Probing the active site of homoserine trans-succinylase

AbstractHomoserine trans-succinylase is the first enzyme in methionine biosynthesis of Escherichia coli and catalyzes the activation of homoserine via a succinylation reaction. The in vivo activity of this enzyme is subject to tight regulation by several mechanisms, including repression and activation of gene expression, feedback inhibition, temperature regulation and proteolysis. This complex regulation reflects the key role of this enzyme in bacterial metabolism. Here, we demonstrate – using proteomics and high-resolution mass spectrometry – that succinyl is covalently bound to one of the two adjacent lysine residues at positions 45 and 46. Replacing these lysine residues by alanine abolished the enzymatic activity. These findings position the lysine residues, one of which is conserved, at the active site

Transfer of noncoding DNA drives regulatory rewiring in bacteria

Understanding the mechanisms that generate variation is a common pursuit unifying the life sciences. Bacteria represent an especially striking puzzle, because closely related strains possess radically different metabolic and ecological capabilities. Differences in protein repertoire arising from gene transfer are currently considered the primary mechanism underlying phenotypic plasticity in bacteria. Although bacterial coding plasticity has been extensively studied in previous decades, little is known about the role that regulatory plasticity plays in bacterial evolution. Here, we show that bacterial genes can rapidly shift between multiple regulatory modes by acquiring functionally divergent nonhomologous promoter regions. Through analysis of 270,000 regulatory regions across 247 genomes, we demonstrate that regulatory “switching” to nonhomologous alternatives is ubiquitous, occurring across the bacterial domain. Using comparative transcriptomics, we show that at least 16% of the expression divergence between Escherichia coli strains can be explained by this regulatory switching. Further, using an oligonucleotide regulatory library, we establish that switching affects bacterial promoter architecture. We provide evidence that regulatory switching can occur through horizontal regulatory transfer, which allows regulatory regions to move across strains, and even genera, independently from the genes they regulate. Finally, by experimentally characterizing the fitness effect of a regulatory transfer on a pathogenic E. coli strain, we demonstrate that regulatory switching elicits important phenotypic consequences. Taken together, our findings expose previously unappreciated regulatory plasticity in bacteria and provide a gateway for understanding bacterial phenotypic variation and adaptation.National Science Foundation (U.S.) (Grant DEB-0936234

A Photolyase-Like Protein from Agrobacterium tumefaciens with an Iron-Sulfur Cluster

Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD) photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD) as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF) as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster

Host Imprints on Bacterial Genomes—Rapid, Divergent Evolution in Individual Patients

Bacteria lose or gain genetic material and through selection, new variants become fixed in the population. Here we provide the first, genome-wide example of a single bacterial strain's evolution in different deliberately colonized patients and the surprising insight that hosts appear to personalize their microflora. By first obtaining the complete genome sequence of the prototype asymptomatic bacteriuria strain E. coli 83972 and then resequencing its descendants after therapeutic bladder colonization of different patients, we identified 34 mutations, which affected metabolic and virulence-related genes. Further transcriptome and proteome analysis proved that these genome changes altered bacterial gene expression resulting in unique adaptation patterns in each patient. Our results provide evidence that, in addition to stochastic events, adaptive bacterial evolution is driven by individual host environments. Ongoing loss of gene function supports the hypothesis that evolution towards commensalism rather than virulence is favored during asymptomatic bladder colonization

AGEs Secreted by Bacteria Are Involved in the Inflammatory Response

Advanced Glycated End Products (AGEs) are formed by non-enzymatic protein glycation and are implicated in several physiological aspects including cell aging and diseases. Recent data indicate that bacteria – although short lived – produce, metabolize and accumulate AGEs. Here we show that Escherichia coli cells secret AGEs by the energy-dependent efflux pump systems. Moreover, we show that in the presence of these AGEs there is an upshift of pro-inflammatory cytokins by mammalian cells. Thus, we propose that secretion of AGEs by bacteria is a novel avenue of bacterial-induced inflammation which is potentially important in the pathophysiology of bacterial infections. Moreover, the sensing of AGEs by the host cells may constitute a warning system for the presence of bacteria

The urgent need for microbiology literacy in society

Contains fulltext : 204933.pdf (publisher's version ) (Open Access

Microbiological sciences: a European perspective

Les ciències microbiològiques, que semblava que després del descobriment dels antibiòtics haguessin quedat obsoletes, es troben ara entre les ciències biològiques més importants. Aquest ressorgiment de la microbiologia es deu en gran part a l'emergència i ràpida propagació de la resistència als antibiòtics. El fet que molts dels bacteris patogènics siguin ara resistents a la majoria dels antibiòtics dóna lloc a la necessitat crítica i urgent d'entendre les bases de la virulència bacteriana per poder tractar i prevenir les malalties. Una altra millora important ha estat la disponibilitat de tecnologies de genòmica i bases de dades que permeten explorar una gran diversitat de genomes bacterians i utilitzar-los per a interessants desenvolupaments tecnològics. Aquests dos aspectes de la microbiologia moderna seran discutits amb èmfasi a la perspectiva Europea. També es descriuran el desenvolupament i les activitats de la Federació Europea de Societats Microbiològiques (FEMS), a la qual ara s'uneixen els esforços de 47 societats microbiològiques.Microbiological sciences, which appeared to be obsolete after the discovery of antibiotics, are now ranked among the most important biological sciences. The revival of microbiology has been due mainly to the emergence and rapid spread of antibiotic resistance. The fact that many pathogenic bacteria are now resistant to most of the antibiotics currently in use has resulted in a critical and urgent need to understand the basis of bacterial virulence in order to prevent and treat the respective diseases. Another important development has been the availability of genomic technologies and databases, which have made it possible to explore the vast biodiversity of bacterial genomes and to use the information for biotechnological applications. This article discusses these two aspects of modern microbiology, with special focus on the European perspective. In addition, the development and activities of the Federation of European Microbiological Societies (FEMS), which unites the efforts of 47 microbiological societies, is described