Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
Correspondence
Manuel Carmona
(mcarmona{at}cib.csic.es)
In contrast to the aerobic catabolism of aromatic compounds for which so much information is available, only very little is known about the genes involved in the anaerobic catabolism of aromatics in bacteria. The genes of the central pathway responsible for the anaerobic catabolism of benzoate have been only described in the denitrifying -Proteobacteria Azoarcus evansii and Thauera aromatica (Breese et al., 1998
; Harwood et al., 1999
) and in the photosynthetic
-proteobacterium Rhodopseudomonas palustris (Egland et al., 1997
; Harwood et al., 1999
), and they are organized in catabolic clusters. In all three bacteria, the anaerobic degradation of benzoate begins with its activation to benzoyl-CoA by a benzoateCoA ligase. However, the subsequent ring reduction to a non-aromatic compound, which is carried out by a four-subunit benzoyl-CoA reductase, and the
-oxidation system that transforms the de-aromatized intermediate by the action of a hydratase, dehydrogenase and ring-cleavage hydrolase, differ between a denitrifying bacterium, T. aromatica, and the photosynthetic bacterium R. palustris. Thus, whereas the T. aromatica pathway converts benzoyl-CoA to 3-hydroxypimelyl-CoA in just four enzymic steps, seven steps are needed through the R. palustris pathway (Harwood et al., 1999
; Gibson & Harwood, 2002
). The benzoate pathway of A. evansii appears to be similar to that of T. aromatica (Ebenau-Jehle et al., 2003
; Harwood et al., 1999
). At this point, some questions arise. Is the Thauera-type pathway and the Rhodopseudomonas-type pathway a landmark of anaerobic benzoate catabolism in bacteria with denitrifying and photosynthetic metabolism, respectively? Or, on the contrary, is there a relationship between the type of benzoate degradation pathway and the phylogenetic mark of the organism? To provide some clues as to the answers to the above questions, we decided to expand our knowledge on the genetic determinants responsible for the anaerobic degradation of benzoate in bacteria. We performed an in silico search on the (un)finished microbial genome database at NCBI (http://www.ncbi.nlm.nih.gov/blast/) for genes homologous to those responsible for benzoyl-CoA catabolism in Thauera (Breese et al., 1998
), Azoarcus (Schühle et al., 2003
) and Rhodopseudomonas (Egland et al., 1997
) strains. The only significant match was found with the unfinished genome sequence of Magnetospirillum magnetotacticum MS-1T, which was a surprising finding since this denitrifying strain has not been reported to degrade aromatic compounds (Blakemore et al., 1979
). The whole set of genes encoding the putative benzoyl-CoA reductase (bcrCBAD), ferredoxin (fdx), hydratase (dch), dehydrogenase (had), ring-cleavage hydrolase (oah), ferredoxin-reducing enzyme (korAB) and benzoateCoA ligase (bclA) proteins (Breese et al., 1998
; Dörner & Boll, 2003
; Schühle et al., 2003
) were found in the genome of M. magnetotacticum MS-1T (Fig. 1a
). It is worth noting that korAB and oahhad are located at the ends of two different contigs in the current genome sequence of M. magnetotacticum and, therefore, it could be possible that such genes are, as in T. aromatica, adjacent to the dch and bcr genes once the complete genome sequence becomes assembled. Interestingly, although the gene encoding the benzoateCoA ligase is not physically associated with the rest of the genes of the cluster in T. aromatica and M. magnetotacticum, this gene is located within the cluster in R. palustris and A. evansii (Fig. 1a
).
|
RT-PCR experiments with M. magnetotacticum MS-1T cells growing under denitrifying conditions using benzoate or succinate as sole carbon sources revealed that bcrA expression was induced when the cells were grown on benzoate but not on succinate (Fig. 1c), which strongly suggests that the bcr gene cluster functions in benzoate metabolism under denitrifying conditions and that expression of such genes is inducible by benzoate. Although anaerobic catabolism of benzoate has been shown in the putative Aquaspirillum sp. strain CC-26 (Shinoda et al., 2000
), the set of genes presented in this work constitutes the first one reported for the anaerobic catabolism of benzoate in denitrifying members of the
-Proteobacteria, and it allows us to expand our current knowledge of the anaerobic catabolism of aromatics by denitrifying bacteria, which has been restricted to the
-proteobacterial genera Azoarcus and Thauera before now.
The G+C content of the gene cluster involved in benzoate degradation in M. magnetotacticum MS-1T averaged 63·8 mol%, a value that is very close to the mean G+C content (64 mol%) of the genome, suggesting that this set of genes has been imprisoned within the chromosome of this bacterium over a long period of evolution. However, the G+C content (64·5 mol%) of the gene clusters involved in anaerobic benzoate degradation in A. evansii and T. aromatica is slightly lower than that of the host genomes (about 67 mol%) (Anders et al., 1995), which might reflect that the evolutionary origin of such genes in these
-Proteobacteria could be their putative horizontal transfer from an organism with a lower G+C content, such as a Magnetospirillum sp. strain.
Interestingly, comparisons of the global gene arrangement and the deduced amino acid sequences among the anaerobic catabolic clusters reported so far indicate higher similarity between the set of genes from M. magnetotacticum and T. aromatica, two denitrifying micro-organisms, than between genes from M. magnetotacticum and R. palustris (phototrophic bacterium), two members of the -Proteobacteria (Fig. 1a
). Since genetic similarities usually reflect equivalent pathways, our results suggest that it is the type of the electron-accepting system rather than the taxonomic position of the organism that determines the type of anaerobic benzoate degradation pathway, which is in agreement with the observation that the catabolic strategy depends largely on the energy situation of the organism involved and the redox potentials of the electron acceptors that it can use (Peters et al., 2004
; Schink et al., 2000
). This work also constitutes an example of how in silico analysis of the current genome-sequencing projects is a suitable approach to identify functions/activities that have not been reported previously, for example by revealing M. magnetotacticum as a new model system to study catabolic and regulatory features of the anaerobic metabolism of aromatic compounds.
Acknowledgements
The technical work of Irene Alonso, Eloisa Cano and Francisca Morente is greatly appreciated. The authors are grateful to M. A. Prieto for her critical reading of the manuscript. This work was supported by Grants 07M/0076/2002 and 07M/0127/2000 from the Comunidad Autónoma de Madrid and by Grants BIO2000-1076, BIO2003-01482 and VEM2003-20075-C02-02 from the Comisión Interministerial de Ciencia y Tecnología. M. J. L. B. is a recipient of a predoctoral fellowship from the Plan Nacional de Formación de Personal Investigador-MCYT, and M. C. is a holder of the Ramón y Cajal Program of the Spanish Ministerio de Ciencia y Tecnología.
REFERENCES
Anders, H. J., Kaetzke, A., Kämpfer, P., Ludwig, W. & Fuchs, G. (1995). Taxonomic position of aromatic-degrading denitrifying pseudomonad strains K 172 and KB 740 and their description as new members of the genera Thauera, as Thauera aromatica sp. nov., and Azoarcus, as Azoarcus evansii sp. nov., respectively, members of the beta subclass of the Proteobacteria. Int J Syst Bacteriol 45, 327333.[Abstract]
Blakemore, R. P., Maratea, D. & Wolfe, R. S. (1979). Isolation and pure culture of a freshwater magnetic spirillum in chemically defined medium. J Bacteriol 140, 720729.[Medline]
Breese, K., Boll, M., Alt-Mörbe, J., Schägger, H. & Fuchs, G. (1998). Genes coding for the benzoyl-CoA pathway of anaerobic aromatic metabolism in the bacterium Thauera aromatica. Eur J Biochem 256, 148154.[Abstract]
Dörner, E. & Boll, M. (2003). Properties of 2-oxoglutarate : ferredoxin oxidoreductase from Thauera aromatica and its role in enzymatic reduction of the aromatic ring. J Bacteriol 184, 39753983.[CrossRef]
Ebenau-Jehle, C. M., Boll, M. & Fuchs, G. (2003). 2-Oxoglutarate : NADP+ oxidoreductase in Azoarcus evansii: properties and function in electron transfer reactions in aromatic ring reduction. J Bacteriol 185, 61196129.
Egland, P. G., Pelletier, D. A., Dispensa, M., Gibson, J. & Harwood, C. S. (1997). A cluster of bacterial genes for anaerobic benzene ring biodegradation. Proc Natl Acad Sci U S A 94, 64846489.
Gibson, J. & Harwood, C. S. (2002). Metabolic diversity in aromatic compound utilization by anaerobic microbes. Annu Rev Microbiol 56, 345369.[CrossRef][Medline]
Harwood, C. S., Burchhardt, G., Herrmann, H. & Fuchs, G. (1999). Anaerobic metabolism of aromatic compounds via the benzoyl-CoA pathway. FEMS Microbiol Rev 22, 439458.[CrossRef]
Peters, F., Rother, M. & Boll, M. (2004). Selenocysteine-containing proteins in anaerobic benzoate metabolism of Desulfococcus multivorans. J Bacteriol 186, 21562163.
Schink, B., Philipp, B. & Müller, J. (2000). Anaerobic degradation of phenolic compounds. Naturwissenschaften 87, 1223.[CrossRef][Medline]
Schühle, K., Gescher, J., Feil, U., Paul, M., Jahn, M., Schägger, H. & Fuchs, G. (2003). Benzoate-coenzyme A ligase from Thauera aromatica: an enzyme acting in anaerobic and aerobic pathways. J Bacteriol 185, 49204929.
Shinoda, Y., Sakai, Y., Ue, M., Hiraishi, A. & Kato, N. (2000). Isolation and characterization of new denitrifying spirillum capable of anaerobic degradation of phenol. Appl Environ Microbiol 66, 12861291.
Zeyer, J. & Kearney, P. C. (1982). Microbial degradation of para-chloroaniline as sole carbon and nitrogen source. Pestic Biochem Physiol 17, 215223.
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