Adaptive remodeling of the bacterial proteome by specific ribosomal modification regulates Pseudomonas infection and niche colonisation

Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome

A Minimal Threshold of c-di-GMP Is Essential for Fruiting Body Formation and Sporulation in Myxococcus xanthus

Generally, the second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) regulates the switch between motile and sessile lifestyles in bacteria. Here, we show that c-di-GMP is an essential regulator of multicellular development in the social bacterium Myxococcus xanthus. In response to starvation, M. xanthus initiates a developmental program that culminates in formation of spore-filled fruiting bodies. We show that c-di-GMP accumulates at elevated levels during development and that this increase is essential for completion of development whereas excess c-di-GMP does not interfere with development. MXAN3735 (renamed DmxB) is identified as a diguanylate cyclase that only functions during development and is responsible for this increased c-di-GMP accumulation. DmxB synthesis is induced in response to starvation, thereby restricting DmxB activity to development. DmxB is essential for development and functions downstream of the Dif chemosensory system to stimulate exopolysaccharide accumulation by inducing transcription of a subset of the genes encoding proteins involved in exopolysaccharide synthesis. The developmental defects in the dmxB mutant are non-cell autonomous and rescued by co-development with a strain proficient in exopolysaccharide synthesis, suggesting reduced exopolysaccharide accumulation as the causative defect in this mutant. The NtrC-like transcriptional regulator EpsI/Nla24, which is required for exopolysaccharide accumulation, is identified as a c-diGMP receptor, and thus a putative target for DmxB generated c-di-GMP. Because DmxB can be—at least partially—functionally replaced by a heterologous diguanylate cyclase, these results altogether suggest a model in which a minimum threshold level of c-di-GMP is essential for the successful completion of multicellular development in M. xanthus

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

We're in this Together: Sensation of the Host Cell Environment by Endosymbiotic Bacteria

Bacteria inhabit diverse environments, including the inside of eukaryotic cells. While a bacterial invader may initially act as a parasite or pathogen, a subsequent mutualistic relationship can emerge in which the endosymbiotic bacteria and their host share metabolites. While the environment of the host cell provides improved stability when compared to an extracellular environment, the endosymbiont population must still cope with changing conditions, including variable nutrient concentrations, the host cell cycle, host developmental programs, and host genetic variation. Furthermore, the eukaryotic host can deploy mechanisms actively preventing a bacterial return to a pathogenic state. Many endosymbionts are likely to use two-component systems (TCSs) to sense their surroundings, and expanded genomic studies of endosymbionts should reveal how TCSs may promote bacterial integration with a host cell. We suggest that studying TCS maintenance or loss may be informative about the evolutionary pathway taken toward endosymbiosis, or even toward endosymbiont-to-organelle conversion.Peer reviewe

Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion

Pseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal c-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here we identified an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase WarB. We established that WarA binds to c-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bi-functional Escherichia coli LPS O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.BBSRC & Wellcome Trus

Growth curves for SBW25 WT and <i>ΔrccR</i>, <i>ΔhexR</i>, and <i>ΔrccRΔhexR</i> mutants.

<p><b>2A</b>: Growth was measured in KB and <b>2B</b>: LB rich media as well as in <b>2C</b>: M9 0.4% glucose, <b>2D</b>: M9 0.4% glycerol, <b>2E</b>: M9 0.4% pyruvate, <b>2F</b>: M9 0.4% acetate and <b>2G</b>: M9 0.4% succinate. Marked differences in growth rate were seen between WT and <i>ΔrccR</i> in glucose (<b>C</b>) and glycerol (<b>D</b>), and between WT and <i>ΔhexR</i> mutants in pyruvate (<b>E</b>), acetate (<b>F</b>), and succinate (<b>G</b>). Experiments were repeated at least three times independently and a representative plot is shown in each case.</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

Screening for the RccR effector.

<p><b>9A</b>: Percentage of normalized response (%Rmax) for RccR binding to the <i>rccR</i>, <b>9B</b>: <i>aceA</i> and <b>9C</b>: <i>aceE</i> consensus sequences in the presence of KDPG (effector) and PEP (negative control) at different concentrations (1-10-100 μM).</p

A model for RccR regulation of primary carbon metabolism.

<p>The figure shows a schematic representation of the metabolic pathways of glucose, glycerol, pyruvate and acetate through the Krebs cycle and the glyoxylate shunt. The protein products of the RccR gene targets are shown: PntAA/PFLU0112/B are subunits of the NAD(P) transhydrogenase membrane protein complex; PckA: phosphoenolpyruvate carboxykinase; AceE/F: pyruvate dehydrogenase subunits; Gap: glyceraldehyde-3-phosphate dehydrogenase; AceA: isocitrate lyase; GlcB: malate synthase G. RccR-regulated carbon transitions are marked in red. HexR-regulated carbon transitions are marked in blue.</p