The whole-genome-sequenced rhizobacterium Bacillus amyloliquefaciens FZB42T (Chen et al., 2007) and other plant-associated strains of the genus Bacillus described as belonging to the species Bacillus amyloliquefaciens or Bacillus subtilis are used commercially to promote the growth and improve the health of crop plants. Previous investigations revealed that a group of strains represented a distinct ecotype related to B. amyloliquefaciens; however, the exact taxonomic position of this group remains elusive (Reva et al., 2004). In the present study, we demonstrated the ability of a group of Bacillus strains closely related to strain FZB42T to colonize Arabidopsis roots. On the basis of their phenotypic traits, the strains were similar to Bacillus amyloliquefaciens DSM 7T but differed considerably from this type strain in the DNA sequences of genes encoding 16S rRNA, gyrase subunit A (gyrA) and histidine kinase (cheA). Phylogenetic analysis performed with partial 16S rRNA, gyrA and cheA gene sequences revealed that the plant-associated strains of the genus Bacillus, including strain FZB42T, formed a lineage, which could be distinguished from the cluster of strains closely related to B. amyloliquefaciens DSM 7T. DNA–DNA hybridizations (DDH) performed with genomic DNA from strains DSM 7T and FZB42T yielded relatedness values of 63.7–71.2 %. Several methods of genomic analysis, such as direct whole-genome comparison, digital DDH and microarray-based comparative genomichybridization (M-CGH) were used as complementary tests. The group of plant-associated strains could be distinguished from strain DSM 7T and the type strain of B. subtilis by differences in the potential to synthesize non-ribosomal lipopeptides and polyketides. Based on the differences found in the marker gene sequences and the whole genomes of these strains, we propose two novel subspecies, designated B. amyloliquefaciens subsp. plantarum subsp. nov., with the type strain FZB42T ( = DSM 23117T = BGSC 10A6T), and B. amyloliquefaciens subsp. amyloliquefaciens subsp. nov., with the type strain DSM 7T( = ATCC 23350T = Fukumoto Strain FT), for plant-associated and non-plant-associated representatives, respecitvely. This is in agreement with results of DDH and M-CGH tests and the MALDI-TOF MS of cellular components, all of which suggested that the ecovars represent two different subspecies.

BackgroundNine gene clusters dedicated to nonribosomal synthesis of secondary metabolites with possible antimicrobial action, including polymyxin and fusaricidin, were detected within the whole genome sequence of the plant growth-promoting rhizobacterium (PGPR) Paenibacillus polymyxa M-1. To survey the antimicrobial compounds expressed by M-1 we analyzed the active principle suppressing phytopathogenic Erwinia spp.ResultsP. polymyxa M-1 suppressed the growth of phytopathogenic Erwinia amylovora Ea 273, and E. carotovora, the causative agents of fire blight and soft rot, respectively. By MALDI-TOF mass spectrometry and reversed-phase high-performance liquid chromatography (RP-HPLC), two antibacterial compounds bearing molecular masses of 1190.9 Da and 1176.9 Da were detected as being the two components of polymyxin P, polymyxin P1 and P2, respectively. The active principle acting against the two Erwinia strains was isolated from TLC plates and identified by postsource decay (PSD)-MALDI-TOF mass spectrometry as polymyxin P1 and polymyxin P2. These findings were corroborated by domain structure analysis of the polymyxin (pmx) gene cluster detected in the M-1 chromosome which revealed that corresponding to the chemical structure of polymyxin P, the gene cluster is encoding D-Phe in position 6 and L-Thr in position 7.ConclusionsIdentical morphological changes in the cell wall of the bacterial phytopathogens treated with either crude polymyxin P or culture supernatant of M-1 corroborated that polymyxin P is the main component of the biocontrol effect exerted by strain M-1 against phytopathogenic Erwinia spp.

The genome of Paenibacillus polymyxa M-1 consisted of a 5.8-Mb chromosome and a 360-kb plasmid. Nine sites were dedicated to nonribosomal synthesis of lipopeptides and polyketides. Eight of them were located at the chromosome, while one gene cluster predicted to encode an unknown secondary metabolite was present on the plasmid.Plant growth-promoting rhizobacteria (PGPR) have been applied as environmentally friendly alternatives to agrochemicals to improve crop yield and quality (16). The PGPR strains belonging to Paenibacillus polymyxa (1) promote plant growth by producing indole-3-acetic acid (IAA) (9) and volatile compounds (14). They are also known to suppress fungal phytopathogens (2, 5-7, 13, 31, 34) and plant-parasitic nematodes (12,29). Due to its action as a biocontrol agent, P. polymyxa produces several peptide antibiotics (3, 8, 10, 11, 15, 18, 20, 22-24, 26-28, 32) which might be important in control of plant pathogens (25).Strain M-1, isolated from surface-sterilized wheat root tissues, was identified by 16S rRNA gene sequencing and by physiological and biochemical analysis as being P. polymyxa (33). It is capable of colonizing root surfaces of wheat, promoting wheat growth, and suppressing wheat sharp eyespot disease. In addition, it acts antagonistically against several phytopathogens in vitro by producing antibiotics, including fusaricidin and polymyxin, and by secreting hydrolytic enzymes, for example, endo-␤-1,3-glucanase.Genomic DNA prepared from M-1 was used for construction of a 3-kb-long paired-end library with a GS FLX library preparation kit in combination with GS FLX paired-end adaptors (both from Roche, Mannheim, Germany) according to the manufacturer's protocol. The reads were assembled using the GS De Novo Assembler software program, and the resulting scaffolds were oriented based on the occurrence of unique single nucleotide polymorphisms (SNPs) in the repetitive rRNA (RRN) contigs. In total, 869,907 reads, including 312,451 paired reads, were assembled with a total of 185,008,620 bp. Utilization of the paired-end information allowed scaffolding of the 55 contigs larger than 500 bp into 16 scaffolds containing 45 contigs. Gap closure was done by long-range PCR (using Phusion polymerase; New England BioLabs, Frankfurt [Main], Germany) and subsequent Sanger sequencing (IIT Biotech, Bielefeld, Germany). Prediction of protein-encoding sequences was initially accomplished with the REGANOR server (17). Manual and automatic annotation was done using the annotation software program GenDB 2.4 (19).The complete genome sequence of M-1 consisted of a circular 5,864,546-bp chromosome and a 366,576-bp plasmid, with GϩC values of 54.58% and 37.61%, respectively. Five thousand sixty-one genes (CDS), 14 rRNA operons, and 110 tRNAs resided in the chromosome, while 345 genes were located on the plasmid. Many important genes were found to be plasmid linked, such as those encoding ribosomal proteins and genes involved in replication, repair and methylation, transcription, translation initiation, metabol...

The genome of the rhizobacterium Bacillus amyloliquefaciens subsp. plantarum CAU B946 was 4.02 Mb in size and harbored 3,823 genes (coding sequences [CDS]). Nine giant gene clusters were dedicated to nonribosomal synthesis of antimicrobial compounds. Remarkably, strain CAU B946 possessed a gene cluster involved in synthesis of iturin A.

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The genome of rhizobacterium Bacillus amyloliquefaciens subsp. plantarum strain NAU-B3 is 4,196,170 bp in size and harbors 4,001 genes. Nine giant gene clusters are dedicated to the nonribosomal synthesis of antimicrobial lipopeptides and polyketides. Remarkably, NAU_B3 contains a large inversion within the central portion of the genome.