Plant species is considered to be one of the most important factors in shaping rhizobacterial communities, but specific plant–microbe interactions in the rhizosphere are still not fully understood. Arabidopsis thaliana, for which a large number of naturally occurring ecotype accessions exist, lacks mycorrhizal associations and is hence an ideal model for rhizobacterial studies. Eight Arabidopsis accessions were found to exert a marked selective influence on bacteria associated with their roots, as determined by terminal-restriction fragment length polymorphism (T-RFLP) and ribosomal intergenic spacer analysis (RISA). Community differences in species composition and relative abundance were both significant (P <0.001). The eight distinct and reproducible accession-dependent community profiles also differed from control bulk soil. Root exudates of these variants were analysed by high performance liquid chromatography (HPLC) to try to establish whether the unique rhizobacterial assemblages among accessions could be attributed to plant-regulated chemical changes in the rhizosphere. Natural variation in root exudation patterns was clearly exhibited, suggesting that differences in exudation patterns among accessions could be influencing bacterial assemblages. Other factors such as root system architecture are also probably involved. Finally, to investigate the Arabidopsis rhizosphere further, the phylogenetic diversity of rhizobacteria from accession Cvi-0 is described.
The rhizosphere is strongly influenced by plant-derived phytochemicals exuded by roots and plant species exert a major selective force for bacteria colonizing the root-soil interface. We have previously shown that rhizobacterial recruitment is tightly regulated by plant genetics, by showing that natural variants of Arabidopsis thaliana support genotype-specific rhizobacterial communities while also releasing a unique blend of exudates at six weeks post-germination. To further understand how exudate release is controlled by plants, changes in rhizobacterial assemblages of two Arabidopsis accessions, Cvi and Ler where monitored throughout the plants' life cycle. Denaturing gradient gel electrophoresis (DGGE) fingerprints revealed that bacterial communities respond to plant derived factors immediately upon germination in an accession-specific manner. Rhizobacterial succession progresses differently in the two accessions in a reproducible manner. However, as plants age, rhizobacterial and control bulk soil communities converge, indicative of an attenuated rhizosphere effect, which coincides with the expected slow down in the active release of root exudates as plants reach the end of their life cycle. These data strongly suggest that exudation changes during plant development are highly genotype-specific, possibly reflecting the unique, local co-evolutionary communication processes that developed between Arabidopsis accessions and their indigenous microbiota. IntroductionPlant roots are an active component of soil, creating the rhizosphere by physically changing soil characteristics as they elongate and branch out, and dramatically altering the chemical milieu through exudation of hundreds of phytochemicals and sloughedoff cells. 1,2 Root-derived compounds mediate a plethora of biological processes. The plant-bacterial relationships that occur at the root-soil interface are probably a combination of opportunistic interactions, as ubiquitous organisms exploit the nutrients present, and highly regulated communication events. The latter interactions are mediated by root-exuded signalling molecules that elicit specific responses. The type and amounts of the various exudate compounds synthesized and released by roots are under the plant's genetic control, making plant genetic make-up one of the most significant determinants of rhizobacterial selection. Even opportunistic use of root exudates as metabolic fuel will attract only those bacteria equipped to make use of the nutrients that are extant and at the same time capable of adapting to the rhizosphere environment. The unique exudate cocktails released by different plant species therefore signal to and attract a specific bacterial assemblage in a reproducible manner, explaining the well established role of plant species in rhizobacterial community selection. 3,4 Using various Arabidopsis thaliana accessions, we have previously shown that the plant genetic control of rhizobacterial recruitment is so tightly regulated that even genetic variants of the same plant species se...
Fluorescently-labelled bacteria (FLB) were used to study the feeding strategies of a natural assemblage of estuarine protozoans and to examine whether the protozoan grazing could account for the in situ size structure of the bacterioplankton. The FLB, DTAF-stained enterococci, ranging in volume from 0.01 to 0.30 × 10(-1) µm(3), were added to a natural planktonic assemblage at a density of 5.5% of the natural bacterioplankton. Initial densities (individuals ml(-1)) were as follows: total natural bacteria, 2.2 × 10(6); FLB, 1.2 × 10(5); pigmented flagellates, 300; colorless flagellates, 250; and ciliates, 30. FLB consumption rates were determined by examining the contents of protozoan food vacuoles, and the long-term effect of grazing (over a period of 100 hours) was determined by monitoring the decline in the FLB density in experimental vessels. The average consumption rates of FLB by pigmented flagellates were similar to those by flagellates that lacked chloroplasts (0.9 and 0.6 FLB protozoan(-1) hour(-1), respectively). The ciliates consumed bacteria at an average rate that was 17-fold higher (per cell) than flagellates, and they displayed a greater preference for larger bacteria than did the flagellates. FLB of the mid-size classes (0.025-0.100 µm(3)) were heavily grazed by the entire protozoan assemblage; the smallest (<0.025 µm(3)) and the largest (>0.100 µm(3)) FLB escaped protozoan grazing. This had a profound effect on the resulting size distribution of FLB. At the end of a 100-hour incubation, the percentage of mid-size FLB (0.025 to 0.100 µm(3)) decreased 2.0-2.2-fold, while the percentage of the smallest and the largest FLB increased 2.0-2.5-fold. Resultant densities of FLB were consistent with initial clearance rates determined for the protozoan groups. The grazing rates of protozoans on FLB were species-specific; whereas some species consumed FLB, others did not demonstrate bacterivory. The results suggest that protozoan grazing has a major effect on the size distribution of coastal bacterioplankton. By selectively feeding on a particular size-class of bacteria, planktonic ciliates may consume 15-90% day(-1) of the standing stock of largest size classes of bacterioplankton. Thus, ciliates, which were present in low abundance in the field, could not balance the production of the entire bacterial community, but they may strongly influence the portion of the bacterial community represented by the largest bacterial class. The direct effect of flagellates (e.g., grazing) was limited to smaller bacteria.
A modified cloning procedure was used to obtain large DNA insertions (20 to 30 kb) from Pseudomonas putida NCIB 9816 that expressed polycyclic aromatic hydrocarbon (PAH) transformation activity in Escherichia coli HB101. Four subclones (16 [in both orientations], 12, and 8.5 kb in size) were constructed from the initial clones. Naphthalene, fluorene, and phenanthrene transformations were investigated in these eight NCIB 9816 clones by a simple agar plate assay method, which was developed to detect and identify potential PAH metabolites. Results indicated that the necessary genes encoding the initial ring fission of the three PAHs in E. coli cells are located in an 8.5-kb EcoRl-XhoI portion, but the lower-pathway genes are not present in a 38-kb neighborhood region. These NCIB 9816 clones could transform naphthalene and phenanthrene to salicylic acid and 1-hydroxy-2-naphthoic acid, respectively. With the same clones, fluorene was degraded to 9-hydroxyfluorene, 9-fluorenone, and two unidentified compounds. Genetic similarity between the NAH7' upper-pathway genes and the cloned NCIB 9816 genes was confirmed by Southern blot DNA-DNA hybridization. In spite of this genetic similarity, the abilities of the two clusters to transform multiple PAHs were different. Under our experimental conditions, only the metabolites from naphthalene transformation by the NAH7 clone (pE317) were detected, whereas the NCIB 9816 clones produced metabolites from all three PAHs.The biodegradation of polycyclic aromatic hydrocarbons (PAHs) has been intensively studied. Many bacterial strains have been shown to degrade naphthalene, fluorene, or phenanthrene, often using these PAHs as sole carbon sources (3, 5). One naphthalene degradation system, NAH7, which was isolated from Pseudomonas putida PpG7, has been well characterized both genetically and biochemically (25). Another system, isolated from P. putida NCIB 9816, has been examined by several investigators (4, 22a).Other systems capable of transforming naphthalene, fluorene, or phenanthrene have also been described. A 9.8-kb DNA fragment was cloned and sequenced from a soil isolate that degrades naphthalene and dibenzothiophene, Pseudomonas sp. strain C18 (8). confusion has arisen in the literature because several variants derived from NCIB 9816 have been used by different investigators (25). These strains vary in their regulation of the naphthalene degradation pathway and in plasmid numbers and plasmid sizes. Yen and Serdar (25) resolved their pedigree and assigned to each of these variants a reference number. Genetic analysis was conducted with NCIB 9816-2 by Connors and Barnsley (6), with NCIB 9816-3 (P. putida PGB1) by Cane and Williams (4), and with NCIB 9816-4 by Serdar (22a). The genes encoding the dioxygenase enzyme complex were cloned and sequenced by Kurkelar and coworkers (15) from an unspecified NCIB 9816 strain and by Simon and coworkers (23) from NCIB 9816-4.Here, we report the preliminary characterization of multiple PAH degradation by a single gene cluster cloned from the...
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