Differential regulation of genes for cyclic-di-GMP metabolism orchestrates adaptive changes during rhizosphere colonization by Pseudomonas fluorescens

Bacteria belonging to the Pseudomonas genus are highly successful colonizers of the plant rhizosphere. The ability of different Pseudomonas species to live either commensal lifestyles or to act as agents of plant-growth promotion or disease is reflected in a large, highly flexible accessory genome. Nevertheless, adaptation to the plant environment involves a commonality of phenotypic outputs such as changes to motility, coupled with synthesis of nutrient uptake systems, stress-response molecules and adherence factors including exopolysaccharides. Cyclic-di-GMP (cdG) is a highly important second messenger involved in the integration of environmental signals with appropriate adaptive responses and is known to play a central role in mediating effective rhizosphere colonization. In this study, we examined the transcription of multiple, reportedly plant-upregulated cdG metabolism genes during colonization of the wheat rhizosphere by the plant-growth-promoting strain P. fluorescens SBW25. While transcription of the tested genes generally increased in the rhizosphere environment, we additionally observed a tightly orchestrated response to environmental cues, with a distinct transcriptional pattern seen for each gene throughout the colonization process. Extensive phenotypical analysis of deletion and overexpression strains was then conducted and used to propose cellular functions for individual cdG signaling genes. Finally, in-depth genetic analysis of an important rhizosphere colonization regulator revealed a link between cdG control of growth, motility and stress response, and the carbon sources available in the rhizosphere

One ligand, two regulators and three binding sites: How KDPG controls primary carbon metabolism in Pseudomonas

Effective regulation of primary carbon metabolism is critically important for bacteria to successfully adapt to different environments. We have identified an uncharacterised transcriptional regulator; RccR, that controls this process in response to carbon source availability. Disruption of rccR in the plant-associated microbe Pseudomonas fluorescens inhibits growth in defined media, and compromises its ability to colonise the wheat rhizosphere. Structurally, RccR is almost identical to the Entner-Doudoroff (ED) pathway regulator HexR, and both proteins are controlled by the same ED-intermediate; 2-keto-3-deoxy-6-phosphogluconate (KDPG). Despite these similarities, HexR and RccR control entirely different aspects of primary metabolism, with RccR regulating pyruvate metabolism (aceEF), the glyoxylate shunt (aceA, glcB, pntAA) and gluconeogenesis (pckA, gap). RccR displays complex and unusual regulatory behaviour; switching repression between the pyruvate metabolism and glyoxylate shunt/gluconeogenesis loci depending on the available carbon source. This regulatory complexity is enabled by two distinct pseudo-palindromic binding sites, differing only in the length of their linker regions, with KDPG binding increasing affinity for the 28 bp aceA binding site but decreasing affinity for the 15 bp aceE site. Thus, RccR is able to simultaneously suppress and activate gene expression in response to carbon source availability. Together, the RccR and HexR regulators enable the rapid coordination of multiple aspects of primary carbon metabolism, in response to levels of a single key intermediate

Polyamines: emerging players in bacteria-host interactions

Polyamines are small polycationic molecules found in almost all cells and associated with a wide variety of physiological processes. In recent years it has become increasingly clear that, in addition to core physiological functions, polyamines play a crucial role in bacterial pathogenesis. Considerable evidence has built up that bacteria have evolved mechanisms to turn these molecules to their own advantage and a novel standpoint to look at host-bacterium interactions emerges from the interplay among polyamines, host cells and infecting bacteria. In this review, we highlight how human bacterial pathogens have developed their own resourceful strategies to exploit polyamines or manipulate polyamine-related processes to optimize their fitness within the host. Besides contributing to a better understanding of the complex relationship between a pathogen and its host, acquisitions in this field have a significant potential towards the development of novel antibacterial therapeutic approaches

Shedding of genes that interfere with the pathogenic lifestyle: the Shigella model

Pathoadaptive mutations are evolutionary events leading to the silencing of specific anti-virulence loci. This reshapes the core genome of a novel pathogen, adapts it to the host and boosts its harmful potential. A paradigmatic case is the emergence of Shigella, the causative agent of bacillary dysentery, from its innocuous Escherichia coli ancestor. Here we summarize current views on how pathoadaptation has allowed Shigella to progressively increase its virulence. In this context, modification of the polyamine pattern emerges as a crucial step towards full expression of the virulence program in Shigella. (c) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved

Molecular and Functional Profiling of the Polyamine Content in Enteroinvasive E. coli : Looking into the Gap between Commensal E. coli and Harmful Shigella.

Polyamines are small molecules associated with a wide variety of physiological functions. Bacterial pathogens have developed subtle strategies to exploit polyamines or manipulate polyamine-related processes to optimize fitness within the host. During the transition from its innocuous E. coli ancestor, Shigella, the aetiological agent of bacillary dysentery, has undergone drastic genomic rearrangements affecting the polyamine profile. A pathoadaptation process involving the speG gene and the cad operon has led to spermidine accumulation and loss of cadaverine. While a higher spermidine content promotes the survival of Shigella within infected macrophages, the lack of cadaverine boosts the pathogenic potential of the bacterium in host tissues. Enteroinvasive E. coli (EIEC) display the same pathogenicity process as Shigella, but have a higher infectious dose and a higher metabolic activity. Pathoadaption events affecting the cad locus have occurred also in EIEC, silencing cadaverine production. Since EIEC are commonly regarded as evolutionary intermediates between E. coli and Shigella, we investigated on their polyamine profile in order to better understand which changes have occurred along the path to pathogenicity. By functional and molecular analyses carried out in EIEC strains belonging to different serotypes, we show that speG has been silenced in one strain only, favouring resistance to oxidative stress conditions and survival within macrophages. At the same time, we observe that the content of spermidine and putrescine, a relevant intermediate in the synthesis of spermidine, is higher in all strains as compared to E. coli. This may represent an evolutionary response to the lack of cadaverine. Indeed, restoring cadaverine synthesis decreases the expression of the speC gene, whose product affects putrescine production. In the light of these results, we discuss the possible impact of pathoadaptation events on the evolutionary emergence of a polyamine profile favouring to the pathogenic lifestyle of Shigella and EIEC

Multifactor Regulation of the MdtJI Polyamine Transporter in Shigella.

The polyamine profile of Shigella, the etiological agent of bacillary dysentery in humans, differs markedly from that of E. coli, its innocuous commensal ancestor. Pathoadaptive mutations such as the loss of cadaverine and the increase of spermidine favour the full expression of the virulent phenotype of Shigella. Spermidine levels affect the expression of the MdtJI complex, a recently identified efflux pump belonging to the small multi-drug resistance family of transporters. In the present study, we have addressed the regulation of the mdtJI operon in Shigella by asking which factors influence its expression as compared to E. coli. In particular, after identifying the mdtJI promoter by primer extension analysis, in vivo transcription assays and gel-retardation experiments were carried out to get insight on the silencing of mdtJI in E. coli. The results indicate that H-NS, a major nucleoid protein, plays a key role in repressing the mdtJI operon by direct binding to the regulatory region. In the Shigella background mdtJI expression is increased by the high levels of spermidine typically found in this microorganism and by VirF, the plasmid-encoded regulator of the Shigella virulence regulatory cascade. We also show that the expression of mdtJI is stimulated by bile components. Functional analyses reveal that MdtJI is able to promote the excretion of putrescine, the spermidine precursor. This leads us to consider the MdtJI complex as a possible safety valve allowing Shigella to maintain spermidine to a level optimally suited to survival within infected macrophages and, at the same time, prevent toxicity due to spermidine over-accumulation

Cadaverine interferes with <i>speC</i> expression.

<p>Experiments were performed using strains lacking only the <i>cadC</i> gene (EIEC HN280, 4608 and 53638 and <i>E. coli</i> K-12 ULS655) or the entire <i>cad</i> operon (EIEC 13.80 and 6.81). Strains were transformed with pCC55 (a pACYC184 derivative containing a functional <i>cadC</i> gene) or pACYC184. (A) Transcription of <i>speC</i> in the presence or absence of cadaverine as monitored by Real-Time PCR. At least three wells were run for each sample and the error bars display the calculated maximum (RQMax) and minimum (RQMin) levels that represent standard error of the mean expression level (RQ value). * denotes 0.05>p≥0.01; ** denotes p<0.01 (B) Ornithine decarboxylase (ODC) activity in the presence or absence of cadaverine. ODC activity was measured in total cell extracts by assaying putrescine production. The synthesized putrescine was made fluorescent through chemical modification and separated by TLC. The fluorimetric data were normalized against the total protein content of each sample. Results are shown as variation (percentage) in strains carrying pCC55 (<i>cadC</i>) vs the corresponding wt strain carrying the backbone construct. * denotes 0.05>p≥0.01; ** denotes p<0.01. (C) Transcription of <i>speC</i> in <i>E. coli</i> K-12 (MG1655); ** denotes p<0.01. Transcription in strains producing or lacking cadaverine was monitored by Real-Time PCR as in panel A.</p

Analysis of polyamine content in different EIEC <i>and S. flexneri</i> strains.

<p>Values reported are in nmol per 10⁸ cells and represent the average ± standard deviations. <i>E. coli</i> K-12 MG1655 has been used as reference. N-SPD: Acetyl spermidine; PUT: Putrescine; CAD: Cadaverine; SPD: spermidine. Student's t tests were performed comparing PUT, SPD, NSPD and CAD concentration in EIEC and <i>Shigella</i> strains with the polyamine concentration detected in <i>E. coli</i> K12 MG1655.</p><p>*denotes 0.05>p≥0.01;</p><p>**denotes p<0.01.</p><p>Analysis of polyamine content in different EIEC <i>and S. flexneri</i> strains.</p

RccR and HexR are highly similar and important for wheat rhizosphere colonisation.

<p><b>1A</b>: Sequence alignment for <i>rccR</i> and <i>hexR</i> from <i>P</i>. <i>fluorescens</i> SBW25. Important amino acid residues for DNA and ligand interactions are marked in blue and red respectively. <b>1B</b>: 3D homology model of the RccR protein structure. Arg-53 and -56 (blue) are the predicted DNA interaction partners in the helix-turn-helix domain. Ser-139 and -183 (red) are located in the predicted effector binding site. <b>1C</b>: Rhizosphere colonisation competition assays. The graph shows the ratio of SBW25 WT or <i>ΔrccR</i>/<i>ΔhexR</i> mutants to WT-<i>lacZ</i> colony forming units (CFU) recovered from the rhizospheres of wheat plants seven days post-inoculation. Each dot represents the ratio of CFUs recovered from an individual plant. In each case, differences between SBW25 and <i>ΔrccR</i> or <i>ΔhexR</i> strains are statistically significant (p < 0.05, Mann-Whitney U test).</p