Advancement of DNA sequencing technology allows the routine use of genome sequences in the various fields of microbiology. The information held in genome sequences proved to provide objective and reliable means in the taxonomy of prokaryotes. Here, we describe the minimal standards for the quality of genome sequences and how they can be applied for taxonomic purposes.

There is a need to clarify relationships within the actinobacterial genus Micromonospora, the type genus of the family Micromonosporaceae, given its biotechnological and ecological importance. Here, draft genomes of 40 Micromonospora type strains and two non-type strains are made available through the Genomic Encyclopedia of Bacteria and Archaea project and used to generate a phylogenomic tree which showed they could be assigned to well supported phyletic lines that were not evident in corresponding trees based on single and concatenated sequences of conserved genes. DNA G+C ratios derived from genome sequences showed that corresponding data from species descriptions were imprecise. Emended descriptions include precise base composition data and approximate genome sizes of the type strains. antiSMASH analyses of the draft genomes show that micromonosporae have a previously unrealised potential to synthesize novel specialized metabolites. Close to one thousand biosynthetic gene clusters were detected, including NRPS, PKS, terpenes and siderophores clusters that were discontinuously distributed thereby opening up the prospect of prioritising gifted strains for natural product discovery. The distribution of key stress related genes provide an insight into how micromonosporae adapt to key environmental variables. Genes associated with plant interactions highlight the potential use of micromonosporae in agriculture and biotechnology.

Our current knowledge of plant-microbe interactions indicate that populations inhabiting a host plant are not restricted to a single microbial species but comprise several genera and species. No one knows if communities inside plants interact, and it has been speculated that beneficial effects are the result of their combined activities. During an ecological study of nitrogen-fixing bacterial communities from Lupinus angustifolius collected in Spain, significant numbers of orangepigmented actinomycete colonies were isolated from surface-sterilized root nodules. The isolates were analysed by BOX-PCR fingerprinting revealing an unexpectedly high genetic variation. Selected strains were chosen for 16S rRNA gene sequencing and phylogenetic analyses confirmed that all strains isolated belonged to the genus Micromonospora and that some of them may represent new species. To determine the possibility that the isolates fixed atmospheric nitrogen, chosen strains were grown in nitrogen-free media, obtaining in some cases, significant growth when compared with the controls. These strains were further screened for the presence of the nifH gene encoding dinitrogenase reductase, a key enzyme in nitrogen fixation. The partial nifH-like gene sequences obtained showed a 99% similarity with the sequence of the nifH gene from Frankia alni ACN14a, an actinobacterium that induces nodulation and fixes nitrogen in symbiosis with Alnus. In addition, in situ hybridization was performed to determine if these microorganisms inhabit the inside of the nodules. This study strongly suggests that Micromonospora populations are natural inhabitants of nitrogen-fixing root nodules.

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Endophytic microorganisms live inside plants for at least part of their life cycle. According to their life strategies, bacterial endophytes can be classified as “obligate” or “facultative”. Reports that members of the genus Micromonospora, Gram-positive Actinobacteria, are normal occupants of nitrogen-fixing nodules has opened up a question as to what is the ecological role of these bacteria in interactions with nitrogen-fixing plants and whether it is in a process of adaptation from a terrestrial to a facultative endophytic life. The aim of this work was to analyse the genome sequence of Micromonospora lupini Lupac 08 isolated from a nitrogen fixing nodule of the legume Lupinus angustifolius and to identify genomic traits that provide information on this new plant-microbe interaction. The genome of M. lupini contains a diverse array of genes that may help its survival in soil or in plant tissues, while the high number of putative plant degrading enzyme genes identified is quite surprising since this bacterium is not considered a plant-pathogen. Functionality of several of these genes was demonstrated in vitro, showing that Lupac 08 degraded carboxymethylcellulose, starch and xylan. In addition, the production of chitinases detected in vitro, indicates that strain Lupac 08 may also confer protection to the plant. Micromonospora species appears as new candidates in plant-microbe interactions with an important potential in agriculture and biotechnology. The current data strongly suggests that a beneficial effect is produced on the host-plant.

The rhizobia-legume, root-nodule symbiosis provides the most efficient source of biologically fixed ammonia fertilizer for agricultural crops. Its development involves pathways of specificity, infectivity, and effectivity resulting from expressed traits of the bacterium and host plant. A key event of the infection process required for development of this root-nodule symbiosis is a highly localized, complete erosion of the plant cell wall through which the bacterial symbiont penetrates to establish a nitrogen-fixing, intracellular endosymbiotic state within the host. This process of wall degradation must be delicately balanced to avoid lysis and destruction of the host cell. Here, we describe the purification, biochemical characterization, molecular genetic analysis, biological activity, and symbiotic function of a cell-bound bacterial cellulase (CelC2) enzyme from Rhizobium leguminosarum bv. trifolii, the clover-nodulating endosymbiont. The purified enzyme can erode the noncrystalline tip of the white clover host root hair wall, making a localized hole of sufficient size to allow wild-type microsymbiont penetration. This CelC2 enzyme is not active on root hairs of the nonhost legume alfalfa. Microscopy analysis of the symbiotic phenotypes of the ANU843 wild type and CelC2 knockout mutant derivative revealed that this enzyme fulfils an essential role in the primary infection process required for development of the canonical nitrogen-fixing R. leguminosarum bv. trifolii-white clover symbiosis.nitrogen fixation ͉ nodulation ͉ clover ͉ root hair ͉ cellulose A central event in development of the Rhizobium-legume root-nodule symbiosis is the localized erosion of a cellulosic plant wall through which the bacterial symbiont passes to establish a nitrogen-fixing, intracellular endosymbiotic state within its legume host. Plant cell wall-degrading enzymes are predicted to participate in two steps of this infection process: during primary infection of host root hairs leading to infection thread formation (Inf) and later during bacterial release (Bar) from infection threads within host nodule cells. This process of plant cell wall degradation must be delicately balanced to allow the localized penetration of the bacterial symbiont into the host cell without its overt lysis and destruction.Several studies indicate that rhizobia produce enzymes capable of degrading plant cell wall polymers (1-14), but little is known about their molecular properties, none have previously been purified to homogeneity, and their specific role (if any) in symbiosis is undefined. The relatively low activities of these rhizobial enzymes have hampered research progress in this area. Using improved assays with increased sensitivity that reliably detect these enzyme activities, we established that cellulases are produced by wild-type strains of Rhizobium leguminosarum (biovars trifolii, phaseoli, and viciae), Bradyrhizobium japonicum, Mesorhizobium loti, and Sinorhizobium meliloti (7, 9). Further studies using R. leguminosarum bv. trifolii ANU843 indic...

The nodulation of legumes has for more than a century been considered an exclusive capacity of a group of microorganisms commonly known as rhizobia and belonging to the ␣-Proteobacteria. However, in the last 3 years four nonrhizobial species, belonging to ␣ and ␤ subclasses of the Proteobacteria, have been described as legume-nodulating bacteria. In the present study, two fast-growing strains, LUP21 and LUP23, were isolated from nodules of Lupinus honoratus. The phylogenetic analysis based on the 16S and 23S rRNA gene sequences showed that the isolates belong to the genus Ochrobactrum. The strains were able to reinfect Lupinus plants. A plasmid profile analysis showed the presence of three plasmids. The nodD and nifH genes were located on these plasmids, and their sequences were obtained. These sequences showed a close resemblance to the nodD and nifH genes of rhizobial species, suggesting that the nodD and nifH genes carried by strain LUP21 T were acquired by horizontal gene transfer. A polyphasic study including phenotypic, chemotaxonomic, and molecular features of the strains isolated in this study showed that they belong to a new species of the genus Ochrobactrum for which we propose the name Ochrobactrum lupini sp. nov. Strain LUP21 T (LMG 20667 T ) is the type strain.Plants from the family Leguminosae are usually capable of dinitrogen fixation because of their symbiotic interaction with nodulating bacteria belonging to the order Rhizobiales. Most bacteria that establish a symbiosis with legume plants, including some nonrhizobial species of Methylobacterium (16, 45) and Devosia (40, 41), belong to the ␣ subclass of Proteobacteria, although some species from genera of the ␤ subclass, such as Ralstonia and Burkholderia, can also nodulate legumes (6,32,53). In the last few years there has been an increasing amount of research focused on bacteria that nodulate stems or roots of tropical legume species. However, the identity of many of the endosymbionts of temperate legumes still remains unknown. The genus Lupinus groups up to 200 species of herbs and small shrubs, broadly distributed in the Mediterranean area and in the American continent, where they colonize very different environments. Despite the agronomic and ecological interest of Lupinus, this plant has been poorly studied with respect to its symbionts. Plants from the genus Lupinus are nodulated by fast-and slow-growing rhizobia; however, slow-growing rhizobia are more frequently isolated from this legume (3,5,21,30). The data obtained from the small-subunit (SSU) rRNA gene indicate a very close relationship between some bradyrhizobia isolated from Lupinus and Bradyrhizobium japonicum (3,12,30). However, bacterial strains nodulating Lupinus plants have been poorly characterized thus far, and fast-growing species nodulating this legume have not been not officially described; nevertheless, in the past the species Rhizobium lupini was proposed (15) and was later abandoned (14).During a study of rhizobia nodulating Lupinus plants in several geographical regi...

An actinomycete strain, NAR01 T , was isolated from root nodules of a Coriaria plant. The 16S rRNA gene sequence of strain NAR01 T showed most similarity to the type strains of Micromonospora endolithica (98?94 %) and Micromonospora chersina (98?4 %). The chemotaxonomic results obtained confirmed the taxonomic position of the isolate within the genus Micromonospora, and revealed differences at the species level. Physiological and biochemical tests showed that strain NAR01 T could be clearly distinguished from its closest phylogenetic neighbours, while DNA-DNA hybridization results indicated that the isolate represents a novel species. On the basis of these results, strain NAR01 T (=DSM 44875 T =LMG 23557 T ) is proposed as the type strain of the novel species Micromonospora coriariae sp. nov.The number of novel species of Micromonospora Ørskov 1923 described has increased significantly in the last year Thawai et al., 2005a, b;Trujillo et al., 2005) and the genus currently holds 27 species with validly published names. While most strains have been isolated from soil collected in diverse geographical regions, members of the genus Micromonospora also appear to be closely associated with plant roots (Coombs & Franco, 2003; Valdés et al., 2005; M. E. Trujillo, unpublished observations). In the present paper, we report on the isolation of a novel Micromonospora strain, NAR01 T , from surfacesterilized, nitrogen-fixing root nodules obtained from the plant Coriaria myrtifolia, which was collected in Salamanca, Spain.The root nodules used for the isolation of strain NAR01 T were washed several times with sterile distilled water and were then surface sterilized in HgCl 2 (2?5 % w/v) for 2 min. The nodules were rinsed several times with sterile distilled water and then crushed using a sterile glass rod. The homogenized plant tissue was inoculated onto yeast extract/ mannitol agar (Vincent, 1970) and the plates were incubated at 28 uC for 10 days. The isolation plates were then examined under a stereoscopic microscope: several small orange colonies were readily observed. Strain NAR01 T was selected because of its capacity to degrade xylan, as shown by the ability of the micro-organism to grow on XED medium (Rivas et al., 2003), which contained this carbohydrate as the sole carbon source.The cultural characteristics of strain NAR01 T were studied on several media, namely yeast extract-malt extract agar (ISP 2), Bennett's agar (Jones, 1949), oatmeal agar (ISP 3), SA1 agar (Trujillo et al., 2005) and yeast extract/mannitol agar. Abundant growth was observed on Bennett's, ISP 2 and SA1 agars, while moderate growth was obtained on ISP 3 and yeast extract/mannitol agar media. Colonies were an intense orange colour, folded and raised, turning darker after 3 weeks; neither aerial hyphae nor diffusible pigments were produced. Differences in substrate-mycelium colour found between NAR01 T and Micromonospora species are presented in Table 1.Phase-contrast observations (Opti-Phot microscope, 6100; Nikon) were performed using cells ob...