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Journal of Bacteriology, January 2002, p. 468-478, Vol. 184, No. 2
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.2.468-478.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Phylogeny and Functional Expression of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Autotrophic Ammonia-Oxidizing Bacterium Nitrosospira sp.Isolate 40KI

Janne B. Utåker,* Kjell Andersen, Ågot Aakra, Birgitte Moen, and Ingolf F. Nes

Laboratory of Microbial Gene Technology, Department of Chemistry and Biotechnology, Agricultural University of Norway, N-1432 Ås, Norway

Received 12 July 2001/ Accepted 11 October 2001

The autotrophic ammonia-oxidizing bacteria (AOB), which play an important role in the global nitrogen cycle, assimilate CO2 by using ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we describe the first detailed study of RubisCO (cbb) genes and proteins from the AOB. The cbbLS genes from Nitrosospira sp. isolate 40KI were cloned and sequenced. Partial sequences of the RubisCO large subunit (CbbL) from 13 other AOB belonging to the ß and {gamma} subgroups of the class Proteobacteria are also presented. All except one of the ß-subgroup AOB possessed a red-like type I RubisCO with high sequence similarity to the Ralstonia eutropha enzyme. All of these new red-like RubisCOs had a unique six-amino-acid insert in CbbL. Two of the AOB, Nitrosococcus halophilus Nc4 and Nitrosomonas europaea Nm50, had a green-like RubisCO. With one exception, the phylogeny of the AOB CbbL was very similar to that of the 16S rRNA gene. The presence of a green-like RubisCO in N. europaea was surprising, as all of the other ß-subgroup AOB had red-like RubisCOs. The green-like enzyme of N. europaea Nm50 was probably acquired by horizontal gene transfer. Functional expression of Nitrosospira sp. isolate 40KI RubisCO in the chemoautotrophic host R. eutropha was demonstrated. Use of an expression vector harboring the R. eutropha cbb control region allowed regulated expression of Nitrosospira sp. isolate 40KI RubisCO in an R. eutropha cbb deletion strain. The Nitrosospira RubisCO supported autotrophic growth of R. eutropha with a doubling time of 4.6 h. This expression system may allow further functional analysis of AOB cbb genes.

* Corresponding author. Mailing address: Laboratory of Microbial Gene Technology, Department of Chemistry and Biotechnology, Agricultural University of Norway, P.O. Box 5051, N-1432 Ås, Norway. Phone: 47 64949471. Fax: 47 64948596. E-mail: janne-beate.utaker{at}ikb.nlh.no.

Journal of Bacteriology, January 2002, p. 468-478, Vol. 184, No. 2
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.2.468-478.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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