Maarten Vercruysse

Stress response regulators identified through genome-wide transcriptome analysis of the (p)ppGpp-dependent response in Rhizobium etli

BackgroundThe alarmone (p)ppGpp mediates a global reprogramming of gene expression upon nutrient limitation and other stresses to cope with these unfavorable conditions. Synthesis of (p)ppGpp is, in most bacteria, controlled by RelA/SpoT (Rsh) proteins. The role of (p)ppGpp has been characterized primarily in Escherichia coli and several Gram-positive bacteria. Here, we report the first in-depth analysis of the (p)ppGpp-regulon in an α-proteobacterium using a high-resolution tiling array to better understand the pleiotropic stress phenotype of a relA/rsh mutant.ResultsWe compared gene expression of the Rhizobium etli wild type and rsh (previously rel) mutant during exponential and stationary phase, identifying numerous (p)ppGpp targets, including small non-coding RNAs. The majority of the 834 (p)ppGpp-dependent genes were detected during stationary phase. Unexpectedly, 223 genes were expressed (p)ppGpp-dependently during early exponential phase, indicating the hitherto unrecognized importance of (p)ppGpp during active growth. Furthermore, we identified two (p)ppGpp-dependent key regulators for survival during heat and oxidative stress and one regulator putatively involved in metabolic adaptation, namely extracytoplasmic function sigma factor EcfG2/PF00052, transcription factor CH00371, and serine protein kinase PrkA.ConclusionsThe regulatory role of (p)ppGpp in R. etli stress adaptation is far-reaching in redirecting gene expression during all growth phases. Genome-wide transcriptome analysis of a strain deficient in a global regulator, and exhibiting a pleiotropic phenotype, enables the identification of more specific regulators that control genes associated with a subset of stress phenotypes. This work is an important step toward a full understanding of the regulatory network underlying stress responses in α-proteobacteria.

A Comparative Transcriptome Analysis of Rhizobium etli Bacteroids: Specific Gene Expression During Symbiotic Nongrowth

Rhizobium etli occurs either in a nitrogen-fixing symbiosis with its host plant, Phaseolus vulgaris, or free-living in the soil. During both conditions, the bacterium has been suggested to reside primarily in a nongrowing state. Using genome-wide transcriptome profiles, we here examine the molecular basis of the physiological adaptations of rhizobia to nongrowth inside and outside of the host. Compared with exponentially growing cells, we found an extensive overlap of downregulated growth-associated genes during both symbiosis and stationary phase, confirming the essentially nongrowing state of nitrogen-fixing bacteroids in determinate nodules that are not terminally differentiated. In contrast, the overlap of upregulated genes was limited. Generally, actively growing cells have hitherto been used as reference to analyze symbiosis-specific expression. However, this prevents the distinction between differential expression arising specifically from adaptation to a symbiotic lifestyle and features associated with nongrowth in general. Using stationary phase as the reference condition, we report a distinct transcriptome profile for bacteroids, containing 203 induced and 354 repressed genes. Certain previously described symbiosis-specific characteristics, such as the downregulation of amino acid metabolism genes, were no longer observed, indicating that these features are more likely due to the nongrowing state of bacteroids rather than representing bacteroid-specific physiological adaptations.

Pleiotropic effects of a rel mutation on stress survival of Rhizobium etli CNPAF512

BackgroundThe rel gene of Rhizobium etli (relRet), the nodulating endosymbiont of the common bean plant, determines the cellular level of the alarmone (p)ppGpp and was previously shown to affect free-living growth and symbiosis. Here, we demonstrate its role in cellular adaptation and survival in response to various stresses.ResultsGrowth of the R. etli relRetmutant was strongly reduced or abolished in the presence of elevated NaCl levels or at 37°C, compared to the wild type. In addition, depending on the cell density, decreased survival of exponentially growing or stationary phase relRetmutant cells was obtained after H2O2, heat or NaCl shock compared to the wild-type strain. Survival of unstressed stationary phase cultures was differentially affected depending on the growth medium used. Colony forming units (CFU) of relRetmutant cultures continuously decreased in minimal medium supplemented with succinate, whereas wild-type cultures stabilised at higher CFU levels. Microscopic examination of stationary phase cells indicated that the relRetmutant was unable to reach the typical coccoid morphology of the wild type in stationary phase cultures. Assessment of stress resistance of re-isolated bacteroids showed increased sensitivity of the relRetmutant to H2O2 and a slightly increased resistance to elevated temperature (45°C) or NaCl shock, compared to wild-type bacteroids.ConclusionThe relRetgene is an important factor in regulating rhizobial physiology, during free-living growth as well as in symbiotic conditions. Additionally, differential responses to several stresses applied to bacteroids and free-living exponential or stationary phase cells point to essential physiological differences between the different states.

Canonical and non‐canonical EcfG sigma factors control the general stress response in Rhizobium etli

A core component of the α‐proteobacterial general stress response (GSR) is the extracytoplasmic function (ECF) sigma factor EcfG, exclusively present in this taxonomic class. Half of the completed α‐proteobacterial genome sequences contain two or more copies of genes encoding σEcfG‐like sigma factors, with the primary copy typically located adjacent to genes coding for a cognate anti‐sigma factor (NepR) and two‐component response regulator (PhyR). So far, the widespread occurrence of additional, non‐canonical σEcfG copies has not satisfactorily been explained. This study explores the hierarchical relation between Rhizobium etli σEcfG1 and σEcfG2, canonical and non‐canonical σEcfG proteins, respectively. Contrary to reports in other species, we find that σEcfG1 and σEcfG2 act in parallel, as nodes of a complex regulatory network, rather than in series, as elements of a linear regulatory cascade. We demonstrate that both sigma factors control unique yet also shared target genes, corroborating phenotypic evidence. σEcfG1 drives expression of rpoH2, explaining the increased heat sensitivity of an ecfG1 mutant, while katG is under control of σEcfG2, accounting for reduced oxidative stress resistance of an ecfG2 mutant. We also identify non‐coding RNA genes as novel σEcfG targets. We propose a modified model for GSR regulation in R. etli, in which σEcfG1 and σEcfG2 function largely independently. Based on a phylogenetic analysis and considering the prevalence of α‐proteobacterial genomes with multiple σEcfG copies, this model may also be applicable to numerous other species.