The magic and menace of metagenomics: prospects for the study of plant growth-promoting rhizobacteria

This article aims to be a pragmatic primer into the field of metagenomics with special emphasis on the prospective contributions of metagenomics to the study of plant growth-promoting rhizobacteria (PGPR). After an introduction into the concepts and methodologies of metagenomics and a discussion of the numerous emerging variations on the basic theme, there will follow a short overview of the success stories in metagenomics (the ‘magic’ in the title of this review), a brief discussion about the technical problems and unrealistic expectations that are sometimes associated with metagenomics (the ‘menace’), and a shortlist of the lessons that can be learned by those that wish to explore the utility of metagenomics in the study of PGPR.

Discovery of a bacterial gene cluster for catabolism of the plant hormone indole 3-acetic acid

The isolation and annotation of an 8994-bp DNA fragment from Pseudomonas putida 1290, which conferred upon P. putida KT2440 the ability to utilize the plant hormone indole 3-acetic acid (IAA) as a sole source of carbon and energy, is described. This iac locus (for indole 3-acetic acid catabolism) was identified through analysis of a plasposon mutant of P. putida 1290 that was no longer able to grow on IAA or indole 3-acetaldehyde and was unable to protect radish roots from stunting by exogenously added IAA. The iac locus consisted of 10 genes with coding similarity to enzymes acting on indole or amidated aromatics and to proteins with regulatory or unknown function. Highly similar iac gene clusters were identified in the genomes of 22 bacterial species. Five of these, i.e. P. putida GB-1, Marinomonas sp. MWYL1, Burkholderia sp. 383, Sphingomonas wittichii RW1 and Rhodococcus sp. RHA1, were tested to confirm that bacteria with IAA-degrading ability have representatives in the Alpha-, Beta- and Gammaproteobacteria and in the Actinobacteria. In P. putida 1290, cat and pca genes were found to be essential to IAA-degradation, suggesting that IAA is channeled via catechol into the β-ketoadipate pathway. Also contributing to the IAA degrading phenotype were genes involved in tricarboxylate cycling, gluconeogenesis, and carbon/nitrogen sensing.

Bioreporters in microbial ecology

Bioreporters are effective research tools for gaining an understanding of a microbe's perception of the world. Fitted with a fusion of an environmentally responsive promoter to a suitable reporter gene, a bacterial or fungal bioreporter is able to communicate its metabolic or transcriptional behavior in a habitat, and furnish us with information on the chemical, physical or biological properties of its immediate surroundings. This review details recent developments in the use of such bioreporters in microbial ecology. Emphasis is placed on reporter genes that allow detection in individual microbial cells, as they provide a high-resolution description of the habitat under investigation. In an outlook on the future of bioreporter technology, this review stresses the need to interpret the activity of a bioreporter within the context of its biology. [KEYWORDS: bioreporter; biosensor; whole cell; single cell; reporter gene; reporter protein; green fluorescent protein; beta-galactosidase; ice nucleation protein]

Bioreporters in microbial ecology

Bioreporters are effective research tools for gaining an understanding of a microbe's perception of the world. Fitted with a fusion of an environmentally responsive promoter to a suitable reporter gene, a bacterial or fungal bioreporter is able to communicate its metabolic or transcriptional behavior in a habitat, and furnish us with information on the chemical, physical or biological properties of its immediate surroundings. This review details recent developments in the use of such bioreporters in microbial ecology. Emphasis is placed on reporter genes that allow detection in individual microbial cells, as they provide a high-resolution description of the habitat under investigation. In an outlook on the future of bioreporter technology, this review stresses the need to interpret the activity of a bioreporter within the context of its biology. [KEYWORDS: bioreporter; biosensor; whole cell; single cell; reporter gene; reporter protein; green fluorescent protein; beta-galactosidase; ice nucleation protein

Bacterial mycophagy: definition and diagnosis of a unique bacterial-fungal interaction

This review analyses the phenomenon of bacterial mycophagy, which we define as a set of phenotypic behaviours that enable bacteria to obtain nutrients from living fungi and thus allow the conversion of fungal into bacterial biomass. We recognize three types of bacterial strategies to derive nutrition from fungi: necrotrophy, extracellular biotrophy and endocellular biotrophy. Each is characterized by a set of uniquely sequential and differently overlapping interactions with the fungal target. We offer a detailed analysis of the nature of these interactions, as well as a comprehensive overview of methodologies for assessing and quantifying their individual contributions to the mycophagy phenotype. Furthermore, we discuss future prospects for the study and exploitation of bacterial mycophagy, including the need for appropriate tools to detect bacterial mycophagy in situ in order to be able to understand, predict and possibly manipulate the way in which mycophagous bacteria affect fungal activity, turnover, and community structure in soils and other ecosystems

Phyllosphere microbiology

Aerial plant surfaces harbor large numbers of microbes, some of which are deleterious to plants whereas others are benign or beneficial. Commercial formulations of bacteria antagonistic to plant pathogenic microbes and ice nucleation active bacteria have been utilized as an environmentally safe method to manage plant disease and to prevent frost damage. Molecular genetic tools, microscopic examination and whole-cell bacterial biosensors have provided extensive information on these microbes, their complex associations and their habitat. The aerial habitat influenced by plants, termed the phyllosphere, is particularly amenable to studies of microbial ecology and the information gained should lead to more effective means of plant protection. [KEYWORDS: SYRINGAE PV. SYRINGAE, SUGAR-BEET PHYTOSPHERE, CONJUGAL GENE-TRANSFER, BACTERIAL LEAF STREAK, UV-B RADIATION, PSEUDOMONAS-SYRINGAE, BIOLOGICAL-CONTROL, FIRE BLIGHT, PANTOEA-AGGLOMERANS, POPULATION-SIZE]

The mechanics of bacterial cluster formation on plant leaf surfaces as revealed by bioreporter technology

Bacteria that colonize the leaves of terrestrial plants often occur in clusters whose size varies from a few to thousands of cells. For the formation of such bacterial clusters, two non-mutually exclusive but very different mechanisms may be proposed: aggregation of multiple cells or clonal reproduction of a single cell. Here we assessed the contribution of both mechanisms on the leaves of bean plants that were colonized by the bacterium Pantoea agglomerans. In one approach, we used a mixture of green and red fluorescent P. agglomerans cells to populate bean leaves. We observed that this resulted in clusters made up of only one colour as well as two-colour clusters, thus providing evidence for both mechanisms. Another P. agglomerans bioreporter, designed to quantify the reproductive success of bacterial colonizers by proxy to the rate at which green fluorescent protein is diluted from dividing cells, revealed that during the first hours on the leaf surface, many bacteria were dividing, but not staying together and forming clusters, which is suggestive of bacterial relocation. Together, these findings support a dynamic model of leaf surface colonization, where both aggregative and reproductive mechanisms take place. The bioreporter-based approach we employed here should be broadly applicable towards a more quantitative and mechanistic understanding of bacterial colonization of surfaces in general.