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.

Proteomic analysis of a hom-disrupted, cephamycin C overproducing Streptomyces clavuligerus.

BACKGROUND: Streptomyces clavuligerus is prolific producer of cephamycin C, a medically important antibiotic. In our former study, cephamycin C titer was 2-fold improved by disrupting homoserine dehydrogenase (hom) gene of aspartate pahway in Streptomyces clavuligerus NRRL 3585. OBJECTIVE: In this article, we aimed to provide a comprehensive understanding at the proteome level on potential complex metabolic changes as a consequence of hom disruption in Streptomyces clavuligerus AK39. METHODS: A comparative proteomics study was carried out between the wild type and its hom disrupted AK39 strain by 2 Dimensional Electrophoresis-Matrix Assisted Laser Desorption and Ionization Time-Of-Flight Mass Spectrometry (2DE MALDI-TOF/MS) and Nanoscale Liquid Chromatography- Tandem Mass Spectrometry (nanoLC-MS/MS) analyses. Clusters of Orthologous Groups (COG) database was used to determine the functional categories of the proteins. The theoretical pI and Mw values of the proteins were calculated using Expasy pI/Mw tool. RESULTS: "Hypothetical/Unknown" and "Secondary Metabolism" were the most prominent categories of the differentially expressed proteins. Upto 8.7-fold increased level of the positive regulator CcaR was a key finding since CcaR was shown to bind to cefF promoter thereby direcly controlling its expression. Consistently, CeaS2, the first enzyme of CA biosynthetic pathway, was 3.3- fold elevated. There were also many underrepresented proteins associated with the biosynthesis of several Non-Ribosomal Peptide Synthases (NRPSs), clavams, hybrid NRPS/Polyketide synthases (PKSs) and tunicamycin. The most conspicuously underrepresented protein of amino acid metabolism was 4-Hydroxyphenylpyruvate dioxygenase (HppD) acting in tyrosine catabolism. The levels of a Two Component System (TCS) response regulator containing a CheY-like receiver domain and an HTH DNA-binding domain as well as DNA-binding protein HU were elevated while a TetR-family transcriptional regulator was underexpressed. CONCLUSION: The results obtained herein will aid in finding out new targets for further improvement of cephamycin C production in Streptomyces clavuligerus.

The catabolism of 3,3'-thiodipropionic acid in Variovorax paradoxus strain TBEA6: A proteomic analysis.

Variovorax paradoxus strain TBEA6 is one of the few organisms known to utilize 3,3'-thiodipropionate (TDP) as the only source of carbon and energy. It cleaves TDP to 3-mercaptopropionate (3MP), which is a direct precursor for polythioester synthesis. To establish this process in V. paradoxus TBEA6, it is crucial to unravel its TDP metabolism. Therefore, a proteomic approach with subsequent deletion of interesting genes in the bacterium was chosen. Cells were cultivated with D-gluconate, TDP or 3-sulfinopropionate as the only carbon sources. Proteins with high abundances in gels of cells cultivated with either of the organic sulfur compounds were analyzed further. Thereby, we did not only confirm parts of the already postulated TDP metabolism, but also eight new protein candidates for TDP degradation were detected. Deletions of the corresponding genes (two enoyl-CoA hydratases (Ech-20 and Ech-30), an FK506-binding protein, a putative acetolactate synthase, a carnitinyl-CoA dehydratase, and a putative crotonase family protein) were obtained. Only the deletions of both Ech-20 and Ech-30 led to a TDP negative phenotype. The deletion mutant of VPARA_05510, which encodes the putative crotonase family protein showed reduced growth with TDP. The three genes are located in one cluster with genes proven to be involved in TDP metabolism. Thermal shift assays showed an increased stability of Ech-20 with TDP-CoA but not with TDP. These results indicate that Ech-20 uses TDP-CoA as a substrate instead of TDP. Hence, we postulate a new putative pathway for TDP metabolism. Ech-30 interacts with neither TDP-CoA nor TDP but might interact with other CoA-activated intermediates of the proposed pathway. Further enzyme characterization is necessary to unravel the complete pathway from TDP to 3MP.

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.

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.

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.

Proteome-wide alterations in an industrial clavulanic acid producing strain of Streptomyces clavuligerus.

The usefulness of genetic/metabolic engineering for further improvement of industrial strains is subject of discussion because of the general lack of knowledge on genetic alterations introduced by iterative cycles of random mutagenesis in such strains. An industrial clavulanic acid (CA)-overproducer Streptomyces clavuligerus DEPA was assessed to understand proteome-wide changes that have occurred in a local industrial CA overproducer developed through succesive mutagenesis programs. The proteins that could be identified corresponded to 33 distinct ORFs for underrepresented ones and 60 ORFs for overrepresented ones. Three CA biosynthetic enzymes were overrepresented in S. clavuligerus DEPA; carboxyethylarginine synthase (Ceas2), clavaldehyde dehydrogenase (Car) and carboxyethyl-arginine beta-lactam-synthase (Bls2) whereas the enzymes of two other secondary metabolites were underrepresented along with two important global regulators [two-component system (TCS) response regulator (SCLAV_2102) and TetR-family transcriptional regulator (SCLAV_3146)] that might be related with CA production and/or differentiation. γ-butyrolactone biosynthetic protein AvaA2 was 2.6 fold underrepresented in S. clavuligerus DEPA. The levels of two glycolytic enzymes, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase and phosophoglycerate kinase were found decreased while those of dihydrolipoyl dehydrogenase (E3) and isocitrate dehydrogenase, with two isoforms were found as significantly increased. A decrease of amino acid metabolism, methionine biosynthesis in particular, as well as S-adenosylmethionine synthetase appeared as one of the prominent mechanisms of success of S. clavuligerus DEPA strain as a prolific producer of CA. The levels of two enzymes of shikimate pathway that leads to the production of aromatic amino acids and aromatic secondary metabolites were also underrepresented. Some of the overrepresented stress proteins in S. clavuligerus DEPA included polynucleotide phosphorylase/polyadenylase (PNPase), ATP-dependent DNA helicase, two isoforms of an anti-sigma factor and thioredoxin reductase. Downregulation of important proteins of cell wall synthesis and division was recorded and a protein with ß-lactamase domain (SCLAV_p1007) appeared in 12 isoforms, 5 of which were drastically overrepresented in DEPA strain. These results described herein provide useful information for rational engineering to improve CA production in Streptomyces clavuligerus.

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.

Small RNA mediated repression of subtilisin production in Bacillus licheniformis.

The species Bacillus licheniformis includes important strains that are used in industrial production processes. Currently the physiological model used to adapt these processes is based on the closely related model organism B. subtilis. In this study we found that both organisms reveal significant differences in the regulation of subtilisin, their main natural protease and a product of industrial fermentation processes. We identified and characterized a novel antisense sRNA AprAs, which represents an RNA based repressor of apr, the gene encoding for the industrial relevant subtilisin protease. Reduction of the AprAs level leads to an enhanced proteolytic activity and an increase of Apr protein expression in the mutant strain. A vector based complementation of the AprAs deficient mutant confirmed this effect and demonstrated the necessity of cis transcription for full efficiency. A comparative analysis of the corresponding genome loci from B. licheniformis and B. subtilis revealed the absence of an aprAs promoter in B. subtilis and indicates that AprAs is a B. licheniformis species specific phenomenon. The discovery of AprAs is of great biotechnological interest since subtilisin Carlsberg is one of the main products of industrial fermentation by B. licheniformis.

Parvalbumin-Expressing GABAergic Neurons in Primary Motor Cortex Signal Reaching.

The control of targeted reaching is thought to be shaped by distinct subtypes of local GABAergic inhibitory neurons in primary forelimb motor cortex (M1). However, little is known about their action potential firing dynamics during reaching. To address this, we recorded the activity of parvalbumin-expressing (PV+) GABAergic neurons identified from a larger population of fast-spiking units and putative excitatory regular-spiking units in layer 5 of the mouse forelimb M1 during an M1-dependent, sensory-triggered reaching task. PV+ neurons showed short latency responses to the acoustic cue and vibrotactile trigger stimulus and an increase in firing at reaching onset that scaled with the amplitude of reaching. Unexpectedly, PV+ neurons fired before regular-spiking units at reach onset and showed high overall firing rates during both sensory-triggered and spontaneous reaches. Our data suggest that increasing M1 PV+ neuron firing rates may play a role in the initiation of voluntary reaching.

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.

Proteomic analysis of organic sulfur compound utilisation in Advenella mimigardefordensis strain DPN7T.

2-Mercaptosuccinate (MS) and 3,3´-ditiodipropionate (DTDP) were discussed as precursor substance for production of polythioesters (PTE). Therefore, degradation of MS and DTDP was investigated in Advenella mimigardefordensis strain DPN7T, applying differential proteomic analysis, gene deletion and enzyme assays. Protein extracts of cells cultivated with MS, DTDP or 3-sulfinopropionic acid (SP) were compared with those cultivated with propionate (P) and/or succinate (S). The chaperone DnaK (ratio DTDP/P 9.2, 3SP/P 4.0, MS/S 6.1, DTDP/S 6.2) and a Do-like serine protease (DegP) were increased during utilization of all organic sulfur compounds. Furthermore, a putative bacterioferritin (locus tag MIM_c12960) showed high abundance (ratio DTDP/P 5.3, 3SP/P 3.2, MS/S 4.8, DTDP/S 3.9) and is probably involved in a thiol-specific stress response. The deletion of two genes encoding transcriptional regulators (LysR (MIM_c31370) and Xre (MIM_c31360)) in the close proximity of the relevant genes of DTDP catabolism (acdA, mdo and the genes encoding the enzymes of the methylcitric acid cycle; prpC,acnD, prpF and prpB) showed that these two regulators are essential for growth of A. mimigardefordensis strain DPN7T with DTDP and that they most probably regulate transcription of genes mandatory for this catabolic pathway. Furthermore, proteome analysis revealed a high abundance (ratio MS/S 10.9) of a hypothetical cupin-2-domain containing protein (MIM_c37420). This protein shows an amino acid sequence similarity of 60% to a newly identified MS dioxygenase from Variovorax paradoxus strain B4. Deletion of the gene and the adjacently located transcriptional regulator LysR, as well as heterologous expression of MIM_c37420, the putative mercaptosuccinate dioxygenase (Msdo) from A. mimigardefordensis, showed that this protein is the key enzyme of MS degradation in A. mimigardefordensis strain DPN7T (KM 0.2 mM, specific activity 17.1 µmol mg-1 min-1) and is controlled by LysR (MIM_c37410).

Genome and Proteome Analysis of Rhodococcus erythropolis MI2: Elucidation of the 4,4´-Dithiodibutyric Acid Catabolism.

Rhodococcus erythropolis MI2 has the extraordinary ability to utilize the xenobiotic 4,4´-dithiodibutyric acid (DTDB). Cleavage of DTDB by the disulfide-reductase Nox, which is the only verified enzyme involved in DTDB-degradation, raised 4-mercaptobutyric acid (4MB). 4MB could act as building block of a novel polythioester with unknown properties. To completely unravel the catabolism of DTDB, the genome of R. erythropolis MI2 was sequenced, and subsequently the proteome was analyzed. The draft genome sequence consists of approximately 7.2 Mbp with an overall G+C content of 62.25% and 6,859 predicted protein-encoding genes. The genome of strain MI2 is composed of three replicons: one chromosome and two megaplasmids with sizes of 6.45, 0.4 and 0.35 Mbp, respectively. When cells of strain MI2 were cultivated with DTDB as sole carbon source and compared to cells grown with succinate, several interesting proteins with significantly higher expression levels were identified using 2D-PAGE and MALDI-TOF mass spectrometry. A putative luciferase-like monooxygenase-class F420-dependent oxidoreductase (RERY_05640), which is encoded by one of the 126 monooxygenase-encoding genes of the MI2-genome, showed a 3-fold increased expression level. This monooxygenase could oxidize the intermediate 4MB into 4-oxo-4-sulfanylbutyric acid. Next, a desulfurization step, which forms succinic acid and volatile hydrogen sulfide, is proposed. One gene coding for a putative desulfhydrase (RERY_06500) was identified in the genome of strain MI2. However, the gene product was not recognized in the proteome analyses. But, a significant expression level with a ratio of up to 7.3 was determined for a putative sulfide:quinone oxidoreductase (RERY_02710), which could also be involved in the abstraction of the sulfur group. As response to the toxicity of the intermediates, several stress response proteins were strongly expressed, including a superoxide dismutase (RERY_05600) and an osmotically induced protein (RERY_02670). Accordingly, novel insights in the catabolic pathway of DTDB were gained.

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.

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).

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.

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.

Cell physiology of the biotechnological relevant bacterium Bacillus pumilus-an omics-based approach.

Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on exoproteome analysis, B. pumilus strain Jo2, possessing a high secretion capability, was chosen for an omics-based investigation. The proteome and metabolome of B. pumilus cells growing either in minimal or complex medium was analyzed. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43% of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC-MS, IP-LC/MS and H NMR methods numerous metabolites were analyzed and assigned to reconstructed metabolic pathways. In the genome sequence a functional secretion system including the components of the Sec- and Tat-secretion machinery was found. Analysis of the exoproteome revealed secretion of about 70 proteins with predicted secretion signals. In addition, selected production-relevant genome features such as restriction modification systems and NRPS clusters of B. pumilus Jo2 are discussed.

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.

A proteomic view of cell physiology of the industrial workhorse Bacillus licheniformis.

Bacillus licheniformis is known for its high protein secretion capacity and is being applied extensively as a host for the industrial production of enzymes such as proteases and amylases. In its natural environment as well as in fermentation processes the bacterium is often facing adverse conditions such as oxidative or osmotic stress or starvation for nutrients. During the last years detailed proteome and transcriptome analyses have been performed to study the adaptation of B. licheniformis cells to various stresses (heat, ethanol, oxidative or salt stress) and starvation conditions (glucose, nitrogen or phosphate starvation). A common feature of the response to all tested conditions is the downregulation of many genes encoding house-keeping proteins and, consequently, a reduced synthesis of the corresponding proteins. Induction of the general stress response (σ(B) regulon) is only observed in cells subjected to heat, ethanol or salt stress. This paper summarizes our current knowledge on general and specific stress and starvation responses of this important industrial bacterium. The importance of selected marker genes and proteins for the monitoring and optimization of B. licheniformis based fermentation processes is discussed.