation of ribosomal protein operons]

Signals of translation initiation of operons of Haemophilus influenzae ribosomal proteins were predicted. This process is regulated by the formation of secondary RNA structures to which one of the proteins encoded in a particular operon binds. In some cases, these structures imitate the region of protein binding to rRNA. Predictions are made by comparing with homologous operons of Escherichia coli and analogous regions of rRNA and by estimating the energy of secondary structure formation. It is shown that this regulatory mechanism occurs: in operons L11, S10, S15, spc, and alpha of H.influenzae and, probably, in operon S15 of Helicobacter pylori, Bacillus subtilis, and Mycoplasma genitalium.     

Characterization of the virB operon of an Agrobacterium tumefaciens Ti plasmid: nucleotide sequence and protein analysis

The virulence regulon of the Agrobacterium tumefaciens TiC58 plasmid is composed of six operons, virA, virB, virG, virC, virD and virE, which direct the transfer of T-DNA into plant cells. The 9.5 kbp virB operon is the largest of these operons and its entire nucleotide sequence was determined and found to contain eleven open reading frames (ORFs). Gene fusions of each VirB ORF to T7 phi 10 were made and overexpressed in Escherichia coli to confirm that they encode proteins of predicted size. Hydrophobic analysis of these peptide sequences revealed nine proteins that contain hydrophobic spanning regions including signal-peptide-like sequences. These data suggest that the majority of VirB proteins may associate with bacterial cell membranes, while the two additional proteins possess a potential ATP-binding site. Strong homologies in amino acid sequences were observed between nopaline- and octopine-type plasmids. Specific differences in amino acid sequence encoded by VirB ORFs of nopaline and octopine Ti plasmid and a functional role of the gene products are discussed.     

Sequence heterogeneities among 16S ribosomal RNA sequences, and their effect on phylogenetic analyses at the species level

We have analyzed what phylogenetic signal can be derived by small subunit rRNA comparison for bacteria of different but closely related genera (enterobacteria) and for different species or strains within a single genus (Escherichia or Salmonella), and finally how similar are the ribosomal operons within a single organism (Escherichia coli). These sequences have been analyzed by neighbor-joining, maximum likelihood, and parsimony. The robustness of each topology was assessed by bootstrap. Sequences were obtained for the seven rrn operons of E. coli strain PK3. These data demonstrated differences located in three highly variable domains. Their nature and localization suggest that since the divergence of E. coli and Salmonella typhimurium, most point mutations that occurred within each gene have been propagated among the gene family by conversions involving short domains, and that homogenization by conversions may not have affected the entire sequence of each gene. We show that the differences that exist between the different operons are ignored when sequences are obtained either after cloning of a single operon or directly from polymerase chain reaction (PCR) products. Direct sequencing of PCR products produces a mean sequence in which mutations present in the most variable domains become hidden. Cloning a single operon results in a sequence that differs from that of the other operons and of the mean sequence by several point mutations. For identification of unknown bacteria at the species level or below, a mean sequence or the sequence of a single nonidentified operon should therefore be avoided. Taking into account the seven operons and therefore mutations that accumulate in the most variable domains would perhaps increase tree resolution. However, if gene conversions that homogenize the rRNA multigene family are rare events, some nodes in phylogenetic trees will reflect these recombination events and these trees may therefore be gene trees rather than organismal trees.