Draft Genome Sequence of Herbaspirillum lusitanum P6-12, an Endophyte Isolated from Root Nodules of Phaseolus vulgaris

Abstract

Herbaspirillum lusitanum strain P6-12 (DSM 17154) is, so far, the only species of Herbaspirillum isolated from plant root nodules. Here we report a draft genome sequence of this organism.

GENOME ANNOUNCEMENT

Herbaspirillum lusitanum strain P6-12 was isolated from root nodules of Phaseolus vulgaris plants in northeastern Portugal (7). Members of the genus Herbaspirillum are nitrogen-fixing betaproteobacteria capable of endophytically colonizing cereals of economic relevance (3, 5).

The genome sequence of H. lusitanum P6-12 was determined by using a combination of fragment and mate-paired libraries on a SOLiD4 sequencer (Life Technologies), producing a total of 4,460,595 fragment and 107,836,914 mate-paired reads 50 bp in length. These libraries were used for independent de novo genome assembly using Velvet v.1.2.03 (8). Gap closure was achieved by combination of the two assemblies.

The draft genome of H. lusitanum P6-12 contains 37 scaffolds and has an estimated size of 4.9 Mb with a coverage of 214-fold and 60.2% G+C content. Automatic annotation using RAST (2) revealed 5,240 open reading frames covering 84% of the chromosome, 38 tRNAs, and a single 16S-23S-5S rRNA operon. This annotation was curated by using an in-house-developed platform named GAAT (Genome Analysis and Annotation Tool) available at www.genopar.org.

H. lusitanum was originally determined to be a nitrogen-fixing bacterium by means of pellicle formation on nitrogen-free medium and nifD gene amplification (7). However, no nitrogen-fixing (nif) genes were found in the genome. Furthermore, in our study, this strain was incapable of reducing acetylene in semisolid medium. Type I and II protein secretion systems were found. The type III secretion system, suggested to be involved in plant-bacterial interaction and present in the endophytes H. seropedicae SmR1 and H. rubrisubalbicans M1 (4), is absent from the H. lusitanum P6-12 genome. A nodD-like gene is present, although the nodulation (nod) genes were not found. The lack of nif and nod genes suggests that H. lusitanum is an opportunistic bacterium capable of colonizing root nodules, as well as other plant tissues. H. lusitanum P6-12 has the complete Entner-Doudoroff, pentose phosphate, and tricarboxylic acid cycle pathways. All of the genes coding for the Embden-Meyerhof-Parnas pathway are also present, although it lacks the classical 6-phosphofructokinase (EC 2.7.1.11) gene, as in H. seropedicae SmR1. An interesting finding is the presence of a gene coding for a protein very similar (83%) to the large ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) subunit, a key enzyme involved in photosynthetic carbon fixation (6). RuBisCO-like proteins have been reported in other heterotrophic bacteria (e.g., Bacillus subtilis) and were suggested to participate in a methionine salvage pathway (1). The gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase was found, indicating probable contributions to plant development under stress conditions. Finally, the lack of nif genes raises questions regarding the ability of H. lusitanum P6-12 to fix nitrogen. Further analysis of the H. lusitanum P6-12 genome will help to improve the understanding of how bacteria may associate and interact with plants.

Nucleotide sequence accession numbers.

This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under accession no. AJHH00000000. The version described in this paper is the first version, AJHH01000000.