Pathogenic bacteria have to cope with adverse conditions, such as the host environment and host defense reactions. To adapt quickly to environmental changes, pathogens have developed complex regulatory networks that ensure adequate expression of their virulence genes. Recent evidence suggests that Fis, an abundant nucleoid-associated protein transiently produced during early exponential growth, plays a major role in these networks in several pathogenic bacteria. This review focuses on two enterobacteria, Salmonella enterica and Dickeya dadantii, that inhabit distinct ecological niches to illustrate how Fis uses different strategies to coordinate virulence gene expression, depending on the bacterial lifestyle.
The pectinolytic Dickeya spp. are soft-rot Gram-negative bacteria that cause severe disease in a wide range of plant species. In recent years, there has been an increase in the damage caused by Dickeya in potato crops in Europe. Soft-rot symptoms are due to the production and secretion of degradative enzymes that destroy the plant cell wall. However, an efficient colonization of the host plant requires many additional bacterial factors, including elements in the early stages allowing for the adhesion and penetration of the bacteria in the plant and different elements in the intermediate stages, involved in the adaptation to the new growth conditions encountered in the host. Dickeya pathogenicity is clearly a multifactorial process, and successful infection by these bacteria requires a temporal coordination of survival and virulence gene expression. This involves the ancestral nucleoid-associated proteins, Fis and H-NS, and modifications of DNA topology, as well as various specific regulatory systems, including a new quorum-sensing pathway and regulators that sense the bacterial metabolic status or environmental stresses. This review presents new information concerning the ecology of Dickeya and the strategies used by this bacterium to coordinate its survival and virulence programmes during infection.
Bacteria are colonizers of various environments and host organisms, and they are often subjected to drastic temperature variations. Dickeya dadantii is a pathogen infecting a wide range of plant species. Soft rot, the visible symptom, is mainly due to the production of pectate lyases (Pels) that destroy plant cell walls. The production of Pels is controlled by a complex regulation system that responds to various stimuli, such as the presence of pectin, growth phase and temperature. Despite numerous regulatory studies, the thermoregulation mechanism of Pel production remains unexplained. Here, we show that PecT, a previously identified repressor, modulates pel gene expression in a temperature-dependent manner, and we demonstrate that PecT binding on pel promoters increases concomitantly with temperature. High temperatures relax the DNA in D.?dadantii, and remarkably, artificial relaxation of DNA at low temperatures increases PecT binding to DNA. Deletion of pecT augmented the capacity of D.?dadantii to initiate soft-rot symptoms at high temperatures. These results reveal that DNA topology and PecT act in concert to fine-tune D.?dadantii virulence in response to temperature. This novel combination between DNA topology and a conventional transcriptional regulator extends our understanding of the thermoregulation mechanisms involved in bacterial virulence.
Successful infection of a pathogen relies on the coordinated expression of numerous virulence factor-encoding genes. In plant-bacteria interactions, this control is very often achieved through the integration of several regulatory circuits controlling cell-cell communication or sensing environmental conditions. Dickeya dadantii (formerly Erwinia chrysanthemi), the causal agent of soft rot on many crops and ornamentals, provokes maceration of infected plants mainly by producing and secreting a battery of plant cell wall-degrading enzymes. However, several other virulence factors have also been characterized. During Arabidopsis infection, most D. dadantii virulence gene transcripts accumulated in a coordinated manner during infection. This activation requires a functional GacA-GacS two-component regulatory system but the Gac system is not involved in the growth phase dependence of virulence gene expression. Here we show that, contrary to Pectobacterium, the AHL-mediated ExpIR quorum-sensing system does not play a major role in the growth phase-dependent control of D. dadantii virulence genes. On the other hand, the global regulator PecS participates in this coordinated expression since, in a pecS mutant, an early activation of virulence genes is observed both in vitro and in planta. This correlated with the known hypervirulence phenotype of the pecS mutant. Analysis of the relationship between the regulatory circuits governed by the PecS and GacA global regulators indicates that these two regulators act independently. PecS prevents a premature expression of virulence genes in the first stages of colonization whereas GacA, presumably in conjunction with other regulators, is required for the activation of virulence genes at the onset of symptom occurrence.
Thirty N,N-disubstituted imidazolium salts have been synthesized and evaluated as LuxR antagonists. Substitution on one of the imidazolium nitrogen atoms includes benzhydryl, fluorenyl or cyclopentyl substituent, and alkyl chains of various lengths on the second one. Most of these compounds displayed antagonist activity, with IC(50) reaching the micromolar range for the most active ones. The disubstituted imidazolium scaffold is thus shown to be a new pertinent pharmacophore in the field of AHL dependent QS inhibition.
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.
Dickeya dadantii is a plant-pathogenic enterobacterium responsible for the soft rot disease of many plants of economic importance. We present here the sequence of strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria.
Pathogenicity of Dickeya dadantii is a process involving several factors, such as plant cell wall-degrading enzymes and adaptation systems to adverse conditions encountered in the apoplast. Regulators of the MarR family control a variety of biological processes, including adaptation to hostile environments and virulence. Analysis of the members of this family in D. dadantii led to the identification of a new regulator, MfbR, which controls virulence. MfbR represses its own expression but activates genes encoding plant cell wall-degrading enzymes. Purified MfbR increases the binding of RNA polymerase at the virulence gene promoters and inhibits transcription initiation at the mfbR promoter. MfbR activity appeared to be modulated by acidic pH, a stress encountered by pathogens during the early stages of infection. Expression of mfbR and its targets, during infection, showed that MfbR is unable to activate virulence genes in acidic conditions at an early step of infection. In contrast, alkalinization of the apoplast, during an advanced stage of infection, led to the potentialization of MfbR activity resulting in plant cell wall degrading enzyme production. This report presents a new example of how pathogens adjust virulence-associated factors during the time-course of an infection.
We have previously reported the implication of Bacillus in the production of pectinolytic enzymes during cocoa fermentation. The objective of this work was to identify the Bacillus strains isolated from cocoa fermentation and study their ability to produce pectate lyase (PL) in various growth conditions. Ninety-eight strains were analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Four different banding patterns were obtained leading to the clustering of the bacterial isolates into 4 distinct ARDRA groups. A subset of representative isolates for each group was identified by 16S rRNA gene partial sequencing. Six species were identified: Bacillus subtilis, Bacillus pumilus, Bacillus sphaericus, Bacillus cereus, Bacillus thuringiensis, together with Bacillus fusiformis which was isolated for the first time from cocoa fermentation. The best PL producers, yielding at least 9 U/mg of bacterial dry weight, belonged to B. fusiformis, B. subtilis, and B. pumilus species while those belonging to B. sphaericus, B. cereus and B. thuringiensis generally showed a low level of activity. Two kinds of PL were produced, as revealed by isoelectrofocusing: one with a pI of 9.8 produced by B. subtilis and B. fusiformis, the other with a pI of 10.5 was produced by B. pumilus. Strains yielded about 2 fold more PL in a pectic compound medium than in glucose medium and maximum enzyme production occurred in the late stationary bacterial growth phase. Together all these results indicate that PL production in the bacilli studied is modulated by the growth phase and by the carbon source present in the medium.
A dynamic mathematical model has been developed and validated to describe the synthesis of pectate lyases (Pels), the major virulence factors in Dickeya dadantii. This work focuses on the simultaneous modeling of the metabolic degradation of pectin by Pel enzymes and the genetic regulation of pel genes by 2-keto-3-deoxygluconate (KDG), a catabolite product of pectin that inactivates KdgR, one of the main repressors of pel genes. This modeling scheme takes into account the fact that the system is composed of two time-varying compartments: the extracellular medium, where Pel enzymes cleave pectin into oligomers, and the bacterial cytoplasm where, after internalization, oligomers are converted to KDG. Using the quasi-stationary state approximations, the model consists of some nonlinear differential equations for which most of the parameters could be estimated from the literature or from independent experiments. The few remaining unknown parameters were obtained by fitting the model equations against a set of Pel activity data. Model predictions were verified by measuring the time courses of bacterial growth, Pel production, pel mRNA accumulation, and pectin consumption under various growth conditions. This work reveals that pectin is almost totally consumed before the burst of Pel production. This paradoxical behavior can be interpreted as an evolutionary strategy to control the diffusion process so that as soon as a small amount of pectin is detected by the bacteria in its surroundings, it anticipates more pectin to come. The model also predicts the possibility of bistable steady states in the presence of constant pectin compounds.
Pectinolytic enzymes play an important role in cocoa fermentation. In this study, we characterized three extracellular pectate lyases (Pels) produced by bacilli isolated from fermenting cocoa beans. These enzymes, named Pel-22, Pel-66, and Pel-90, were synthesized by Bacillus pumilus BS22, Bacillus subtilis BS66, and Bacillus fusiformis BS90, respectively. The three Pels were produced under their natural conditions and purified from the supernatants using a one-step chromatography method. The purified enzymes exhibited optimum activity at 60 degrees C, and the half-time of thermoinactivation at this temperature was approximately 30 min. Pel-22 had a low specific activity compared with the other two enzymes. However, it displayed high affinity for the substrate, about 2.5-fold higher than those of Pel-66 and Pel-90. The optimum pHs were 7.5 for Pel-22 and 8.0 for Pel-66 and Pel-90. The three enzymes trans-eliminated polygalacturonate in a random manner to generate two long oligogalacturonides, as well as trimers and dimers. A synergistic effect was observed between Pel-22 and Pel-66 and between Pel-22 and Pel-90, but not between Pel-90 and Pel-66. The Pels were also strongly active on highly methylated pectins (up to 60% for Pel-66 and Pel-90 and up to 75% for Pel-22). Fe(2+) was found to be a better cofactor than Ca(2+) for Pel-22 activity, while Ca(2+) was the best cofactor for Pel-66 and Pel-90. The amino acid sequences deduced from the cloned genes showed the characteristics of Pels belonging to Family 1. The pel-66 and pel-90 genes appear to be very similar, but they are different from the pel-22 gene. The characterized enzymes form two groups, Pel-66/Pel-90 and Pel-22; members of the different groups might cooperate to depolymerize pectin during the fermentation of cocoa beans.
Dickeya dadantii is a plant pathogen that secretes cell wall-degrading enzymes (CWDE) that are responsible for soft-rot symptoms. Virulence genes are expressed in a concerted manner and culminate when bacterial multiplication slows. We identify a 25 kb vfm cluster required for D.?dadantii CWDE production and pathogenesis. The vfm cluster encodes proteins displaying similarities both with enzymes involved in amino acid activation and with enzymes involved in fatty acid biosynthesis. These similarities suggest that the vfm genes direct the production of a metabolite. Cell-free supernatant from the D.?dadantii wild-type strain restores CWDE production in vfm mutants. Collectively, our results indicate that vfm genes direct the synthesis of an extracellular signal and constitute a new quorum sensing system. Perception of the signal is achieved by the two-component system VfmH-VfmI, which activates the expression of the vfmE gene encoding an AraC regulator. VfmE then activates both the transcription of the CWDE genes and the expression of the vfm operons. The vfm gene cluster does not seem to be widespread among bacterial species but is conserved in other Dickeya species and could have been laterally transferred to Rahnella. This work highlights that entirely new families of bacterial languages remain to be discovered.
Bacteria use biofilm structures to colonize surfaces and to survive in hostile conditions, and numerous bacteria produce cellulose as a biofilm matrix polymer. Hence, expression of the bcs operon, responsible for cellulose biosynthesis, must be finely regulated in order to allow bacteria to adopt the proper surface-associated behaviours. Here we show that in the phytopathogenic bacterium, Dickeya dadantii, production of cellulose is required for pellicle-biofilm formation and resistance to chlorine treatments. Expression of the bcs operon is growth phase-regulated and is stimulated in biofilms. Furthermore, we unexpectedly found that the nucleoid-associated protein and global regulator of virulence functions, Fis, directly represses bcs operon expression by interacting with an operator that is absent from the bcs operon of animal pathogenic bacteria and the plant pathogenic bacterium Pectobacterium. Moreover, production of cellulose enhances plant surface colonization by D. dadantii. Overall, these data suggest that cellulose production and biofilm formation may be important factors for surface colonization by D. dadantii and its subsequent survival in hostile environments. This report also presents a new example of how bacteria can modulate the action of a global regulator to co-ordinate basic metabolism, virulence and modifications of lifestyle.
Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity.
Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and ?-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates.
Dickeya dadantii is a pathogen infecting a wide range of plant species. Soft rot, the visible symptom, is mainly due to the production of pectate lyases (Pels) that can destroy the plant cell walls. Previously we found that the pel gene expression is modulated by H-NS and FIS, two nucleoid-associated proteins (NAPs) modulating the DNA topology. Here, we show that relaxation of the DNA in growing D. dadantii cells decreases the expression of pel genes. Deletion of fis aggravates, whereas that of hns alleviates the negative impact of DNA relaxation on pel expression. We further show that H-NS and FIS directly bind the pelE promoter and that the response of D. dadantii pel genes to stresses that induce DNA relaxation is modulated, although to different extents, by H-NS and FIS. We infer that FIS acts as a repressor buffering the negative impact of DNA relaxation on pel gene transcription, whereas H-NS fine-tunes the response of virulence genes precluding their expression under suboptimal conditions of supercoiling. This novel dependence of H-NS effect on DNA topology expands our understanding of the role of NAPs in regulating the global bacterial gene expression and bacterial pathogenicity.
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