The 70S prokaryotic ribosome is composed of a 30S small subunit and a 50S large subunit. Ribosomal protein composition is very conserved among prokaryotes. The 30S subunit may contain 22 ribosomal proteins, S1 to S22, 19 of which (S2 to S20) are obligatory (Lecompte et al., 2002). The 50S subunit may contain 33 ribosomal proteins, L1 to L6, L9 to L25 and L27 to L36. Ribosomal proteins L25 and L30 are the only optional components of the large subunit in some prokaryotes. The C. violaceum genome contains the genes for all ribosomal proteins, except S22.
Aminoacyl-tRNA synthesis provides the interface between nucleotide sequence and the amino acid sequence, and therefore is crucial for translation accuracy. The attachment of the correct amino acid to a specific tRNA is carried out by aminoacyl-tRNA synthetases in a two-step process of ATP-dependent activation, followed by transfer of the amino acid to the tRNA (Ibba and Söll, 2000). This process involves preferential binding of the correct amino acid and editing of the incorrect one, and is capable of discriminating very similar amino acids. Most organisms, like E. coli, possess 20 aminoacyl-transferases, one for each of the 20 amino acids (Ibba and Söll, 2001). Many prokaryotes, however, synthesize some of their aminoacyl-tRNAs by enzymatic modification of a mischarged amino acid. Some archea synthesize in this way up to 5 of the 21 aminoacyl-tRNAs needed, such as formylmethionyl-tRNAfMet, glutaminyl-tRNAGln, asparaginyl-tRNAAsp and selenocysteinyl-tRNASec (Blanquet et al., 2000).
C. violaceum contains the genes of all aminoacyl-tRNA synthetases, except asparaginyl-tRNAAsp. This observation is in agreement with what was previously described for other b-proteobacteria, such as Neisseria spp (Ibba and Söll, 2001). Asparaginyl-tRNAAsn is probably obtained through enzymatic modification of a mischarged aspartyl-tRNAAsn by a glutamyl-tRNAGln amidotransferase, the genes of which (gatA, gatB and gatC) were also found in the C. violaceum genome. It has been found that glutamyl-tRNAGln amidotransferases can also function as aspartyl-tRNAAsn amidotransferases (Salazar et al., 2001). In fact, synthesis of asparaginyl-tRNAAsn by amidotransferase reactions is so widespread in bacteria that it has been speculated that asparagine was the last amino acid recruited for protein synthesis (Ibba and Söll, 2001). In addition, three aminoacyl-tRNA synthetase-related protein genes were found, which in other organisms have been shown to have functions unrelated to aminoacylation (Schimmel and De Pouplana, 2000); they are glutamyl-tRNA synthetase-related protein, histidyl-tRNA synthetase-related protein and alanyl-tRNA synthetase-related protein. The function of these genes in C. violaceum is unclear.
Translation is divided into three steps, initiation, elongation and termination, which are controlled by specific factors. These factors, IF-1, IF-2, IF-3, EF-Ts, EF-Tu, EF-G, RF-1, RF-2 and RF-3, are all present in the C. violaceum genome, including the ribosome recycling factor. Some proteins require a modified amino acid, selenocysteine, which is incorporated at the time of peptide chain elongation (Bock, 2000). There is no codon for this modified amino acid, which is inserted at an in-frame UGA termination codon recognized by a selenocysteinyl-tRNASec-specific translation factor called SelB. The selB gene was not found, suggesting that C. violaceum cannot synthesize selenoproteins.
tmRNA is an abundant, small RNA, universally present in bacteria (Williams, 2002), which has a dual nature, having characteristics of both tRNA and mRNA (Williams and Bartel, 1996). It was first described in E. coli as being encoded by the ssrA gene, and is also called 10Sa RNA or SsrA RNA (Ray and Apirion, 1979; Chauhan and Apirion, 1989; Oh et al., 1990). tmRNA plays, together with SmpB (small protein B), a small basic protein that binds specifically to it, a role in the process of trans-translation. When ribosomes reach the 3’ end of an mRNA that lacks a termination codon, they become stalled. Trans-translation is the mechanism whose function is to recycle the stalled ribosomes, adding a C-terminal peptide tag to the unfinished protein, targeting it for proteolysis (Tu et al., 1995; Keiler et al., 1996; Vioque and de la Cruz, 2003). Trans-translation also comes into play when bacteria are placed in sub-lethal concentrations of protein synthesis inhibitors (Vioque and de la Cruz, 2003).
The tmRNA of C. violaceum has already been described in strains ATCC07461 and ATCC12472 by Felden et al. (2001) and by Williams (2002), respectively. There are two different sequence versions that are available at the tmRNA Web site
(http://www.indiana.edu/~tmrna) and the three differing nucleotides possibly reflect intraspecies variation. The SmpB protein gene is also present in the C. violaceum genome. Other proteins are described to associate less tightly with tmRNA: ribosomal protein S1, RNase R (a member of the RNase II family), PrsA (an enzyme required for de novo synthesis of nucleotides, tryptophan and histidine) and SAF (a possible formyltransferase) (Karzai and Sauer, 2001). Both the ribosomal protein S1 and a member of the RNase II family are also present in the C. violaceum genome. In general, tmRNA is not essential for cell viability under standard lab conditions, but charged tmRNA seems to be essential for Neisseria gonorrhoeae viability (Huang et al., 2000). It would be interesting to know if charged tmRNA is also essential to C. violaceum.
TRANSCRIPTIONAL REGULATION
Bacteria have one housekeeping s and a variable number of alternative s which possess different promoter-recognition properties. A repertoire of different s can be used by the cell to modify the transcription program in response to stress.
Free-living microorganisms are exposed to a series of variable environmental conditions, such as alterations in the abundance of nutrients, changes in temperature and toxic compounds. These environmental variations require fast adaptive responses, usually triggering transcriptional activation of specific genes. Alternative s are specialized in controlling some regulons that are activated by specific stress conditions, growth transitions, and morphological changes. In addition, transcription is modulated by activators and repressors, most of which alter the expression of transcription of the housekeeping s.
The s70 family has been divided, based on similarity, into four major groups. Group 1 includes s70 and its orthologs, such as RpoD, responsible for the transcription of housekeeping genes. Group 2 members, such as sS (RpoS), the general stress regulator of E. coli and the most studied member of this group, are not necessary for growth. Group 3 members are more divergent in sequence, and can be divided, with related proteins, into groups with similar functions, such as heat shock (s32 or RpoH), flagellar biosynthesis (sF or FliA) and sporulation. Group 4 members, originally called extracytoplasmic function (Ecf) family members, such as sE (RpoE) and FecI, have reduced sequence similarity to the other s70 groups. The superscript numbers on s indicate the molecular weight. Escherichia coli has a pool of six alternative s, such as the s70 family, s70 (RpoD), sS (RpoS), s32 (RpoH), sF (FliA), sE (RpoE), and FecI. In C. violaceum, ORFs corresponding to s70 (RpoD), sS or s38 (RpoS), s32 (RpoH), sF or s28 (FliA) and sE or s24 (RpoE) were found.
Most alternative s are related in sequence and structure to s70. However, many bacteria also have a second distinct type of s called sN (also called s54 or RpoN), which shares no sequence similarity with the s70 family. s54 family members utilize an enhancer sequence and ATP to form an open complex, while s70 members do not (Studholme and Buck, 2000). Although most bacteria contain multiple members of the s70 family, they usually have no more than one representative of the s54 family. Different from other alternative s70, the s54 or sN is correlated with the expression of gene products that have a broad range of functions. The diverse types of functions of genes that are under RNA polymerase and sN control also vary within groups of bacteria; this situation does not suggest any clear set of specific functions. Functions described to be under s54 control are nitrogen assimilation, fixation and related regulatory functions, glutamine transport, nodulation, phage shock, and others (Gruberl and Gross, 2003). Moreover, the distribution of s54 family members varies among microorganisms. It is present in N. meningitidis, Yersinia pestis, Salmonella typhi, and absent in Haemophilus influenzae. The genome of C. violaceum contains s54 (rpoN), although the set of functions regulated by this protein is unknown.
Anti-sigma factors modulate the regulons controlled by some s at the level of transcription, by inhibitory binding. The broad range of cell processes regulated by anti-sigma factors includes bacteriophage growth, sporulation, stress response, flagellar biosynthesis, pigment production, ion transport, and virulence (Helmann, 1999). We found an ORF that potentially codes for anti-sigma 28 (FlgM) in the C. violaceum genome.
Transcriptional activators and repressors belong to a variety of well-defined families of transcription factors, which interact with alternative sigma factors involved in bacterial cell response to stressors. More than 60% of the ORFs related to transcriptional regulators identified in C. violaceum show high similarity scores (e value of at least -17) to transcription factors described for E. coli. One hundred and seventy ORFs were identified as transcription factors that bind DNA through helix-turn-helix motifs and were tentatively classified into families. The largest group of transcription regulators found in C. violaceum appears to belong to the LysR and AraC families, as has also been described in other bacteria (Schell, 1993; Martin and Rosner, 2001). An interesting feature of LysR transcriptional regulators is that some of them behave as sensors of physiological changes and can bind directly to different kinds of molecules. The AraC family is known by their homology to a 99-amino acid segment described for the first transcriptional activator in E. coli B (Martin and Rosner, 2001). In Ralstonia solanacearum, which can be found free-living in soil, a large number of transcription factor genes have also been reported (Salanoubat et al., 2002). The large number of transcriptional regulators found in the C. violaceum genome suggests considerable adaptive capability in patterns of gene expression in response to changes in the environment.
CONCLUDING REMARKS
The genome sequencing of C. violaceum, a bacterium found in tropical and subtropical regions, has provided a large amount of information about how this free-living organism can adapt to different environments. Similarity searches and comparison analyses of the ORFs related to gene expression and regulation identified in this organism showed the expected conservation in these systems. The protein components of transcription, RNA processing, translation, and regulation are very similar to that of other members of b-proteobacteria, such as R. solanacearum and Neisseria spp., and show a considerable versatility in gene expression. This preliminary analysis may be used as the starting point for further studies to improve our understanding of the gene expression mechanisms of C. violaceum.
ACKNOWLEDGMENTS
This study was undertaken within the context of the Brazilian National Genome Program, a consortium funded in December 2000 by the Ministério da Ciência e Tecnologia (MCT) through the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
REFERENCES
Blanquet, S., Mechulam, Y. and Schmitt, E. (2000). The many routes of bacterial transfer RNAs after aminoacylation. Curr. Opin. Struct. Biol. 10: 95-101.
Bock, A. (2000). Biosynthesis of selenoproteins - an overview. Biofactors 11: 77-78.
Caldas, L.R. (1990). Um pigmento nas águas negras. Cienc. Hoje 11: 56-67.
Chauhan, A.K. and Apirion, D. (1989). The gene for a small stable RNA (10Sa RNA) of Escherichia coli. Mol. Microbiol. 3: 1481-1485.
Dreyfus, M. and Regnier, P. (2002). The poly(A) tail of mRNAs: bodyguard in eukaryotes. Cell 111: 611-613.
Felden, B., Massire, C., Westhof, E., Atkins, J.F. and Gesteland, R.F. (2001). Phylogenetic analysis of tmRNA genes within a bacterial subgroup reveals a specific structural signature. Nucleic Acids Res. 29: 1602-1607.
Gruberl, T.M. and Gross, C.A. (2003). Sigma subunit and the partitioning of bacterial transcription space. Annu. Rev. Microbiol. 57: 441-466.
Gusarov, I. and Nudler, E. (2001). Control of intrinsic transcription termination by N and NusA: the basic mechanisms. Cell 107: 437-449.
Helmann, J.D. (1999). Anti-sigma factors. Curr. Opin. Microbiol. 2: 135-141.
Huang, C., Wolfgang, M.C., Withey, J., Koomey, M. and Friedman, D.I. (2000). Charged tmRNA but not tmRNA-mediated proteolysis is essential for Neisseria gonorrhoeae viability. EMBO J. 19: 1098-1107.
Ibba, M. and Söll, D. (2000). Aminoacyl-tRNA synthesis. Annu. Rev. Biochem. 69: 617-650.
Ibba, M. and Söll, D. (2001). The renaissance of aminoacyl-tRNA synthesis. EMBO Reports 21: 382-387.
Karzai, A.W. and Sauer, R.T. (2001). Protein factors associated with the SsrA-SmpB tagging and ribosome rescue complex. Proc. Natl. Acad. Sci. USA 98: 3040-3044.
Keiler, K.C., Waller, P.R.H. and Sauer, R.T. (1996). Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271: 990-993.
Knowlton, J.R., Bubunenko, M., Andrykovitch, M., Guo, W., Routzahn, K.M., Waugh, D.S., Court, D.L. and Ji, X. (2003). A spring-loaded state of NusG in its functional cycle is suggested by X-ray crystallography and supported by site-directed mutants. Biochemistry 42: 2275-2281.
Koulich, D., Nikiforov, V. and Borukhov, S. (1998). Distinct functions of N- and C-terminal domains of GreA, an Escherichia coli transcript cleavage factor. Mol. Biol. 276: 379-389.
Lecompte, O., Ripp, R., Thierry, J.-C., Moras, D. and Poch, O. (2002). Comparative analysis of ribosomal proteins in complete genomes: an example of reductive evolution at the domain scale. Nucleic Acids Res. 30: 5382-5390.
Martin, R.G. and Rosner, J.L. (2001). The AraC transcriptional activators. Curr. Op. Microbiol. 4: 132-137.
Oh, B.K., Chauhan, A.K., Isono, K. and Apirion, D. (1990). Location of a gene (ssrA) for a small, stable RNA (10Sa RNA) in the Escherichia coli chromosome. J. Bacteriol. 172: 4708-4709.
Paget, M.S. and Helmann, J.D. (2003). The sigma70 family of sigma factors. Genome Biol. 4: 203.
Ray, B.K. and Apirion, D. (1979). Characterization of 10S RNA: a new stable RNA molecule from Escherichia coli. Mol. Gen. Genet. 174: 25-32.
Salanoubat, M., Genin, S., Artiguenave, F., Gouzy, J., Mangenot, S., Ariat, M., Billaut, A., Brottlier, P., Camus, J.C., Cattolico, L., Chandler, M., Cholsne, N., Claudel-Renard, C., Cunnac, S., Demange, N., Gaspin, C., Lavie, M., Moisan, A., Robert, C., Saurin, W., Schlex, T., Siguler, P., Thébault, P., Whalen, M., Wincker, P., Levy, M., Weissenbach, J. and Boucher, C.A. (2002). Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415: 497-501.
Salazar, J.C., Zúñiga, R., Raczniak, G., Becker, H., Söll, D. and Orellana, O. (2001). A dual-specific Glu-tRNAGln and Asp-tRNAAsn amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferroxidans. FEBS Lett. 500: 129-131.
Schell, M.A. (1993). Molecular biology of the LysR family of transcriptional regulators. Annu. Rev. Microbiol. 47: 597-626.
Schimmel, P. and De Pouplana, L.R. (2000). Footprints of aminoacyl-tRNA synthetases are everywhere. Trends Biochem. Sci. 25: 207-209.
Studholme, D.J. and Buck, M. (2000). The biology of enhancer-dependent transcriptional regulation in bacteria: insights from genome sequences. FEMS Microbiol. Lett. 186: 1-9.
Tettelin, H., Saunders, N.J., Heidelberg, J., Jeffries, A.C., Nelson, K.E., Eisen, J.A., Ketchum, K.A., Hood, D.W., Peden, J.F., Dodson, R.J., Nelson, W.C., Gwinn, M.L., DeBoy, R., Peterson, J.D., Hickey, E.K., Haft, D.H., Salzberg, S.L., White, O., Fleischmann, R.D., Dougherty, B.A., Mason, T., Ciecko, A., Parksey, D.S., Blair, E., Cittone, H., Clark, E.B., Cotton, M.D., Utterback, T.R., Khouri, H., Qin, H., Vamathevan, J., Gill, J., Scarlato, V., Masignani, V., Pizza, M., Grandi, G., Sun, L., Smith, H.O., Fraser, C.M., Moxon, E.R., Rappuoli, R. and Venter, J.C. (2000). Complete genome sequence of Neisseria meningitides serogroup B strain MC58. Science 287: 1809-1815.
Tu, G.-F., Reid, G.E., Zhang, J.-G., Moritz, R.L. and Simpson, R.J. (1995). C-teminal extension of truncated recombinant proteins in Escherichia coli with a 10as RNA decapeptide. J. Biol. Chem. 270: 9322-9326.
Vasconcelos, A.T.R., Almeida, D.F., Hungria, M., Guimarães, C.T., Antônio, R.V., Almeida, F.C., Almeida, L.G.P., Almeida, R., Alves-Gomes, J.A., Andrade, E.M., Araripe, J., Araújo, M.F.F., Astolfi-Filho, S., Azevedo, V., Baptista, A.J., Bataus, L.A.M., Batista, J.S., Beló, A., van den Berg, C., Bogo, M., Bonatto, S., Bordignon, J., Brigido, M.M., Brito, C.A., Brocchi, M., Burity, H.A., Camargo, A.A., Cardoso, D.D.P., Carneiro, N.P., Carraro, D.M., Carvalho, C.M.B., Cascardo, J.C.M., Cavada, B.S., Chueire, L.M.O., Creczynski-Pasa, T.B., Fagundes, N., Falcão, C.L., Fantinatti, F., Farias, I.P., Felipe, M.S.S., Ferrari, L.P., Ferro, J.A., Ferro, M.I.T., Franco, G.R., Freitas, N.S.A., Furlan, L.R., Gazzinelli, R.T., Gomes, E.A., Gonçalves, P.R., Grangeiro, T.B., Grattapaglia, D., Grisard, E.C., Hanna, E.S., Jardim, S.N., Laurino, J., Leoi, L.C.T., Lima, L.F.A., Loureiro, M.F., Lyra, M.C.C.P., Madeira, H.M.F., Manfio, G.P., Maranhão, A.Q., Martins, W.S., Mauro, S.M.Z., Medeiros, S.R.B., Meissner, R.V., Moreira, M.A.M., Nascimento, F.F., Nicolás, M.F., Oliveira, J.G., Oliveira, S.C., Paixão, R.F.C., Parente, J.A., Pedrosa, F.O., Pena, S.D.J., Pereira, J.O., Pereira, M., Pinto, L.S.R.C., Pinto, L.S., Porto, J.I.R., Potrich, D.P., Ramalho-Neto, C.E., Reis, A.M.M., Rigo, L.U., Rondinelli, E., Santos, E.B.P., Santos, F.R., Schneider, M.P.C., Seuanez, H.N., Silva, A.M.R., Silva, A.L.C., Silva, D.W., Silva, R., Simões, I.C., Simon, D., Soares, C.M.A., Soares, R.B.A., Souza, E.M., Souza, K.R.L., Souza, R.C., Steffens, M.B.R., Steindel, M., Teixeira, S.R., Ürményi, T.P., Vettore, A., Wassem, R., Zaha, A. and Simpson, A.J.G. (2003). The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proc. Natl. Acad. Sci. USA 100: 11660-11665.
Vioque, A. and de la Cruz, J. (2003). Trans-translation and protein synthesis inhibitors. FEMS Microb. Lett. 218: 9-14.
Williams, K.P. (2002). The tmRNA Website: invasion by an intron. Nucleic Acids Res. 30: 179-182.
Williams, K.P. and Bartel, D.P. (1996). Phylogenetic analysis of tmRNA secondary structure. RNA 2: 1306-1310.
Woese, C.R., Kandler, O. and Wheelis, M.L. (1990). Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci. USA 87: 4576-4579.