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Journal of Bacteriology, August 2004, p. 5031-5039, Vol. 186, No. 15
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.15.5031-5039.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus

Juan G. Arrieta,* Mailin Sotolongo, Carmen Menéndez, Dubiel Alfonso, Luis E. Trujillo, Melvis Soto, Ricardo Ramírez, and Lázaro Hernández

Plant-Microbe Interactions Lab, Plant Division, Center for Genetic Engineering and Biotechnology, Havana 10600, Cuba

Received 24 November 2003/ Accepted 30 April 2004

The endophytic diazotroph Gluconacetobacter diazotrophicus secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose. LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway. We identified an unusually organized gene cluster encoding at least the components LsdG, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane. Another open reading frame, designated lsdX, is located between the operon promoter and lsdG, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases. The lsdX, -G, and -O genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane. The downstream genes lsdE, -F, -H, -I, -J, -L, -M, -N, and -D were isolated through chromosomal walking. The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard G. diazotrophicus structural gene. Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized. Targeted disruption of lsdG, lsdO, or lsdF blocked LsdA secretion, and the bacterium failed to grow on sucrose. Replacement of Cys162 by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins. Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (lsdA-lsdB) locus in 14 G. diazotrophicus strains representing 11 genotypes recovered from four different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II pathway for protein secretion in the Acetobacteraceae.

* Corresponding author. Mailing address: Plant-Microbe Interactions Lab, Plant Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba. Phone: (53-7) 271-6022. Fax: (53-7) 331779. E-mail: arrieta{at}cigb.edu.cu.

Journal of Bacteriology, August 2004, p. 5031-5039, Vol. 186, No. 15
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.15.5031-5039.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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