Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons.

Hippocampal GABAergic interneurons are crucial for cortical network function and have been implicated in psychiatric disorders. We show here that Neuregulin 3 (Nrg3), a relatively little investigated low-affinity ligand, is a functionally dominant interaction partner of ErbB4 in parvalbumin-positive (PV) interneurons. Nrg3 and ErbB4 are located pre- and postsynaptically, respectively, in excitatory synapses on PV interneurons in vivo Additionally, we show that ablation of Nrg3 results in a similar phenotype as the one described for ErbB4 ablation, including reduced excitatory synapse numbers on PV interneurons, altered short-term plasticity, and disinhibition of the hippocampal network. In culture, presynaptic Nrg3 increases excitatory synapse numbers on ErbB4+ interneurons and affects short-term plasticity. Nrg3 mutant neurons are poor donors of presynaptic terminals in the presence of competing neurons that produce recombinant Nrg3, and this bias requires postsynaptic ErbB4 but not ErbB4 kinase activity. Furthermore, when presented by non-neuronal cells, Nrg3 induces postsynaptic membrane specialization. Our data indicate that Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4+ interneurons.

The Proteomic Response of Bacillus pumilus Cells to Glucose Starvation.

Since starvation for carbon sources is a common condition for bacteria in nature and it can also occur in industrial fermentation processes due to mixing zones, knowledge about the response of cells to carbon starvation is beneficial. The preferred carbon source for bacilli is glucose. The response of Bacillus pumilus cells to glucose starvation using metabolic labeling and quantitative proteomics was analyzed. Glucose starvation led to an extensive reprogramming of the protein expression pattern in B. pumilus. The amounts of proteins of the central carbon metabolic pathways (glycolysis and TCC) remained stable in starving cells. Proteins for gluconeogenesis were found in higher amounts during starvation. Furthermore, many proteins involved in acquisition and usage of alternative carbon sources were present in elevated amounts in starving cells. Enzymes for fatty acid degradation and proteases and peptidases were also found in higher abundance when cells entered stationary phase. Among the proteins found in lower amounts were many enzymes involved in amino acid and nucleotide synthesis and several NRPS and PKS proteins.

A proteomic analysis of ferulic acid metabolism in Amycolatopsis sp. ATCC 39116.

The pseudonocardiate Amycolatopsis sp. ATCC 39116 is used for the biotechnical production of natural vanillin from ferulic acid. Our laboratory has performed genetic modifications of this strain previously, but there are still many gaps in our knowledge regarding its vanillin tolerance and the general metabolism. We performed cultivations with this bacterium and compared the proteomes of stationary phase cells before ferulic acid feeding with those during ferulic acid feeding. Thereby, we identified 143 differently expressed proteins. Deletion mutants were constructed and characterized to analyze the function of nine corresponding genes. Using these mutants, we identified an active ferulic acid ß-oxidation pathway and the enzymes which constitute this pathway. A combined deletion mutant in which the ß-oxidation as well as non-ß-oxidation pathways of ferulic acid degradation were deleted was unable to grow on ferulic acid as the sole source of carbon and energy. This mutant differs from the single deletion mutants and was unable to grow on ferulic acid. Furthermore, we showed that the non-ß-oxidation pathway is involved in caffeic acid degradation; however, its deletion is complemented even in the double deletion mutant. This shows that both pathways can complement each other. The ß-oxidation deletion mutant produced significantly reduced amounts of vanillic acid (0.12 instead of 0.35 g/l). Therefore, the resulting mutant could be used as an improved production strain. The quinone oxidoreductase deletion mutant (ΔytfG) degraded ferulic acid slower at first but produced comparable amounts of vanillin and significantly less vanillyl alcohol when compared to the parent strain.

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors.

Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phase-specific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests their phase specific function. The distinct temporal or spatial presence of such proteins might have important implications for functional assignments in the future or for use as life-stage specific markers for pathogen analysis.

Bacillus pumilus KatX2 confers enhanced hydrogen peroxide resistance to a Bacillus subtilis PkatA::katX2 mutant strain.

BACKGROUND: Bacillus pumilus cells exhibit a significantly higher resistance to hydrogen peroxide compared to closely related Bacilli like Bacillus subtilis. RESULTS: In this study we analyzed features of the catalase KatX2 of B. pumilus as one of the most important parts of the cellular response to hydrogen peroxide. KatX2, the vegetative catalase expressed in B. pumilus, was compared to the vegetative catalase KatA of B. subtilis. Data of our study demonstrate that B. pumilus can degrade toxic concentrations of hydrogen peroxide faster than B. subtilis. By replacing B. subtilis katA gene by katX2 we could significantly enhance its resistance to H2O2 and its potential to eliminate this toxic compound. Mutant cells showed a 1.5- to 2-fold higher survival to toxic concentrations of hydrogen peroxide compared to wild type cells. Furthermore, we found reversible but also irreversible oxidations of the KatX2 protein which, in contrast to KatA, contains several cysteine residues. CONCLUSIONS: Our study indicates that the catalase KatX2 plays a major role in the increased resistance of B. pumilus to oxidative stress caused by hydrogen peroxide. Resistance to hydrogen peroxide of other Bacilli can be enhanced by exchanging the native catalase in the cells with katX2.

Oligo(cis-1,4-isoprene) aldehyde-oxidizing dehydrogenases of the rubber-degrading bacterium Gordonia polyisoprenivorans VH2.

The actinomycete Gordonia polyisoprenivorans strain VH2 is well-known for its ability to efficiently degrade and catabolize natural rubber [poly(cis-1,4-isoprene)]. Recently, a pathway for the catabolism of rubber by strain VH2 was postulated based on genomic data and the analysis of mutants (Hiessl et al. in Appl Environ Microbiol 78:2874-2887, 2012). To further elucidate the degradation pathway of poly(cis-1,4-isoprene), 2-dimensional-polyacrylamide gel electrophoresis was performed. The analysis of the identified protein spots by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry confirmed the postulated intracellular pathway suggesting a degradation of rubber via ß-oxidation. In addition, other valuable information on rubber catabolism of G. polyisoprenivorans strain VH2 (e.g. oxidative stress response) was provided. Identified proteins, which were more abundant in cells grown with rubber than in cells grown with propionate, implied a putative long-chain acyl-CoA-dehydrogenase, a 3-ketoacyl-CoA-thiolase, and an aldehyde dehydrogenase. The amino acid sequence of the latter showed a high similarity towards geranial dehydrogenases. The expression of the corresponding gene was upregulated > 10-fold under poly(cis-1,4-isoprene)-degrading conditions. The putative geranial dehydrogenase and a homolog were purified and used for enzyme assays. Deletion mutants for five aldehyde dehydrogenases were generated, and growth with poly(cis-1,4-isoprene) was investigated. While none of the mutants had an altered phenotype regarding growth with poly(cis-1,4-isoprene) as sole carbon and energy source, purified aldehyde dehydrogenases were able to catalyze the oxidation of oligoisoprene aldehydes indicating an involvement in rubber degradation.

CodY regulates expression of the Bacillus subtilis extracellular proteases Vpr and Mpr.

UNLABELLED: CodY is a global transcriptional regulator in low-G+C Gram-positive bacteria that is responsive to GTP and branched-chain amino acids. By interacting with its two cofactors, it is able to sense the nutritional and energetic status of the cell and respond by regulating expression of adaptive genetic programs. In Bacillus subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. In this study, we demonstrated that expression of two extracellular proteases, Vpr and Mpr, is negatively controlled by CodY. By gel mobility shift and DNase I footprinting assays, we showed that CodY binds to the regulatory regions of both genes, in the vicinity of their transcription start points. The mpr gene is also characterized by the presence of a second, higher-affinity CodY-binding site located at the beginning of its coding sequence. Using strains carrying vpr- or mpr-lacZ transcriptional fusions in which CodY-binding sites were mutated, we demonstrated that repression of both protease genes is due to the direct effect by CodY and that the mpr internal site is required for regulation. The vpr promoter is a rare example of a sigma H-dependent promoter that is regulated by CodY. In a codY null mutant, Vpr became one of the more abundant proteins of the B. subtilis exoproteome. IMPORTANCE: CodY is a global transcriptional regulator of metabolism and virulence in low-G+C Gram-positive bacteria. In B. subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. However, no role for B. subtilis CodY in regulating expression of extracellular proteases has been established to date. In this work, we demonstrate that by binding to the regulatory regions of the corresponding genes, B. subtilis CodY negatively controls expression of Vpr and Mpr, two extracellular proteases. Thus, in B. subtilis, CodY can now be seen to regulate the entire protein utilization pathway.

High-resolution proteome maps of Bacillus licheniformis cells growing in minimal medium.

Bacillus licheniformis is an important host for the industrial production of enzymes mainly because of its ability to secrete large amounts of protein. We analyzed the proteome of B. licheniformis cells growing in a minimal medium. Beside the cytosolic proteome, the membrane and the extracellular proteome were studied. We could identify 1470 proteins; 1168 proteins were classified as cytosolic proteins, 195 proteins with membrane-spanning domains were classified as membrane proteins, and 107 proteins, with either putative signals peptides or flagellin-like sequences, were classified as secreted proteins. The identified proteins were grouped into functional categories and used to reconstruct cellular functions and metabolic pathways of growing B. licheniformis cells. The largest group was proteins with functions in basic metabolic pathways such as carbon metabolism, amino acid and nucleotide synthesis and synthesis of fatty acids and cofactors. Many proteins detected were involved in DNA replication, transcription, and translation. Furthermore, a high number of proteins employed in the transport of a wide variety of compounds were found to be expressed in the cells. All MS data have been deposited in the ProteomeXchange with identifier PXD000791 (http://proteomecentral.proteomexchange.org/dataset/PXD000791).

Influence of root exudates on the extracellular proteome of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42.

Proteins secreted by Bacillus amyloliquefaciens FZB42, a root-associated plant growth-promoting rhizobacterium, are thought to play an important role in the establishment of beneficial interactions with plants. To investigate the possible role of proteins in this process, extracellular proteome maps of B. amyloliquefaciens FZB42 during the late exponential and stationary growth phases were generated using 2D gel electrophoresis. Out of the 121 proteins identified by MALDI-TOF MS, 61 were predicted to contain secretion signals. A few of the others, bearing no signal peptide, have been described as elicitors of plant innate immunity, including flagellin proteins, cold-shock proteins and the elongation factor Tu, suggesting that B. amyloliquefaciens FZB42 protects plants against disease by eliciting innate immunity. Our reference maps were used to monitor bacterial responses to maize root exudates. Approximately 34 proteins were differentially secreted in response to root exudates during either the late exponential or stationary phase. These were mainly involved in nutrient utilization and transport. The protein with the highest fold change in the presence of maize root exudates during the late exponential growth phase was acetolactate synthase (AlsS), an enzyme involved in the synthesis of the volatile acetoin, known as an inducer of systemic resistance against plant pathogens and as a trigger of plant growth.

Metaproteomics of a gutless marine worm and its symbiotic microbial community reveal unusual pathways for carbon and energy use.

Low nutrient and energy availability has led to the evolution of numerous strategies for overcoming these limitations, of which symbiotic associations represent a key mechanism. Particularly striking are the associations between chemosynthetic bacteria and marine animals that thrive in nutrient-poor environments such as the deep sea because the symbionts allow their hosts to grow on inorganic energy and carbon sources such as sulfide and CO(2). Remarkably little is known about the physiological strategies that enable chemosynthetic symbioses to colonize oligotrophic environments. In this study, we used metaproteomics and metabolomics to investigate the intricate network of metabolic interactions in the chemosynthetic association between Olavius algarvensis, a gutless marine worm, and its bacterial symbionts. We propose previously undescribed pathways for coping with energy and nutrient limitation, some of which may be widespread in both free-living and symbiotic bacteria. These pathways include (i) a pathway for symbiont assimilation of the host waste products acetate, propionate, succinate and malate; (ii) the potential use of carbon monoxide as an energy source, a substrate previously not known to play a role in marine invertebrate symbioses; (iii) the potential use of hydrogen as an energy source; (iv) the strong expression of high-affinity uptake transporters; and (v) as yet undescribed energy-efficient steps in CO(2) fixation and sulfate reduction. The high expression of proteins involved in pathways for energy and carbon uptake and conservation in the O. algarvensis symbiosis indicates that the oligotrophic nature of its environment exerted a strong selective pressure in shaping these associations.

Coping with anoxia: a comprehensive proteomic and transcriptomic survey of denitrification.

Ralstonia eutropha H16 is a denitrifying microorganism able to use nitrate and nitrite as terminal electron acceptors under oxygen deprivation. To identify proteins showing an altered expression pattern in response to oxygen supply, R. eutropha cells grown aerobically and anaerobically were compared in a comprehensive proteome and transcriptome approach. Nearly 700 proteins involved in several processes including respiration, formation of cell appendages, and DNA and cofactor biosynthesis were found to be differentially expressed. A combination of 1D gel-LC and conventional 2D gel analysis of six consecutive sample points covering the entire denitrification sequence revealed a detailed view on the shifting abundance of the key proteins of denitrification. Denitrification- or anaerobiosis-induced alterations of the respiratory chain included a distinct expression pattern for multiple terminal oxidases. Alterations in the central carbon metabolism were restricted to a few key functions including the isoenzymes for aconitase and isocitrate dehydrogenase. Although R. eutropha is a strictly respiratory bacterium, the abundance of certain fermentation enzymes was increased. This work represents a comprehensive survey of denitrification on the proteomic and transcriptomic levels and provides unique insight into how R. eutropha adapts its metabolism to low oxygen conditions.

Mercaptosuccinate metabolism in Variovorax paradoxus strain B4--a proteomic approach.

Variovorax paradoxus B4 was isolated due to its ability to degrade the organic thiol compound mercaptosuccinate, which could be a promising precursor for novel polythioesters. The analysis of the proteome of this Gram-negative bacterium revealed several proteins with significantly increased expression during growth of cells with mercaptosuccinate as carbon source when compared to cells grown with gluconate or succinate. Among those, a large number of proteins involved in amino acid metabolism were identified, e.g., adenosylhomocysteinase and glutamate-ammonia ligase. Additionally, detection of superoxide dismutase strengthened the assumption of enhanced stress levels in mercaptosuccinate-grown cells. Several isoforms of a rhodanese domain-containing protein exhibited particularly increased expression during growth with mercaptosuccinate in comparison to gluconate (factor 14.2, stationary phase) or to succinate (factor 15.4, stationary phase). Besides this, augmented expression of the hypothetical protein VAPA_1c41240 raised attention. VAPA_1c41240 exhibited up to 13.3-fold (mercaptosuccinate vs gluconate) or 9.5-fold (mercaptosuccinate vs succinate) increased expression levels, and in silico searches revealed that this protein might be a thiol dioxygenase. Based on these results, a novel degradation pathway is proposed for mercaptosuccinate. The newly identified putative mercaptosuccinate dioxygenase could convert mercaptosuccinate to sulfinosuccinate by the introduction of two molecules of oxygen. Subsequently, sulfinosuccinate would be cleaved into succinate and sulfite either by a yet unknown enzyme, by spontaneous hydrolysis, or by the putative mercaptosuccinate dioxygenase itself. Succinate could then enter the central metabolism, while detoxification of sulfite could be achieved by the previously identified putative molybdopterin oxidoreductase. Biochemical studies will be done in the future to confirm this pathway.

The response of Bacillus licheniformis to heat and ethanol stress and the role of the SigB regulon.

The heat and ethanol stress response of Bacillus licheniformis DSM13 was analyzed at the transcriptional and/or translational level. During heat shock, regulons known to be heat-induced in Bacillus subtilis 168 are upregulated in B. licheniformis, such as the HrcA, SigB, CtsR, and CssRS regulon. Upregulation of the SigY regulon and of genes controlled by other extracytoplasmic function (ECF) sigma factors indicates a cell-wall stress triggered by the heat shock. Furthermore, tryptophan synthesis enzymes were upregulated in heat stressed cells as well as regulons involved in usage of alternative carbon and nitrogen sources. Ethanol stress led to an induction of the SigB, HrcA, and CtsR regulons. As indicated by the upregulation of a SigM-dependent protein, ethanol also triggered a cell wall stress. To characterize the SigB regulon of B. licheniformis, we analyzed the heat stress response of a sigB mutant. It is shown that the B. licheniformis SigB regulon comprises additional genes, some of which do not exist in B. subtilis, such as BLi03885, encoding a hypothetical protein, the Na/solute symporter gene BLi02212, the arginase homolog-encoding gene BLi00198 and mcrA, encoding a protein with endonuclease activity.

Proteomic profiles and kinetics of development of bacteriophage T4 and its rI and rIII mutants in slowly growing Escherichia coli.

Bacteriophage T4 survival in its natural environment requires adjustment of phage development to the slow bacterial growth rate or the initiation of mechanisms of pseudolysogeny or lysis inhibition (LIN). While phage-encoded RI and probably RIII proteins seem to be crucial players in pseudolysogeny and LIN phenomena, the identity of proteins involved in the regulation of T4 development in slowly growing bacteria has remained unknown. In this work, using a chemostat system, we studied the development of wild-type T4 (T4wt) and its rI (T4rI) and rIII (T4rIII) mutants in slowly growing bacteria, where T4 did not initiate LIN or pseudolysogeny. We determined eclipse periods, phage propagation times, latent periods and burst sizes of T4wt, T4rI and T4rIII. We also compared intracellular proteomes of slowly growing Escherichia coli infected with either T4wt or the mutants. Using two-dimensional PAGE analyses we found 18 differentially expressed proteins from lysates of infected cells. Proteins whose amounts were different in cells harbouring T4wt and the mutants are involved in processes of replication, phage-host interactions or they constitute virion components. Our data indicate that functional RI and RIII proteins - apart from their already known roles in LIN and pseudolysogeny - are also necessary for the regulation of phage T4 development in slowly growing bacteria. This regulation may be more complicated than previously anticipated, with many factors influencing T4 development in its natural habitat.

Fermentation stage-dependent adaptations of Bacillus licheniformis during enzyme production.

BACKGROUND: Industrial fermentations can generally be described as dynamic biotransformation processes in which microorganisms convert energy rich substrates into a desired product. The knowledge of active physiological pathways, reflected by corresponding gene activities, allows the identification of beneficial or disadvantageous performances of the microbial host. Whole transcriptome RNA-Seq is a powerful tool to accomplish in-depth quantification of these gene activities, since the low background noise and the absence of an upper limit of quantification allow the detection of transcripts with high dynamic ranges. Such data enable the identification of potential bottlenecks and futile energetic cycles, which in turn can lead to targets for rational approaches to productivity improvement. Here we present an overview of the dynamics of gene activity during an industrial-oriented fermentation process with Bacillus licheniformis, an important industrial enzyme producer. Thereby, valuable insights which help to understand the complex interactions during such processes are provided. RESULTS: Whole transcriptome RNA-Seq has been performed to study the gene expression at five selected growth stages of an industrial-oriented protease production process employing a germination deficient derivative of B. licheniformis DSM13. Since a significant amount of genes in Bacillus strains are regulated posttranscriptionally, the generated data have been confirmed by 2D gel-based proteomics. Regulatory events affecting the coordinated activity of hundreds of genes have been analyzed. The data enabled the identification of genes involved in the adaptations to changing environmental conditions during the fermentation process. A special focus of the analyses was on genes contributing to central carbon metabolism, amino acid transport and metabolism, starvation and stress responses and protein secretion. Genes contributing to lantibiotics production and Tat-dependent protein secretion have been pointed out as potential optimization targets. CONCLUSIONS: The presented data give unprecedented insights into the complex adaptations of bacterial production strains to the changing physiological demands during an industrial-oriented fermentation. These are, to our knowledge, the first publicly available data that document quantifiable transcriptional responses of the commonly employed production strain B. licheniformis to changing conditions over the course of a typical fermentation process in such extensive depth.

Cell surface proteome of the marine planctomycete Rhodopirellula baltica.

The surface proteome (surfaceome) of the marine planctomycete Rhodopirellula baltica SH1(T) was studied using a biotinylation and a proteinase K approach combined with SDS-PAGE and mass spectrometry. 52 of the proteins identified in both approaches could be assigned to the group of potential surface proteins. Among them are some high molecular weight proteins, potentially involved in cell-cell attachment, that contain domains shown before to be typical for surface proteins like cadherin/dockerin domains, a bacterial adhesion domain or the fasciclin domain. The identification of proteins with enzymatic functions in the R. baltica surfaceome provides further clues for the suggestion that some degradative enzymes may be anchored onto the cell surface. YTV proteins, which have been earlier supposed to be components of the proteinaceous cell wall of R. baltica, were detected in the surface proteome. Additionally, 8 proteins with a novel protein structure combining a conserved type IV pilin/N-methylation domain and a planctomycete-typical DUF1559 domain were identified.

Impact of the core components of the phosphoenolpyruvate-carbohydrate phosphotransferase system, HPr and EI, on differential protein expression in Ralstonia eutropha H16.

In Ralstonia eutropha H16, seven genes encoding proteins being involved in the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified. In order to provide more insights into the poly(3-hydroxybutyrate) (PHB)-leaky phenotype of the HPr/EI deletion mutants H16ΔptsH, H16ΔptsI, and H16ΔptsHI when grown on the non-PTS substrate gluconate, parallel fermentations for comparison of their growth behavior were performed. Samples from the exponential, the early stationary, and late stationary growth phases were investigated by microscopy, gas chromatography and (phospho-) proteome analysis. A total of 71 differentially expressed proteins were identified using 2D-PAGE, Pro-Q Diamond and Coomassie staining, and MALDI-TOF analysis. Detected proteins were classified into five major functional groups: carbon metabolism, energy metabolism, amino acid metabolism, translation, and membrane transport/outer membrane proteins. Proteome analyses revealed enhanced expression of proteins involved in the Entner-Doudoroff pathway and in subsequent reactions in cells of strain H16 compared to the mutant H16ΔptsHI. Furthermore, proteins involved in PHB accumulation showed increased abundance in the wild-type. This expression pattern allowed us to identify proteins affecting carbon metabolism/PHB biosynthesis in strain H16 and translation/amino acid metabolism in strain H16ΔptsHI, and to gain insight into the molecular response of R. eutropha to the deletion of HPr/EI.

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.

Elevated poly(3-hydroxybutyrate) synthesis in mutants of Ralstonia eutropha H16 defective in lipopolysaccharide biosynthesis.

Several independent transposon Tn5-induced mutants of Ralstonia eutropha H16 exhibited a poly(3-hydroxybutyric acid) (PHB) elevated phenotype and accumulated substantial amounts of PHB already in the exponential growth phase. The insertion loci of Tn5 in these six mutants were mapped in the genes hldA (twice), hldC (twice), rfaF2, and rfaF3, which are all involved in the synthesis of lipopolysaccharides (LPS), an important component of the outer membrane (OM) of Gram-negative bacteria. The generated defined deletion mutant ΔhldA confirmed the PHB elevated phenotype. According to the literature,such a truncated LPS may cause an increased permeability of the OM; thereby, the mutations may lead to a facilitated uptake of carbon source from the medium as exemplarily shown for gluconate and succinate. Thus, the ratio of carbon to nitrogen in the cell is increased. Proteome analyses revealed reinforcement of the Entner­Doudoroff pathway and of subsequent reactions that finally may lead to higher concentrations of acetyl-CoA in the cells. Due to the impaired synthesis of complete LPS, intermediates of LPS biosynthesis might be recycled by reactions yielding higher levels of NADPH and acetyl-CoA. Since the latter are precursors for synthesis of PHB, this could explain the elevated synthesis and accumulation of this polymer in case of the LPS mutants.

Human endothelial cells form an endothelium in freestanding collagen hollow filaments fabricated by direct extrusion printing.

Fiber-shaped materials have great potential for tissue engineering applications as they provide structural support and spatial patterns within a three-dimensional construct. Here we demonstrate the fabrication of mechanically stable, meter-long collagen hollow filaments by a direct extrusion printing process. The fibres are permeable for oxygen and proteins and allow cultivation of primary human endothelial cells (ECs) at the inner surface under perfused conditions. The cells show typical characteristics of a well-organized EC lining including VE-cadherin expression, cellular response to flow and ECM production. The results demonstrate that the collagen tubes are capable of creating robust soft tissue filaments. The mechanical properties and the biofunctionality of these collagen hollow filaments facilitate the engineering of prevascularised tissue engineering constructs.