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

Within-host evolution of Staphylococcus aureus during asymptomatic carriage

Background Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus. Principal Findings We characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA). Significance This investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease

Discrete cyclic di-GMP-dependent control of bacterial predation versus axenic growth in Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways

HexR controls the Entner-Doudoroff pathway in <i>P</i>. <i>fluorescens</i>.

<p><b>3A</b>: Schematic organisation of the HexR gene targets. HexR binds to a DNA consensus sequence in the intergenic regions between the <i>zwf/pgl/eda</i> operon and <i>hexR</i> genes, and between the <i>edd/glk/gltR2/gltS</i> operon and the <i>gap-1</i> gene. HexR negatively regulates expression of these gene targets, but not of itself. <b>3B</b>: The HexR regulon. HexR gene targets are involved in the glucose phosphorylative and Entner-Doudoroff pathways in <i>P</i>. <i>fluorescens</i>. Glk: glucokinase; Zwf: glucose 6-P dehydrogenase; Pgl: 6-phosphogluconolactonase; Edd: 6-phosphogluconate dehydratase; Gap-1: glyceraldehyde 3-phosphate dehydrogenase; the blue and light blue stars indicate activation of the glucose transport system, which is positively regulated by the transcriptional regulators GltR2 and GltS. <b>3C</b>: <i>zwf</i>, <i>edd</i>, and <i>gap</i> gene expression in glucose, <b>3D</b>: in glycerol, <b>3E</b>: in pyruvate and <b>3F</b>: in acetate in the <i>hexR</i> mutant background relative to WT (qRT-PCR data). <b>3G</b>: <i>hexR</i> promoter activity in SBW25 Δ<i>hexR</i> relative to WT, determined by β-gal assays tested in glucose, glycerol, pyruvate and acetate conditions. <b>3H</b>: SBW25 <i>hexR</i> gene expression determined by qRT-PCR after media exchange and 30 min growth in glucose, pyruvate or Root Solution (RS; media without carbon sources, used as a negative control).</p

RccR controls expression of pyruvate metabolism, gluconeogenesis and the glyoxylate shunt.

<p><b>5A-C</b>: RccR gene target expression determined by qRT-PCR. Data are shown for SBW25 <i>ΔrccR</i> relative to WT in <b>5A</b>: glucose media, <b>5B</b>: glycerol media, <b>5C</b>: pyruvate media, and <b>5D</b>: acetate media. <b>5E</b>: <i>rccR</i> promoter activity determined by β-gal assay in glucose, glycerol, pyruvate and acetate media conditions. Data are shown for the SBW25 <i>ΔrccR</i> background relative to WT. <b>5F</b>: SBW25 <i>rccR</i> gene expression determined by qRT-PCR after media exchange and 30 min growth in glucose, glycerol, pyruvate, acetate or Root Solution (RS; media without carbon sources, used as a negative control).</p

RccR binds the DNA consensus binding site of its targets.

<p><b>7A</b>: SPR experiments measuring the biomolecular interactions between the RccR protein and indicated DNA consensus sequences. Percentage of normalized response (%Rmax) of RccR (1μM and 0.1 μM concentrations) binding the consensus sequences found by MEME and manual sequence analysis alongside a random sequence DNA control. %Rmax indicates the experimental RccR binding values (Response registered from the SPR machine) normalized on the maximal response (R_max) that can be potentially reached when all ligand binding sites (DNA) are occupied by the analyte (RccR protein). <b>7B</b>: Sensorgrams (up) and fitting (down) curves showing RccR affinity to <i>aceE</i>, <i>aceA</i> and <i>rccR</i> consensus sequences.</p

Mapped reads from the RccR ChIP-seq experiment.

<p><b>4A-H</b>: Locations of genes and operons of interest are shown below each peak. Blue arrows indicate the direction of gene transcription and <i>PFLU</i> gene numbers are indicated in each case. Relative scales are indicated for each panel as well as the gene position in the SBW25 genome. Green and red peaks denote the SBW25 WT datasets, while blue and black show data for the <i>ΔrccR</i> mutant strain. Green and black lines indicate bacterial growth in glycerol, while red and blue indicate bacterial growth in pyruvate.</p

RccR binds the 28bp and the 15bp binding sites.

<p><b>8A</b>: DNaseI footprinting panel of RccR on <i>rccR</i>, <i>aceA</i>, <i>aceE</i> promoters. Radiolabelled promoter probes were incubated with increasing concentrations of purified RccR-His (0, 10, 20, 40, 80, 160 nM of RccR-His from left to right in each panel) before DNaseI digestion and DNA purification. Recovered DNA fragments were subjected to electrophoretic separation along with a Maxam and Gilbert G+A sequence reaction ladder (leftmost lane of each autoradiograph). On the left of each autoradiograph, a schematic representation of the genomic region is reported, with symbols as follows: block arrow represents the coding sequence, bent arrow represents the transcriptional start site identified in this study (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006839#pgen.1006839.s003" target="_blank">S3 Fig</a>), while black box indicates the -10 promoter element. Protected regions are highlighted by a black box on the right of each autoradiograph, while DNaseI hypersensitive sites are evidenced by black arrowheads. <b>8B</b>: mapping of the RccR binding sites on the <i>rccR</i>, <i>aceA</i> and <i>aceE</i> promoter regions. Arrowheads denote hypersensitive sites, protected regions are included in open boxes, and conserved pseudopalindromic sequences are highlighted in light grey. Bent arrow indicates the transcriptional start site identified in this study (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006839#pgen.1006839.s003" target="_blank">S3 Fig</a>) and the first transcribed nucleotide is in bold.</p

RccR has two related, pseudo-palindromic binding sequences.

<p><b>6A</b>: The predicted 28 bp RccR DNA-binding site identified by MEME analysis. This consensus is generated from the sequences identified in each RccR binding region, including the binding site located 292 bp after the <i>pckA</i> start codon (indicated with an *). The relative p- values of each RccR binding sites is indicated alongside the name of the RccR gene target in each case. The manually-identified 29 bp site upstream of <i>pckA</i> is also shown. <b>6B</b>: The predicted 15 bp RccR DNA-binding site identified by MEME analysis. The sequences found in the upstream regions of <i>aceE</i> and <i>rccR</i> are indicated with the relative p-values of each. The <i>aceE</i> upstream region contains two slightly different RccR binding sites 68 bp apart (TGTAGTTTTACTACT and TGTAGTAAAACTACA), both of which were used to generate the consensus sequence.</p