Showing papers by "Johan H. J. Leveau published in 2013"

New insights into the structure and function of phyllosphere microbiota through high-throughput molecular approaches.

Abstract: The phyllosphere is an ecologically and economically important ecosystem that hosts a large and diverse microbial community. Phyllosphere microbiota play a critical role in protecting plants from diseases as well as promoting their growth by various mechanisms. There are serious gaps in our understanding of how and why microbiota composition varies across spatial and temporal scales, the ecology of leaf surface colonizers and their interactions with their host, and the genetic adaptations that enable phyllosphere survival of microorganisms. These gaps are due in large part to past technical limitations, as earlier studies were restricted to the study of culturable bacteria only and used low-throughput molecular techniques to describe community structure and function. The availability of high-throughput and cost-effective molecular technologies is changing the field of phyllosphere microbiology, enabling researchers to begin to address the dynamics and composition of the phyllosphere microbiota across a large number of samples with high, in-depth coverage. Here, we discuss and connect the most recent studies that have used next-generation molecular techniques such as metagenomics, proteogenomics, genome sequencing, and transcriptomics to gain new insights into the structure and function of phyllosphere microbiota and highlight important challenges for future research.

Effects of indole-3-acetic acid on the transcriptional activities and stress tolerance of Bradyrhizobium japonicum.

Abstract: A genome-wide transcriptional profile of Bradyrhizobium japonicum, the nitrogen-fixing endosymbiont of the soybean plant, revealed differential expression of approximately 15% of the genome after a 1 mM treatment with the phytohormone indole-3-acetic acid (IAA). A total of 1,323 genes were differentially expressed (619 up-regulated and 704 down-regulated) at a two-fold cut off with q value ≤ 0.05. General stress response genes were induced, such as those involved in response to heat, cold, oxidative, osmotic, and desiccation stresses and in exopolysaccharide (EPS) biosynthesis. This suggests that IAA is effective in activating a generalized stress response in B. japonicum. The transcriptional data were corroborated by the finding that stress tolerance of B. japonicum in cell viability assays was enhanced when pre-treated with 1 mM IAA compared to controls. The IAA treatment also stimulated biofilm formation and EPS production by B. japonicum, especially acidic sugar components in the total EPS. The IAA pre-treatment did not influence the nodulation ability of B. japonicum. The data provide a comprehensive overview of the potential transcriptional responses of the symbiotic bacterium when exposed to the ubiquitous hormone of its plant host.

Mighty small: Observing and modeling individual microbes becomes big science.

Abstract: Progress in microbiology has always been driven by technological advances, ever since Antonie van Leeuwenhoek discovered bacteria by making an improved compound microscope. However, until very recently we have not been able to identify microbes and record their mostly invisible activities, such as nutrient consumption or toxin production on the level of the single cell, not even in the laboratory. This is now changing with the rapid rise of exciting new technologies for single-cell microbiology (1, 2), which enable microbiologists to do what plant and animal ecologists have been doing for a long time: observe who does what, when, where, and next to whom. Single cells taken from the environment can be identified and even their genomes sequenced. Ex situ, their size, elemental, and biochemical composition, as well as other characteristics can be measured with high-throughput and cells sorted accordingly. Even better, individual microbes can be observed in situ with a range of novel microscopic and spectroscopic methods, enabling localization, identification, or functional characterization of cells in a natural sample, combined with detecting uptake of labeled compounds. Alternatively, they can be placed into fabricated microfluidic environments, where they can be positioned, exposed to stimuli, monitored, and their interactions controlled “in microfluido.” By introducing genetically engineered reporter cells into a fabricated landscape or a microcosm taken from nature, their reproductive success or activity can be followed, or their sensing of their local environment recorded.

Functional characterization of the bacterial iac genes for degradation of the plant hormone indole-3-acetic acid.

Abstract: Pseudomonas putida 1290 is a model organism for the study of bacterial degradation of the plant hormone indole-3-acetic acid (IAA). This property is encoded by the iac gene cluster. Insertional inactivation and/or deletion of individual iac genes and heterologous expression of the gene cluster in Escherichia coli were combined with mass spectrometry to demonstrate that iac-based degradation of IAA is likely to involve 2-hydroxy-IAA, 3-hydroxy-2-oxo-IAA, and catechol as intermediates. The first gene of the cluster, iacA encodes for the first step in the pathway, and also can convert indole to indoxyl to produce the blue pigment indigo. Transcriptional profiling of iac genes in P. putida 1290 revealed that they were induced in the presence of IAA. Based on results with an iacR knockout, we propose that this gene codes for a repressor of iacA expression and that exposure to IAA relieves this repression. Transformation of P. putida KT2440 (which cannot degrade IAA) with the iac gene cluster conferred the ability to grow on IAA as a sole source of carbon and energy, but not the ability to chemotaxi towards IAA. We could show such tactic response for P. putida 1290, thus representing the first demonstration of bacterial chemotaxis towards IAA. We discuss the ecological significance of our findings, and specifically the following question: under what circumstances do bacteria with the ability to degrade, recognize, and move towards IAA have a selective advantage?

Isolation of Arthrobacter species from the phyllosphere and demonstration of their epiphytic fitness

Abstract: Bacteria of the genus Arthrobacter are common inhabitants of the soil environment, but can also be recovered from leaf surfaces (the phyllosphere). Using enrichment cultures on 4-chlorophenol, we succeeded in specifically isolating Arthrobacter bacteria from ground cover vegetation in an apple orchard. Based on 16S rRNA gene sequencing, the isolates were found to belong to at least three different species of Arthrobacter. Compared to the model bacterial epiphyte Pantoea agglomerans, the Arthrobacter isolates performed as well or even better in a standardized laboratory test of phyllosphere fitness. A similar performance was observed with the well-characterized soil isolate Arthrobacter chlorophenolicus A6. These findings suggest that the frequently reported presence of Arthrobacter strains on plant foliage can be explained by the capacity to multiply and persist in the phyllosphere environment. As bacteria from the genus Arthrobacter are known for their ability to degrade a wide variety of organic pollutants, their high phyllosphere competency marks them as a promising group for future studies on phyllosphere-based bioremediation, for example, as foliar bioaugmentation on ground cover or buffer-zone vegetation to prevent pesticides from reaching soil, surface-, or groundwater.

Draft Genome Sequence of the Phyllosphere Model Bacterium Pantoea agglomerans 299R

Abstract: Bacteria belonging to the genus Pantoea are common colonizers of plant leaf surfaces. Here, we present the draft genome sequence of Pantoea agglomerans 299R, a phyllosphere isolate that has become a model strain for studying the ecology of plant leaf-associated bacterial commensals.

Single-cell versus population-level reproductive success of bacterial immigrants to pre-colonized leaf surfaces.

Abstract: We assessed how preemptive inoculation of plant leaves with bacteria affected the establishment of secondary colonizers. We quantified the latter in two ways: (i) at the population level, i.e. as counts of colony-forming units and (ii) at the level of single cells by tracking the reproductive success of individual bacteria. Both analyses showed that the ability of secondary immigrants to establish on the leaf was negatively correlated with the level of pre-population by primary colonizers. This effect was best described by an inverse dose-response curve with an apparent half-point inhibition efficacy of approximately 10(6) cells of primary colonizers per gram leaf. This efficacy was the same whether calculated from population- or average single-cell data. However, single-cell data revealed that even under conditions of heavy pre-population with primary colonizers, a small fraction of secondary immigrants still produced offspring, although the corresponding population measurement showed no increase in total population size. This observation has direct relevance for biocontrol strategies that are based on the principle of preemptive exclusion of foliar bacterial pathogens: even at seemingly saturating levels of primary inoculum, some secondary colonizers may still be able to reproduce and possibly reach a quorum to trigger behaviours that enhance survival or virulence.

Explaining Bacterial Dispersion on Leaf Surfaces with an Individual-Based Model (PHYLLOSIM)

Abstract: We developed the individual-based model PHYLLOSIM to explain observed variation in the size of bacterial clusters on plant leaf surfaces (the phyllosphere). Specifically, we tested how different ‘waterscapes’ impacted the diffusion of nutrients from the leaf interior to the surface and the growth of individual bacteria on these nutrients. In the ‘null’ model or more complex ‘patchy’ models, the surface was covered with a continuous water film or with water drops of equal or different volumes, respectively. While these models predicted the growth of individual bacterial immigrants into clusters of variable sizes, they were unable to reproduce experimentally derived, previously published patterns of dispersion which were characterized by a much larger variation in cluster sizes and a disproportionate occurrence of clusters consisting of only one or two bacteria. The fit of model predictions to experimental data was about equally poor (<5%) regardless of whether the water films were continuous or patchy. Only by allowing individual bacteria to detach from developing clusters and re-attach elsewhere to start a new cluster, did PHYLLOSIM come much closer to reproducing experimental observations. The goodness of fit including detachment increased to about 70–80% for all waterscapes. Predictions of this ‘detachment’ model were further supported by the visualization and quantification of bacterial detachment and attachment events at an agarose-water interface. Thus, both model and experiment suggest that detachment of bacterial cells from clusters is an important mechanism underlying bacterial exploration of the phyllosphere.

region. regulatory oxidative operon, and analysis of the activator of the tcbCDEF chlorocatechol P51 gene tcbR, a LysR-type transcriptional Characterization of the Pseudomonas sp. strain

Abstract: region. regulatory oxidative operon, and analysis of the activator of the tcbCDEF chlorocatechol P51 gene tcbR, a LysR-type transcriptional Characterization of the Pseudomonas sp. strain