Duplication of plasmid-borne nitrite reductase gene nirK in the wheat-associated plant growth-promoting rhizobacterium Azospirillum brasilense Sp245.

In the plant growth-promoting rhizobacterium Azospirillum brasilense Sp245, nitric oxide produced by denitrification could be a signal involved in stimulation of root branching, and the dissimilatory nitrite reductase gene nirK is upregulated on wheat roots. Here, it was found that Sp245 did not contain one copy of nirK but two (named nirK1 and nirK2), localized on two different plasmids, including one plasmid prone to rearrangements. Their deduced protein sequences displayed 99.2% identity but their promoter regions and upstream genetic environment differed. Phylogenetic studies revealed that nirK1 and nirK2 clustered next to most beta-proteobacterial sequences rather than in the vicinity of other Azospirillum spp. and most alpha-proteobacterial sequences, regardless of whether DNA or deduced protein sequences were used. This points to past horizontal gene transfers. Analysis of the number of nonsynonymous and synonymous substitutions per site indicated that nirK has been subjected to neutral selection in bacteria. The use of transcriptional fusions with egfp, encoding an enhanced green fluorescent protein variant, revealed that both nirK1 and nirK2 promoter regions were upregulated in vitro under microaerobiosis or the presence of nitrite as well as on wheat roots. The analysis of nirK1 and nirK2 mutants revealed that the two genes were functional. Overall, results suggest that nirK has been acquired horizontally by A. brasilense Sp245 from a distant relative and underwent subsequent duplication; however, both paralogs remained functional and retained their upregulation by the plant partner.

Bacteriophage prevalence in the genus Azospirillum and analysis of the first genome sequence of an Azospirillum brasilense integrative phage.

The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus Azospirillum. Upon induction by mitomycin C, the release of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (phiAb-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the phiAb-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of phiAb-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd.

Evaluation of rhizobacterial indicators of tobacco black root rot suppressiveness in farmers' fields.

Very few soil quality indicators include disease-suppressiveness criteria. We assessed whether 64 16S rRNA microarray probes whose signals correlated with tobacco black root rot suppressiveness in greenhouse analysis could also discriminate suppressive from conducive soils under field conditions. Rhizobacterial communities of tobacco and wheat sampled in 2 years from four farmers' fields of contrasted suppressiveness status were compared. The 64 previously identified indicator probes correctly classified 72% of 29 field samples, with nine probes for Azospirillum, Gluconacetobacter, Sphingomonadaceae, Planctomycetes, Mycoplasma, Lactobacillus crispatus and Thermodesulforhabdus providing the best prediction. The whole probe set (1033 probes) revealed strong effects of plant, field location and year on rhizobacterial community composition, and a smaller (7% variance) but significant effect of soil suppressiveness status. Seventeen additional probes correlating with suppressiveness status in the field (noticeably for Agrobacterium, Methylobacterium, Ochrobactrum) were selected, and combined with the nine others, they improved correct sample classification from 72% to 79% (100% tobacco and 63% wheat samples). Pseudomonas probes were not informative in the field, even those targeting biocontrol pseudomonads producing 2,4-diacetylphloroglucinol, nor was quantitative polymerase chain reaction for 2,4-diacetylphloroglucinol-synthesis gene phlD. This study shows that a subset of 16S rRNA probes targeting diverse rhizobacteria can be useful as suppressiveness indicators under field conditions.

Phase variation and genomic architecture changes in Azospirillum.

The plant growth-promoting rhizobacterium Azospirillum lipoferum 4B generates in vitro at high frequency a stable nonswimming phase variant designated 4V(I), which is distinguishable from the wild type by the differential absorption of dyes. The frequency of variants generated by a recA mutant of A. lipoferum 4B was increased up to 10-fold. The pleiotropic modifications characteristic of the phase variant are well documented, but the molecular processes involved are unknown. Here, the objective was to assess whether genomic rearrangements take place during phase variation of strain 4B. The random amplified polymorphic DNA (RAPD) profiles of strains 4B and 4V(I) differed. RAPD fragments observed only with the wild type were cloned, and three cosmids carrying the corresponding fragments were isolated. The three cosmids hybridized with a 750-kb plasmid and pulse-field gel electrophoresis analysis revealed that this replicon was missing in the 4V(I) genome. The same rearrangements took place during phase variation of 4BrecA. Large-scale genomic rearrangements during phase variation were demonstrated for two additional strains. In Azospirillum brasilense WN1, generation of stable variants was correlated with the disappearance of a replicon of 260 kb. For Azospirillum irakense KBC1, the variant was not stable and coincided with the formation of a new replicon, whereas the revertant recovered the parental genomic architecture. This study shows large-scale genomic rearrangements in Azospirillum strains and correlates them with phase variation.