lpxC and yafS are the Most Suitable Internal Controls to Normalize Real Time RT-qPCR Expression in the Phytopathogenic Bacteria Dickeya dadantii

Background: Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions. [br/] Results: We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions. [br/] Conclusions: We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria

Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans

<p>Abstract</p> <p>Background</p> <p>Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. <it>Herminiimonas arsenicoxydans </it>has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III) to As(V) as a detoxification mechanism.</p> <p>Results</p> <p>In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of <it>H. arsenicoxydans </it>to As(III). To get insight into the molecular mechanisms of this enzyme activity, a Tn<it>5 </it>transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted <it>aoxR </it>and <it>aoxS </it>genes, showing that the <it>aox </it>operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in <it>rpoN </it>coding for the alternative N sigma factor (σ⁵⁴) of RNA polymerase and in <it>dnaJ </it>coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the <it>rpoN </it>and <it>dnaJ </it>gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the <it>aoxAB </it>operon was determined using rapid amplification of cDNA ends (RACE) and a putative -12/-24 σ⁵⁴-dependent promoter motif was identified upstream of <it>aoxAB </it>coding sequences.</p> <p>Conclusion</p> <p>These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in <it>H. arsenicoxydans</it>. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III) in this microorganism.</p

Genomic Species Are Ecological Species as Revealed by Comparative Genomics in Agrobacterium tumefaciens

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome—one on the circular chromosome and six on the linear chromosome—suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species

Structure, Function, and Evolution of the Thiomonas spp. Genome

Bacteria of the Thiomonas genus are ubiquitous in extreme environments, such as arsenic-rich acid mine drainage (AMD). The genome of one of these strains, Thiomonas sp. 3As, was sequenced, annotated, and examined, revealing specific adaptations allowing this bacterium to survive and grow in its highly toxic environment. In order to explore genomic diversity as well as genetic evolution in Thiomonas spp., a comparative genomic hybridization (CGH) approach was used on eight different strains of the Thiomonas genus, including five strains of the same species. Our results suggest that the Thiomonas genome has evolved through the gain or loss of genomic islands and that this evolution is influenced by the specific environmental conditions in which the strains live

Plasmid‐chromosome cross‐talks

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Quorum Sensing Regulation in Phytopathogenic Bacteria

International audienceQuorum sensing is a type of chemical communication by which bacterial populations control expression of their genes in a coordinated manner. This regulatory mechanism is commonly used by pathogens to control the expression of genes encoding virulence factors and that of genes involved in the bacterial adaptation to variations in environmental conditions. In phytopathogenic bacteria, several mechanisms of quorum sensing have been characterized. In this review, we describe the different quorum sensing systems present in phytopathogenic bacteria, such as those using the signal molecules named N-acyl-homoserine lactone (AHL), diffusible signal factor (DSF), and the unknown signal molecule of the virulence factor modulating (VFM) system. We focus on studies performed on phytopathogenic bacteria of major importance, including Pseudomonas, Ralstonia, Agrobacterium, Xanthomonas, Erwinia, Xylella,Dickeya, and Pectobacterium spp. For each system, we present the mechanism of regulation, the functions targeted by the quorum sensing system, and the mechanisms by which quorum sensing is regulated

A novel plasmid‐transcribed regulatory sRNA, QfsR, controls chromosomal polycistronic gene expression in Agrobacterium fabrum

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Plant-phytopathogen interactions: bacterial responses to environmental and plant stimuli

International audiencePlant pathogenic bacteria attack numerous agricultural crops, causing devastating effects on plant productivity and yield. They survive in diverse environments, both in plants, as pathogens, and also outside their hosts as saprophytes. Hence, they are confronted with numerous changing environmental parameters. During infection, plant pathogens have to deal with stressful conditions, such as acidic, oxidative and osmotic stresses; anaerobiosis; plant defenses; and contact with antimicrobial compounds. These adverse conditions can reduce bacterial survival and compromise disease initiation and propagation. Successful bacterial plant pathogens must detect potential hosts and also coordinate their possibly conflicting programs for survival and virulence. Consequently, these bacteria have a strong and finely tuned capacity for sensing and responding to environmental and plant stimuli. This review summarizes our current knowledge of the signals and genetic circuits that affect survival and virulence factor expression in three important and well-studied plant pathogenic bacteria with wide host ranges and the capacity for long-term environmental survival. These are: Ralstonia solanacerarum, a vascular pathogen that causes wilt disease; Agrobacterium tumefaciens, a biotrophic tumorigenic pathogen responsible for crown gall disease and Dickeya, a brute force apoplastic pathogen responsible for soft-rot disease