Evolutionary resurrection of flagellar motility via rewiring of the nitrogen regulation system

A central process in evolution is the recruitment of genes to regulatory networks. We engineered immotile strains of the bacterium Pseudomonas fluorescens that lack flagella due to deletion of the regulatory gene fleQ. Under strong selection for motility, these bacteria consistently regained flagella within 96 hours via a two-step evolutionary pathway. Step 1 mutations increase intracellular levels of phosphorylated NtrC, a distant homologue of FleQ, which begins to commandeer control of the fleQ regulon at the cost of disrupting nitrogen uptake and assimilation. Step 2 is a switch-of-function mutation that redirects NtrC away from nitrogen uptake and towards its novel function as a flagellar regulator. Our results demonstrate that natural selection can rapidly rewire regulatory networks in very few, repeatable mutational steps

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

Out of the Ashes: Remembrance and Reconstruction in Catholic Shanxi, 1900-Present

LEWI Conference Paper, Hong Kong 201

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

Positive selection inhibits gene mobilization and transfer in soil bacterial communities

Horizontal gene transfer (HGT) between bacterial lineages is a fundamental evolutionary process that accelerates adaptation. Sequence analyses show that conjugative plasmids are principal agents of HGT in natural communities. However, we lack understanding of how the ecology of bacterial communities and their environments affect the dynamics of plasmid-mediated gene mobilization and transfer. Here we show, in simple experimental soil bacterial communities containing a conjugative mercury resistance plasmid, the repeated, independent mobilization of transposon-borne genes from chromosome to plasmid, plasmid to chromosome and, in the absence of mercury selection, interspecific gene transfers from the chromosome of one species to the other via the plasmid. By reducing conjugation, positive selection for plasmid-encoded traits, like mercury resistance, can consequently inhibit HGT. Our results suggest that interspecific plasmid-mediated gene mobilization is most likely to occur in environments where plasmids are infectious, parasitic elements rather than those where plasmids are positively selected, beneficial elements

Defining the Pseudomonas Genus: Where Do We Draw the Line with Azotobacter?

The genus Pseudomonas has gone through many taxonomic revisions over the past 100 years, going from a very large and diverse group of bacteria to a smaller, more refined and ordered list having specific properties. The relationship of the Pseudomonas genus to Azotobacter vinelandii is examined using three genomic sequence-based methods. First, using 16S rRNA trees, it is shown that A. vinelandii groups within the Pseudomonas close to Pseudomonas aeruginosa. Genomes from other related organisms (Acinetobacter, Psychrobacter, and Cellvibrio) are outside the Pseudomonas cluster. Second, pan genome family trees based on conserved gene families also show A. vinelandii to be more closely related to Pseudomonas than other related organisms. Third, exhaustive BLAST comparisons demonstrate that the fraction of shared genes between A. vinelandii and Pseudomonas genomes is similar to that of Pseudomonas species with each other. The results of these different methods point to a high similarity between A. vinelandii and the Pseudomonas genus, suggesting that Azotobacter might actually be a Pseudomonas

A proteogenomic analysis of Shigella flexneri using 2D LC-MALDI TOF/TOF

<p>Abstract</p> <p>Background</p> <p>New strategies for high-throughput sequencing are constantly appearing, leading to a great increase in the number of completely sequenced genomes. Unfortunately, computational genome annotation is out of step with this progress. Thus, the accurate annotation of these genomes has become a bottleneck of knowledge acquisition.</p> <p>Results</p> <p>We exploited a proteogenomic approach to improve conventional genome annotation by integrating proteomic data with genomic information. Using <it>Shigella flexneri </it>2a as a model, we identified total 823 proteins, including 187 hypothetical proteins. Among them, three annotated ORFs were extended upstream through comprehensive analysis against an in-house N-terminal extension database. Two genes, which could not be translated to their full length because of stop codon 'mutations' induced by genome sequencing errors, were revised and annotated as fully functional genes. Above all, seven new ORFs were discovered, which were not predicted in <it>S. flexneri </it>2a str.301 by any other annotation approaches. The transcripts of four novel ORFs were confirmed by RT-PCR assay. Additionally, most of these novel ORFs were overlapping genes, some even nested within the coding region of other known genes.</p> <p>Conclusions</p> <p>Our findings demonstrate that current <it>Shigella </it>genome annotation methods are not perfect and need to be improved. Apart from the validation of predicted genes at the protein level, the additional features of proteogenomic tools include revision of annotation errors and discovery of novel ORFs. The complementary dataset could provide more targets for those interested in <it>Shigella </it>to perform functional studies.</p