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Pseudomonas corrugata crpCDE is part of the cyclic lipopeptide corpeptin biosynthetic gene cluster and is involved in bacterial virulence in tomato and in hypersensitive response in Nicotiana benthamiana
Strano, C.P. ; Bella, P. ; Licciardello, G. ; Fiore, A. ; Piero, A.R. Lo; Fogliano, V. ; Fogliano, V. ; Catara, V. - \ 2015
Molecular Plant Pathology 16 (2015)5. - ISSN 1464-6722 - p. 495 - 506.
syringae pv.-syringae - gram-negative bacteria - quorum-sensing system - chromobacterium-violaceum - putisolvin-ii - syringomycin - lipodepsipeptides - syringopeptin - peptide - cloning
Pseudomonas corrugata CFBP 5454 produces two kinds of cyclic lipopeptides (CLPs), cormycin A and corpeptins, both of which possess surfactant, antimicrobial and phytotoxic activities. In this study, we identified genes coding for a putative non-ribosomal peptide synthetase and an ABC-type transport system involved in corpeptin production. These genes belong to the same transcriptional unit, designated crpCDE. The genetic organization of this locus is highly similar to other Pseudomonas CLP biosynthetic clusters. Matrix-assisted laser desorption ionization-time of flightmass spectrometry (MALDI-TOF-MS) analysis revealed that transporter and synthetase genomic knock-out mutants were unable to produce corpeptins, but continued to produce cormycin A. This suggests that CrpCDE is the only system involved in corpeptin production in P. corrugata CFBP 5454. In addition, phylogenetic analysis revealed that the CrpE ABC transporter clustered with the transporters of CLPs with a long peptide chain. Strains depleted in corpeptin production were significantly less virulent than the wildtype strain when inoculated in tomato plants and induced only chlorosis when infiltrated into Nicotiana benthamiana leaves. Thus, corpeptins are important effectors of P. corrugata interaction with plants. Expression analysis revealed that crpC transcription occurs at high cell density. Two LuxR transcriptional regulators, PcoR and RfiA, have a pivotal role in crpC expression and thus in corpeptin production.
Cysteine Depletion Causes Oxidative Stress and Triggers Outer Membrane Vesicle Release by Neisseria meningitidis Implications for Vaccine Development
Waterbeemd, B. van de; Zomer, G. ; IJssel, J. van den; Keulen, L. van; Eppink, M.H.M. ; Ley, P. de; Pol, L.A. van der - \ 2013
PLoS One 8 (2013)1. - ISSN 1932-6203 - 9 p.
gram-negative bacteria - iron-sulfur clusters - escherichia-coli - serogroup-b - transcriptome analysis - meningococcal disease - chelating-agents - dna microarrays - strains - lipopolysaccharide
Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (61500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteinedependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis.
In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates
Poblete-Castro, I. ; Binger, D. ; Rodrigues, A. ; Becker, J. ; Martins Dos Santos, V.A.P. ; Wittmann, C. - \ 2013
Metabolic Engineering 15 (2013). - ISSN 1096-7176 - p. 113 - 123.
chain-length polyhydroxyalkanoates - gram-negative bacteria - fed-batch culture - genome sequence - escherichia-coli - kt2440 - pathways - acid - poly(3-hydroxyalkanoates) - biosynthesis
Here, we present systems metabolic engineering driven by in-silico modeling to tailor Pseudomonas putida for synthesis of medium chain length PHAs on glucose. Using physiological properties of the parent wild type as constraints, elementary flux mode analysis of a large-scale model of the metabolism of P. putida was used to predict genetic targets for strain engineering. Among a set of priority ranked targets, glucose dehydrogenase (encoded by gcd) was predicted as most promising deletion target. The mutant P. putida ¿gcd, generated on basis of the computational design, exhibited 100% increased PHA accumulation as compared to the parent wild type, maintained a high specific growth rate and exhibited an almost unaffected gene expression profile, which excluded detrimental side effects of the modification. A second mutant strain, P. putida ¿pgl, that lacked 6-phosphogluconolactonase, exhibited a substantially decreased PHA synthesis, as was also predicted by the model. The production potential of P. putida ¿gcd was assessed in batch bioreactors. The novel strain showed an increase of the PHA yield (+80%), the PHA titer (+100%) and cellular PHA content (+50%) and revealed almost unaffected growth and diminished by-product formation. It was thus found superior in all relevant criteria towards industrial production. Beyond the contribution to more efficient PHA production processes at reduced costs that might replace petrochemical plastics in the future, the study illustrates the power of computational prediction to tailor microbial strains for enhanced biosynthesis of added-value compounds
Molecular cloning and expression of two B-defensin and two mucin genes in common carp (Cyprinus carpio L.) and their up-regulation after B-glucan feeding
Marel, M.C. van der; Adamek, M. ; Gonzalez, S.F. ; Frost, P. ; Rombout, J.H.W.M. ; Wiegertjes, G.F. ; Savelkoul, H.F.J. ; Steinhagen, D. - \ 2012
Fish and Shellfish Immunology 32 (2012)3. - ISSN 1050-4648 - p. 494 - 501.
gram-negative bacteria - salmo-salar l - disease resistance - immune-responses - atlantic salmon - secreted mucins - infection - fish - macrophages - microbiota
In this study, we described the partial structure, mRNA tissue distribution and regulation of two carp mucin and two ß-defensin genes. This is the first description of these genes in fish. The genes might provide relevant tools to monitor feed-related improvements of fish health under aquaculture conditions. Carp mucin 2 and mucin 5B genes show a high similarity to their mammalian and avian counterparts. The carp ß-defensin 1 and ß-defensin 2 genes cluster together well with their piscine family members. The influence of a ß-glucan immunomodulant on the expression of these genes in mucosal tissues could be confirmed for the first time. Muc5B expression was significantly increased in the skin. For Muc2 no significant up- or down-regulation could be observed. Significantly higher expression levels of ß-defensin 2 in gills and both ß-defensin genes in skin were found. Thus, the mucosal system can be influenced by the addition of ß-glucans to the food
A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum - Cellular behavior in adaptation to n-butanol
Schwarz, K.M. ; Kuit, W. ; Grimmler, C. ; Ehrenreich, A. ; Kengen, S.W.M. - \ 2012
Journal of Biotechnology 161 (2012)3. - ISSN 0168-1656 - p. 366 - 377.
fatty-acid biosynthesis - 2-component signal-transduction - organic-solvent tolerance - response regulator yycf - beijerinckii ncimb 8052 - gram-negative bacteria - escherichia-coli - bacillus-subtilis - lipid-composition - gene-expression
To gain more insight into the butanol stress response of Clostridium acetobutylicum the transcriptional response of a steady state acidogenic culture to different levels of n-butanol (0.25-1%) was investigated. No effect was observed on the fermentation pattern and expression of typical solvent genes (aad, ctfA/B, adc, bdhA/B, ptb, buk). Elevated levels of butanol mainly affected class I heat-shock genes (hrcA, grpE, dnaK, dnaJ, groES, groEL, hsp90), which were upregulated in a dose- and time-dependent manner, and genes encoding proteins involved in the membrane composition (fab and fad or glycerophospholipid related genes) and various ABC-transporters of unknown specificity. Interestingly, fab and fad genes were embedded in a large, entirely repressed cluster (CAC1988-CAC2019), which inter alia encoded an iron-specific ABC-transporter and molybdenum-cofactor synthesis proteins. Of the glycerophospholipid metabolism, the glycerol-3-phosphate dehydrogenase (glpA) gene was highly upregulated, whereas a glycerophosphodiester ABC-transporter (ugpAEBC) and a phosphodiesterase (ugpC) were repressed. On the megaplasmid, only a few genes showed differential expression, e.g. a rare lipoprotein (CAP0058, repressed) and a membrane protein (CAP0102, upregulated) gene. Observed transcriptional responses suggest that C. acetobutylicum reacts to butanol stress by induction of the general stress response and changing its cell envelope and transporter composition, but leaving the central catabolism unaffected
Increased detection of extended spectrum beta-lactamase producing Salmonella enterica and Escherichia coli isolates from poultry
Dierikx, C.M. ; Essen-Zandbergen, A. van; Veldman, K.T. ; Smith, H.E. ; Mevius, D.J. - \ 2010
Veterinary Microbiology 145 (2010)3-4. - ISSN 0378-1135 - p. 273 - 278.
gram-negative bacteria - resistance plasmid - ctx-m - enterobacteriaceae - animals - cephalosporins - identification - typhimurium - strains - genes
To gain more information on the genetic basis of the rapid increase in the number of isolates exhibiting non-wild type Minimum Inhibitory Concentrations (MICs) for cefotaxime observed since 2003, beta-lactamase genes of 22 Salmonella enterica and 22 Escherichia coli isolates from broilers in 2006 showing this phenotype were characterized by miniaturized micro-array, PCR and DNA-sequencing. Presence and size of plasmids were determined by S1-digest pulsed-field gel electrophoresis and further characterized by PCR-based replicon typing. Transfer of resistance plasmids was tested by conjugation and transformation experiments. To link resistance genes and plasmid type, Southern blot hybridization experiments were conducted. In 42 isolates, five (blaCTX-M-1, blaCTX-M-2, blaTEM-20, blaTEM-52, blaSHV-2) different extended spectrum beta-lactamase (ESBL)-genes and two (blaACC-1, blaCMY-2) AmpC-genes were present. Three of the detected ESBL-genes (blaCTX-M-1, blaTEM-52 and blaCTX-M-2) were located on similar types of plasmids (IncI1 and IncHI2/P) in both E. coli and Salmonella. Two other detected ESBL- and AmpC-genes blaSHV-2 and blaCMY-2 respectively (on IncK plasmids), were only found in E. coli, whereas the AmpC-gene blaACC-1 (on non-typable plasmids), and the ESBL-gene blaTEM-20 (on IncI1 plasmids), were only detected in Salmonella. In two isolates, no ESBL- or AmpC-gene could be detected through these methods. The increase in the number of E. coli and S. enterica isolates from the gastro-intestinal tract of broilers exhibiting non-wild type MICs for cefotaxime is mainly due to an increase in IncI1 plasmids containing blaCTX-M-1. The reason for the successful spread of this plasmid type in these species is not yet understood.
Natural evolution of TEM-1 ß-lactamase: experimental reconstruction and clinical relevance
Salverda, M.L.M. ; Visser, J.A.G.M. de; Barlow, M. - \ 2010
FEMS Microbiology Reviews 34 (2010)6. - ISSN 0168-6445 - p. 1015 - 1036.
hydrolyzing 3rd-generation cephalosporins - inhibitor-resistant mutants - amino-acid substitutions - gram-negative bacteria - extended-spectrum - escherichia-coli - antibiotic-resistance - klebsiella-pneumoniae - in-vitro - directed evolution
TEM-1 ß-lactamase is one of the most well-known antibiotic resistance determinants around. It confers resistance to penicillins and early cephalosporins and has shown an astonishing functional plasticity in response to the introduction of novel drugs derived from these antibiotics. Since its discovery in the 1960s, over 170 variants of TEM-1 – with different amino acid sequences and often resistance phenotypes – have been isolated in hospitals and clinics worldwide. Next to this well-documented ‘natural’ evolution, the in vitro evolution of TEM-1 has been the focus of attention of many experimental studies. In this review, we compare the natural and laboratory evolution of TEM-1 in order to address the question to what extent the evolution of antibiotic resistance can be repeated, and hence might have been predicted, under laboratory conditions. We also use the comparison to gain an insight into the adaptive relevance of hitherto uncharacterized substitutions present in clinical isolates and to predict substitutions not yet observed in nature. Based on new structural insights, we review what is known about substitutions in TEM-1 that contribute to the extension of its resistance phenotype. Finally, we address the clinical relevance of TEM alleles during the past decade, which has been dominated by the emergence of another ß-lactamase, CTX-M
Oviposition responses of Anopheles gambiae s.s. (Diptera: Culicidae) and identification of volatiles from bacteria-containing solutions
Lindh, J.M. ; Kännaste, A. ; Knols, B.G.J. ; Faye, I. ; Borg-Karlson, A.K. - \ 2008
Journal of Medical Entomology 45 (2008)6. - ISSN 0022-2585 - p. 1039 - 1049.
mexican fruit-fly - aedes-albopictus diptera - gram-negative bacteria - culex-quinquefasciatus - gas-chromatography - enterobacter-agglomerans - behavioral-responses - malaria mosquito - pipiens diptera - marine-bacteria
In this study, a dual-choice oviposition bioassay was used to screen responses of gravid An. gambiae toward 17 bacterial species, previously isolated from Anopheles gambiae s.l. (Diptera: Culicidae) midguts or oviposition sites. The 10 isolates from oviposition sites have been identified by phylogenetic analyses of their 16S rRNA genes. Eight of the 10 isolates were gram-positive, out of which six belonged to the Bacilli class. Solid phase microextraction and gas chromatography coupled to mass spectrometry (GC-MS) were used to identify the volatiles emitted from the bacterial isolates. Aromatic and aliphatic alcohols, aliphatic ketones, alkylpyrazines, dimethyl oligosulfides, and indole were among the chemical compounds identified from the headspace above bacteria-containing saline. The mosquitoes laid significantly more eggs in six of the bacteria-containing solutions compared with the sterile solution. These six bacteria did not emit any compounds in common that could explain the positive oviposition response. Instead, the bacteria were grouped according to principal component analysis (PCA) based on the relative amounts of volatiles emitted. The PCA-plots facilitated the identification of 13 putative oviposition attractants for An. gambiae mosquitoes
Modeling Neisseria meningitidis metabolism: from genome to metabolic fluxes
Baart, G.J.E. ; Zomer, B. ; Haan, A. de; Pol, L.A. van der; Beuvery, E.C. ; Tramper, J. ; Martens, D.E. - \ 2007
Genome Biology 8 (2007)7. - ISSN 1474-7596 - p. R136 - R136.
c14 labelled glucose - bombardment mass-spectrometry - biochemical reaction systems - linear constraint relations - serum bactericidal activity - membrane vesicle vaccine - gram-negative bacteria - escherichia-coli - meningococcal disease - genus neisseria
Background - Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years. Results - Using the genomic database of N. meningitidis serogroup B together with biochemical and physiological information in the literature we constructed a genome-scale flux model for the primary metabolism of N. meningitidis. The validity of a simplified metabolic network derived from the genome-scale metabolic network was checked using flux-balance analysis in chemostat cultures. Several useful predictions were obtained from in silico experiments, including substrate preference. A minimal medium for growth of N. meningitidis was designed and tested succesfully in batch and chemostat cultures. Conclusion - The verified metabolic model describes the primary metabolism of N. meningitidis in a chemostat in steady state. The genome-scale model is valuable because it offers a framework to study N. meningitidis metabolism as a whole, or certain aspects of it, and it can also be used for the purpose of vaccine process development (for example, the design of growth media). The flux distribution of the main metabolic pathways (that is, the pentose phosphate pathway and the Entner-Douderoff pathway) indicates that the major part of pyruvate (69%) is synthesized through the ED-cleavage, a finding that is in good agreement with literature.
Energetics and surface properties of Pseudomonas putida DOT-T1E in a two-phase fermentation system with 1-decanol as second phase
Neumann, G. ; Cornelissen, S. ; Breukelen, F.R. van; Hunger, S. ; Lippold, H. ; Loffhagen, N. ; Wick, L.Y. ; Heipieper, H.J. - \ 2006
Applied and Environmental Microbiology 72 (2006)6. - ISSN 0099-2240 - p. 4232 - 4238.
gram-negative bacteria - escherichia-coli - solvent-tolerant - organic-solvents - membrane-vesicles - aeruginosa pao1 - energy-charge - fatty-acids - a-band - growth
The solvent-tolerant strain Pseudomonas putida DOT-TIE was grown in batch fermentations in a 5-liter bioreactor in the presence and absence of 10% (vol/vol) of the organic solvent 1-decanol. The growth behavior and cellular energetics, such as the cellular ATP content and the energy charge, as well as the cell surface hydrophobicity and charge, were measured in cells growing in the presence and absence of 1-decanol. Although the cells growing in the presence of 1-decanol showed an about 10% reduced growth rate and a 48% reduced growth yield, no significant differences were measured either in the ATP and potassium contents or in the energy charge, indicating that the cells adapted completely at the levels of membrane permeability and energetics. Although the bacteria needed additional energy for adaptation to the presence of the solvent, they were able to maintain or activate electron transport phosphorylation, allowing homeostasis of the ATP level and energy charge in the presence of the solvent, at the price of a reduced growth yield. On the other hand, significantly enhanced cell hydrophobicities and more negative cell surface charges were observed in cells grown in the presence of 1-decanol. Both reactions occurred within about 10 min after the addition of the solvent and were significantly different after killing of the cells with toxic concentrations of HgCl2. This adaptation of the surface properties of the bacterium to the presence of solvents seems to be very similar to previously observed reactions on the level of lipopolysaccharides, with which bacteria adapt to environmental stresses, such as heat shock, antibiotics, or low oxygen content. The results give clear physiological indications that the process with P. putida DOT-TIE as the biocatalyst and 1-decanol as the solvent is a stable system for two-phase biotransformations that will allow the production of fine chemicals in economically sound amounts.
The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants
Eede, G. van den; Aarts, H.J.M. ; Buhk, H.J. ; Corthier, G. ; Flint, H.J. ; Hammes, W. ; Jacobsen, B. ; Midtvedt, T. ; Vossen, J. van der; Wright, A. ; Wackernagel, W. ; Wilcks, A. - \ 2004
Food and Chemical Toxicology 42 (2004)7. - ISSN 0278-6915 - p. 1127 - 1156.
agrobacterium-mediated transformation - polymerase-chain-reaction - facilitated illegitimate recombination - high-velocity microprojectiles - crown-gall tumorigenesis - gram-negative bacteria - free transgenic plants - coated gold particles - human fecal flora - e
In 2000, the thematic network ENTRANSFOOD was launched to assess four different topics that are all related to the testing or assessment of food containing or produced from genetically modified organisms (GMOs). Each of the topics was linked to a European Commission (EC)-funded large shared cost action (see http://www.entransfood.com). Since the exchange of genetic information through horizontal (lateral) gene transfer (HGT) might play a more important role, in quantity and quality, than hitherto imagined, a working group dealing with HGT in the context of food and feed safety was established. This working group was linked to the GMOBILITY project (GMOBILITY, 2003) and the results of the deliberations are laid down in this review paper. HGT is reviewed in relation to the potential risks of consuming food or feed derived from transgenic crops. First, the mechanisms for obtaining transgenic crops are described. Next, HGT mechanisms and its possible evolutionary role are described. The use of marker genes is presented in detail as a special case for genes that may pose a risk. Furthermore, the exposure to GMOs and in particular to genetically modified (GM) deoxyribonucleic acid (DNA) is discussed as part of the total risk assessment. The review finishes off with a number of conclusions related to GM food and feed safety. The aim of this paper is to provide a comprehensive overview to assist risk assessors as well as regulators and the general public in understanding the safety issues related to these mechanisms.
Thermodynamics of micellization of cholic acid based facial amphiphiles carrying three permanent ionic head groups
Willemen, H.M. ; Marcelis, A.T.M. ; Sudhölter, E.J.R. - \ 2003
Langmuir 19 (2003)7. - ISSN 0743-7463 - p. 2588 - 2591.
gram-negative bacteria - micelle formation - bile-acids - derivatives - organogelators
This paper describes a series of cholic acid based facial amphiphiles carrying three ionic headgroups. Their micellization behavior in water was studied as a function of spacer length and alkyl tail length: both were found to have a small influence on the critical micellization concentration (cmc). Isothermal titration calorimetry was used to gain information on the thermodynamics of micellization. These surfactants show rather normal temperature-dependent behavior: Gibbs energy of micellization is almost constant, cmc increases, and enthalpy of micellization decreases with temperature, and both entropy and enthalpy contribute to the micellization.