The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

The growth of the soil bacterium Pseudomonas putida KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(glpD-gfp) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of the glp genes. Either deleting glpR (encoding the G3P-responsive transcriptional repressor that controls the expression of the glpFKRD gene cluster) or altering G3P formation (by overexpressing glpK, encoding glycerol kinase) abolished the bimodal glpD expression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision of glpR in trans restored the phenotypes lost in the ΔglpR mutant. The prolonged nongrowth regime of P. putida on glycerol could thus be traced to the regulatory device controlling the transcription of the glp genes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states

Molecular Longitudinal Tracking of Mycobacterium abscessus spp. during Chronic Infection of the Human Lung

<div><p>The <i>Mycobacterium abscessus</i> complex is an emerging cause of chronic pulmonary infection in patients with underlying lung disease. The <i>M. abscessus</i> complex is regarded as an environmental pathogen but its molecular adaptation to the human lung during long-term infection is poorly understood. Here we carried out a longitudinal molecular epidemiological analysis of 178 <i>M. abscessus</i> spp. isolates obtained from 10 cystic fibrosis (CF) and 2 non CF patients over a 13 year period. Multi-locus sequence and molecular typing analysis revealed that 11 of 12 patients were persistently colonized with the same genotype during the course of the infection while replacement of a <i>M. abscessus sensu stricto</i> strain with a <i>Mycobacterium massiliense</i> strain was observed for a single patient. Of note, several patients including a pair of siblings were colonized with closely-related strains consistent with intra-familial transmission or a common infection reservoir. In general, a switch from smooth to rough colony morphology was observed during the course of long-term infection, which in some cases correlated with an increasing severity of clinical symptoms. To examine evolution during long-term infection of the CF lung we compared the genome sequences of 6 sequential isolates of <i>Mycobacterium bolletii</i> obtained from a single patient over an 11 year period, revealing a heterogeneous clonal infecting population with mutations in regulators controlling the expression of virulence factors and complex lipids. Taken together, these data provide new insights into the epidemiology of <i>M. abscessus</i> spp. during long-term infection of the CF lung, and the molecular transition from saprophytic organism to human pathogen.</p></div

Biochemistry, genetics and biotechnology of glycerol utilization in Pseudomonas species

Indexación: Scopus.The use of renewable waste feedstocks is an environment-friendly choice contributing to the reduction of waste treatment costs and increasing the economic value of industrial by-products. Glycerol (1,2,3-propanetriol), a simple polyol compound widely distributed in biological systems, constitutes a prime example of a relatively cheap and readily available substrate to be used in bioprocesses. Extensively exploited as an ingredient in the food and pharmaceutical industries, glycerol is also the main by-product of biodiesel production, which has resulted in a progressive drop in substrate price over the years. Consequently, glycerol has become an attractive substrate in biotechnology, and several chemical commodities currently produced from petroleum have been shown to be obtained from this polyol using whole-cell biocatalysts with both wild-type and engineered bacterial strains. Pseudomonas species, endowed with a versatile and rich metabolism, have been adopted for the conversion of glycerol into value-added products (ranging from simple molecules to structurally complex biopolymers, e.g. polyhydroxyalkanoates), and a number of metabolic engineering strategies have been deployed to increase the number of applications of glycerol as a cost-effective substrate. The unique genetic and metabolic features of glycerol-grown Pseudomonas are presented in this review, along with relevant examples of bioprocesses based on this substrate – and the synthetic biology and metabolic engineering strategies implemented in bacteria of this genus aimed at glycerol valorization.https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.1340

Second-generation functionalized mediumchain- length polyhydroxyalkanoates: the gateway to high-value bioplastic applications

Polyhydroxyalkanoates (PHAs) are biodegradable biocompatible polyesters, which accumulate as granulesin the cytoplasm of many bacteria under unbalanced growth conditions. Medium-chain-length PHAs (mcl-PHAs), characterizedby C6-C14 branched monomer chains and typically produced by Pseudomonas species, are promising thermoelastomers,as they can be further modified by introducing functional groups in the side chains. Functionalized PHAs areobtained either by feeding structurally related substrates processed through the &beta;-oxidation pathway, or using specificstrains able to transform sugars or glycerol into unsaturated PHA by de novo fatty-acid biosynthesis. Functionalized mcl-PHAs provide modified mechanical and thermal properties, and consequently have new processing requirements andhighly diverse potential applications in emergent fields such as biomedicine. However, process development and sampleavailability are limited due to the toxicity of some precursors and still low productivity, which hinder investigation. Conversely,improved mutant strains designed through systems biology approaches and cofeeding with low-cost substratesmay contribute to the widespread application of these biopolymers. This review focuses on recent developments in theproduction of functionalized mcl-PHAs, placing particular emphasis on strain and bioprocess design for cost-effectiveproduction. [Int Microbiol 2013; 16(1):1-15

Chemical interplay and complementary adaptative strategies toggle bacterial antagonism and co-existence.

Bacterial communities are in a continuous adaptive and evolutionary race for survival. In this work we expand our knowledge on the chemical interplay and specific mutations that modulate the transition from antagonism to co-existence between two plant-beneficial bacteria, Pseudomonas chlororaphis PCL1606 and Bacillus amyloliquefaciens FZB42. We reveal that the bacteriostatic activity of bacillaene produced by Bacillus relies on an interaction with the protein elongation factor FusA of P. chlororaphis and how mutations in this protein lead to tolerance to bacillaene and other protein translation inhibitors. Additionally, we describe how the unspecific tolerance of B. amyloliquefaciens to antimicrobials associated with mutations in the glycerol kinase GlpK is provoked by a decrease of Bacillus cell membrane permeability, among other pleiotropic responses. We conclude that nutrient specialization and mutations in basic biological functions are bacterial adaptive dynamics that lead to the coexistence of two primary competitive bacterial species rather than their mutual eradication

E Unibus Plurum: Genomic Analysis of an Experimentally Evolved Polymorphism in Escherichia coli

Microbial populations founded by a single clone and propagated under resource limitation can become polymorphic. We sought to elucidate genetic mechanisms whereby a polymorphism evolved in Escherichia coli under glucose limitation and persisted because of cross-feeding among multiple adaptive clones. Apart from a 29 kb deletion in the dominant clone, no large-scale genomic changes distinguished evolved clones from their common ancestor. Using transcriptional profiling on co-evolved clones cultured separately under glucose-limitation we identified 180 genes significantly altered in expression relative to the common ancestor grown under similar conditions. Ninety of these were similarly expressed in all clones, and many of the genes affected (e.g., mglBAC, mglD, and lamB) are in operons coordinately regulated by CRP and/or rpoS. While the remaining significant expression differences were clone-specific, 93% were exhibited by the majority clone, many of which are controlled by global regulators, CRP and CpxR. When transcriptional profiling was performed on adaptive clones cultured together, many expression differences that distinguished the majority clone cultured in isolation were absent, suggesting that CpxR may be activated by overflow metabolites removed by cross-feeding strains in co-culture. Relative to their common ancestor, shared expression differences among adaptive clones were partly attributable to early-arising shared mutations in the trans-acting global regulator, rpoS, and the cis-acting regulator, mglO. Gene expression differences that distinguished clones may in part be explained by mutations in trans-acting regulators malT and glpK, and in cis-acting sequences of acs. In the founder, a cis-regulatory mutation in acs (acetyl CoA synthetase) and a structural mutation in glpR (glycerol-3-phosphate repressor) likely favored evolution of specialists that thrive on overflow metabolites. Later-arising mutations that led to specialization emphasize the importance of compensatory rather than gain-of-function mutations in this system. Taken together, these findings underscore the importance of regulatory change, founder genotype, and the biotic environment in the adaptive evolution of microbes

Harnessing Interspecies Antagonism to Enhance Antibiotic Efficacy

Beyond genetically encoded mechanisms of resistance, the factors that contribute to antibiotic treatment failure within the host are poorly understood. Traditional susceptibility assays fail to account for extrinsic determinants of antibiotic susceptibility present during infection and are therefore poor predictors of treatment outcome. To maximize the reach of current therapeutics, we must develop a more sophisticated understanding of antibiotic efficacy in the infection environment. Here we demonstrate that interspecies interactions between two important opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, alters S. aureus response to antibiotics. We show that the P. aeruginosa-produced endopeptidase LasA potentiates lysis of S. aureus by vancomycin, rhamnolipids facilitate proton-motive force-independent aminoglycoside uptake, and that small molecule 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) induces multidrug tolerance in S. aureus through respiratory inhibition and reduction of cellular ATP. We further demonstrate rhamnolipid-mediated potentiation of aminoglycoside uptake and killing of S. aureus restores susceptibility to otherwise tolerant persister, biofilm, small colony variant, anaerobic, and resistant S. aureus populations. Furthermore, bacterial pathogens that replicate within the intracellular niche are protected from antibiotics that cannot penetrate the eukaryotic membrane. Identifying and disrupting the pathways used by these pathogens to modify the intracellular niche in order to survive is an alternative strategy for limiting bacterial proliferation. Here, we use Francisella tularensis as a model intracellular bacterial pathogen to identify and describe the bacterial metabolic pathways and host-derived nutrients necessary for intracellular and in vivo growth. These findings reveal potential new therapeutic targets for disrupting bacterial nutrient acquisition that may be broadly applicable for treating other important intracellular pathogens. Overall, the findings presented here suggest that antibiotic susceptibility is contingent on a multitude of factors including interspecies interaction and the physiological replicative niche. Further elucidation of key antibiotic susceptibility determinants in vivo, as well as of strategies to overcome barriers to antibiotic efficacy may lead to a more holistic and personalized approach to therapy that will aid in the resolution of persistent infection.Doctor of Philosoph

Bacterial Lifestyle in a Deep-sea Hydrothermal Vent Chimney Revealed by the Genome Sequence of the Thermophilic Bacterium Deferribacter desulfuricans SSM1

The complete genome sequence of the thermophilic sulphur-reducing bacterium, Deferribacter desulfuricans SMM1, isolated from a hydrothermal vent chimney has been determined. The genome comprises a single circular chromosome of 2 234 389 bp and a megaplasmid of 308 544 bp. Many genes encoded in the genome are most similar to the genes of sulphur- or sulphate-reducing bacterial species within Deltaproteobacteria. The reconstructed central metabolisms showed a heterotrophic lifestyle primarily driven by C1 to C3 organics, e.g. formate, acetate, and pyruvate, and also suggested that the inability of autotrophy via a reductive tricarboxylic acid cycle may be due to the lack of ATP-dependent citrate lyase. In addition, the genome encodes numerous genes for chemoreceptors, chemotaxis-like systems, and signal transduction machineries. These signalling networks may be linked to this bacterium's versatile energy metabolisms and may provide ecophysiological advantages for D. desulfuricans SSM1 thriving in the physically and chemically fluctuating environments near hydrothermal vents. This is the first genome sequence from the phylum Deferribacteres

\u3ci\u3eEx Uno Plures\u3c/i\u3e: Clonal Reinforcement Drives Evolution of a Simple Microbial Community

A major goal of genetics is to define the relationship between phenotype and genotype, while a major goal of ecology is to identify the rules that govern community assembly. Achieving these goals by analyzing natural systems can be difficult, as selective pressures create dynamic fitness landscapes that vary in both space and time. Laboratory experimental evolution offers the benefit of controlling variables that shape fitness landscapes, helping to achieve both goals. We previously showed that a clonal population of E. coli experimentally evolved under continuous glucose limitation gives rise to a genetically diverse community consisting of one clone, CV103, that best scavenges but incompletely utilizes the limiting resource, and others, CV101 and CV116, that consume its overflow metabolites. Because this community can be disassembled and reassembled, and involves cooperative interactions that are stable over time, its genetic diversity is sustained by clonal reinforcement rather than by clonal interference. To understand the genetic factors that produce this outcome, and to illuminate the community’s underlying physiology, we sequenced the genomes of ancestral and evolved clones. We identified ancestral mutations in intermediary metabolism that may have predisposed the evolution of metabolic interdependence. Phylogenetic reconstruction indicates that the lineages that gave rise to this community diverged early, as CV103 shares only one Single Nucleotide Polymorphism with the other evolved clones. Underlying CV103’s phenotype we identified a set of mutations that likely enhance glucose scavenging and maintain redox balance, but may do so at the expense of carbon excreted in overflow metabolites. Because these overflow metabolites serve as growth substrates that are differentially accessible to the other community members, and because the scavenging lineage shares only one SNP with these other clones, we conclude that this lineage likely served as an ‘‘engine’’ generating diversity by creating new metabolic niches, but not the occupants themselves