Reduction of Cell Lysate Viscosity during Processing of Poly(3-Hydroxyalkanoates) by Chromosomal Integration of the Staphylococcal Nuclease Gene in Pseudomonas putida

1999 ◽  
Vol 65 (4) ◽  
pp. 1524-1529 ◽  
Author(s):  
Zhuang L. Boynton ◽  
Joseph J. Koon ◽  
Elaine M. Brennan ◽  
Jeralyn D. Clouart ◽  
Daniel M. Horowitz ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Ralstonia Eutropha ◽  
Bacterial Species ◽  
Downstream Processing ◽  
Staphylococcal Nuclease ◽  
Side Chain ◽  
Chromosomal Dna ◽  
Industrial Processing ◽  
Film Forming ◽  
High Cell Density Cultures

ABSTRACT Poly(3-hydroxyalkanoates) (PHAs) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granules and which are thought to serve as reserves of carbon and energy. Pseudomonas putida accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. Industrial processing of PHA involves purification of the PHA granules from high-cell-density cultures. After the fermentation process, cells are lysed by homogenization and PHA granules are purified by chemical treatment and repeated washings to yield a PHA latex. Unfortunately, the liberation of chromosomal DNA during lysis causes a dramatic increase in viscosity, which is problematic in the subsequent purification steps. Reduction of the viscosity is generally achieved by the supplementation of commercially available nuclease preparations or by heat treatment; however, both procedures add substantial costs to the process. As a solution to this problem, a nuclease-encoding gene fromStaphylococcus aureus was integrated into the genomes of several PHA producers. Staphylococcal nuclease is readily expressed in PHA-producing Pseudomonas strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. During downstream processing, the viscosity of the lysate from a nuclease-integratedPseudomonas strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nuclease gene was also functionally integrated into the chromosomes of other PHA producers, including Ralstonia eutropha.

scholarly journals Comparative Assessment of NMR Probes for the Experimental Description of Protein Folding Pathways with High-Pressure NMR

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 656
Author(s):  
Vincent Van Deuren ◽  
Yin-Shan Yang ◽  
Karine de Guillen ◽  
Cécile Dubois ◽  
Catherine Anne Royer ◽  
...  
Keyword(s):  
High Pressure ◽  
Staphylococcal Nuclease ◽  
Comparative Assessment ◽  
Side Chain ◽  
Folding Pathway ◽  
Hydrophobic Core ◽  
Folding Pathways ◽  
Partial Unfolding ◽  
Protein Folding Pathways ◽  
Similar Description

Multidimensional NMR intrinsically provides multiple probes that can be used for deciphering the folding pathways of proteins: NH amide and CH groups are strategically located on the backbone of the protein, while CH3 groups, on the side-chain of methylated residues, are involved in important stabilizing interactions in the hydrophobic core. Combined with high hydrostatic pressure, these observables provide a powerful tool to explore the conformational landscapes of proteins. In the present study, we made a comparative assessment of the NH, CH, and CH3 groups for analyzing the unfolding pathway of ∆+PHS Staphylococcal Nuclease. These probes yield a similar description of the folding pathway, with virtually identical thermodynamic parameters for the unfolding reaction, despite some notable differences. Thus, if partial unfolding begins at identical pressure for these observables (especially in the case of backbone probes) and concerns similar regions of the molecule, the residues involved in contact losses are not necessarily the same. In addition, an unexpected slight shift toward higher pressure was observed in the sequence of the scenario of unfolding with CH when compared to amide groups.

scholarly journals Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29

2018 ◽  
Vol 293 (47) ◽  
pp. 18296-18308 ◽  
Author(s):  
Chelsea Vickers ◽  
Feng Liu ◽  
Kento Abe ◽  
Orly Salama-Alber ◽  
Meredith Jenkins ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Biological Activities ◽  
Bacterial Species ◽  
Structural Data ◽  
Side Chain ◽  
Glycoside Hydrolase Family ◽  
X Ray Crystallography ◽  
Catalytic Mechanisms ◽  
Thickening Agents ◽  
Hydrolase Family

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

scholarly journals Methylofuran is a prosthetic group of the formyltransferase/hydrolase complex and shuttles one-carbon units between two active sites

2019 ◽  
Vol 116 (51) ◽  
pp. 25583-25590 ◽  
Author(s):  
Jethro L. Hemmann ◽  
Tristan Wagner ◽  
Seigo Shima ◽  
Julia A. Vorholt
Keyword(s):  
Active Sites ◽  
Bacterial Species ◽  
Methanogenic Archaea ◽  
Side Chain ◽  
Strong Interactions ◽  
Prosthetic Group ◽  
Additional Function ◽  
Amide Bonds ◽  
Consecutive Reactions ◽  
The One

Methylotrophy, the ability of microorganisms to grow on reduced one-carbon substrates such as methane or methanol, is a feature of various bacterial species. The prevailing oxidation pathway depends on tetrahydromethanopterin (H4MPT) and methylofuran (MYFR), an analog of methanofuran from methanogenic archaea. Formyltransferase/hydrolase complex (Fhc) generates formate from formyl-H4MPT in two consecutive reactions where MYFR acts as a carrier of one-carbon units. Recently, we chemically characterized MYFR from the model methylotrophMethylorubrum extorquensand identified an unusually long polyglutamate side chain of up to 24 glutamates. Here, we report on the crystal structure of Fhc to investigate the function of the polyglutamate side chain in MYFR and the relatedness of the enzyme complex with the orthologous enzymes in archaea. We identified MYFR as a prosthetic group that is tightly, but noncovalently, bound to Fhc. Surprisingly, the structure of Fhc together with MYFR revealed that the polyglutamate side chain of MYFR is branched and contains glutamates with amide bonds at both their α- and γ-carboxyl groups. This negatively charged and branched polyglutamate side chain interacts with a cluster of conserved positively charged residues of Fhc, allowing for strong interactions. The MYFR binding site is located equidistantly from the active site of the formyltransferase (FhcD) and metallo-hydrolase (FhcA). The polyglutamate serves therefore an additional function as a swinging linker to shuttle the one-carbon carrying amine between the two active sites, thereby likely increasing overall catalysis while decreasing the need for high intracellular MYFR concentrations.

Metabolic engineering of strains of Ralstonia eutropha and Pseudomonas putida for biotechnological production of 2-methylcitric acid

2006 ◽  
Vol 8 (6) ◽  
pp. 587-602 ◽  
Author(s):  
Christian Ewering ◽  
Florian Heuser ◽  
Jens Klaus Benölken ◽  
Christian O. Brämer ◽  
Alexander Steinbüchel
Keyword(s):  
Metabolic Engineering ◽  
Pseudomonas Putida ◽  
Ralstonia Eutropha ◽  
Biotechnological Production

scholarly journals Identification of chromosomally integrated TOL DNA in cured derivatives of Pseudomonas putida PAW1

1982 ◽  
Vol 152 (2) ◽  
pp. 911-914
Author(s):  
P Meulien ◽  
P Broda
Keyword(s):  
Pseudomonas Putida ◽  
Parental Strain ◽  
Chromosomal Dna ◽  
Tol Plasmid ◽  
Derivatives Of

Some plasmid-free Tol- strains derived from Pseudomonas putida PAW1 (which carries the TOL plasmid pWW0) have a segment of TOL DNA located chromosomally. Of three independently isolated strains, PAW86 had an integrated TOL segment of 16 kilobases and PAW85 had two copies of this segment in different chromosomal locations, whereas the chromosomal DNA of PAW82 showed no homology with the TOL plasmid. In cultures of the parental strain, it appears that a 56-kilobase TOL DNA segment is located chromosomally in some cells.

Cloning and molecular analysis of promoter-like sequences isolated from the chromosomal DNA ofLactobacillus acidophilusATCC 4356

1997 ◽  
Vol 43 (1) ◽  
pp. 61-69 ◽  
Author(s):  
G. Djordjevic ◽  
B. Bojovic ◽  
N. Miladinov ◽  
L. Topisirovic
Keyword(s):  
Lactococcus Lactis ◽  
Molecular Analysis ◽  
Lactobacillus Acidophilus ◽  
Promoter Activity ◽  
Lactobacillus Reuteri ◽  
Bacterial Species ◽  
Chromosomal Dna ◽  
Complementary Dna ◽  
E Coli ◽  
Dna Strand

Promoter-like sequences from the chromosomal DNA of thermophilic strain Lactobacillus acidophilus ATCC 4356 were cloned. Analysis of the three DNA fragments showing promoter activity, designated P3, P6, and P15, were performed in Lactobacillus reuteri, Lactococcus lactis, and E. coli. The reporter cat-86 gene was expressed in all three bacterial species under control of the fragments P3 and P6. Fragment P15 showed promoter activity only in Lactobacillus reuteri and E. coli but not in Lactococcus lactis. The three host-specific transcriptional start points (TSPs) were used when transcription of the cat-86 gene was controlled by fragment P3 in Lactobacillus reuteri, E. coli, and Lactococcus lactis. Similarly, fragment P15 initiated transcription of the cat-86 gene at two distinctive sites in Lactobacillus reuteri and E. coli. Only within fragment P6, a common TSP was used in Lactobacillus reuteri and E. coli, but different from that used in Lactococcus lactis. Each TSP was preceded by the putative −35 and −10 hexamers. Computer analysis of the fragment P3 sequence revealed the existence of divergent promoterlike sequence (P3rev) located on the complementary DNA strand. Fragments P6 and P15 were also functional in Lactobacillus acidophilus ATCC 4356 from which chromosomal DNA they were originally cloned.Key words: Lactobacillus acidophilus, promoter-like sequences, regulation.

scholarly journals An alkylaminoquinazoline restores antibiotic activity in Gram-negative resistant isolates

Microbiology ◽  
2011 ◽  
Vol 157 (2) ◽  
pp. 566-571 ◽  
Author(s):  
Abdallah Mahamoud ◽  
Jacqueline Chevalier ◽  
Milad Baitiche ◽  
Elissavet Adam ◽  
Jean-Marie Pagès
Keyword(s):  
Efflux Pump ◽  
Bacterial Species ◽  
Efflux Pumps ◽  
Multidrug Resistant ◽  
Side Chain ◽  
Structural Class ◽  
Gram Negative ◽  
Activity Spectrum ◽  
Efflux Pump Inhibitors ◽  
Drug Efflux Pump

To date, various bacterial drug efflux pump inhibitors (EPIs) have been described. They exhibit variability in their activity spectrum with respect to antibiotic structural class and bacterial species. Among the various 4-alkylaminoquinazoline derivatives synthesized and studied in this work, one molecule, 1167, increased the susceptibility of important human-pathogenic, resistant, Gram-negative bacteria towards different antibiotic classes. This 4-(3-morpholinopropylamino)-quinazoline induced an increase in the activity of chloramphenicol, nalidixic acid, norfloxacin and sparfloxacin, which are substrates of the AcrAB-TolC and MexAB-OprM efflux pumps that act in these multidrug-resistant isolates. In addition, 1167 increased the intracellular concentration of chloramphenicol in efflux pump-overproducing strains. The rate of restoration depended on the structure of the antibiotic, suggesting that different sites in the efflux pumps may be involved. A molecule exhibiting a morpholine functional group and a propyl extension of the side chain was more active.

Polyquinolines: Multifunctional Polymers for Electro-Optic and Light-Emitting Applications

1999 ◽  
Vol 558 ◽  
Author(s):  
Alex K.-Y. Jen ◽  
Hong Ma
Keyword(s):  
Electrical Characterization ◽  
Bright Light ◽  
Side Chain ◽  
Light Emitting ◽  
Electro Optic ◽  
Film Forming ◽  
Hole Transporting ◽  
Broad Variation ◽  
Nlo Chromophores ◽  
Multifunctional Polymers

ABSTRACTA versatile, and generally applicable modular approach for making second-order nonlinear optical (NLO) side-chain aromatic polyquinolines has been developed. This approach emphasizes the ease of incorporating NLO chromophores onto the pendent phenyl moieties of parent polyquinolines at the final stage via mild Mitsunobu reaction. This method provides the synthesis of polyquinolines with a broad variation of the polymer backbones and great flexibility in the selection of NLO chromophores. These side-chain NLO polyquinolines demonstrate high electro-optic (E-O) activity (up to 35 pm/V at 830 nm and 22 pm/V at 1300 nm, respectively) and a good combination of thermal, optical, electrical and mechanical properties.Comparatively, two new electroluminescent (EL) polyquinolines have been prepared via the Friedlander condensation and nucleophilic reaction. The resulting polymers contain a bipolar property with both an efficient hole-transporting moiety, tetraphenyldiaminobiphenyl (TPD), and an electron affinitive light-emitting moiety, bis-quinoline. In addition, they possess high thermal stability, excellent electrochemical reversibility, good thin film-forming ability, and bright light-emitting property. Electrical characterization of two-layer diode devices based on the configurations of ITO/CuPc/TPD-PQ or TPD-PQE/Al showed excellent electroluminescence performance (a rectification ratio greater than 105 and a low turn-on voltage of less than 4 V).

scholarly journals Natural Transformation of Acinetobactersp. Strain BD413 with Cell Lysates of Acinetobacter sp.,Pseudomonas fluorescens, and Burkholderia cepaciain Soil Microcosms

2000 ◽  
Vol 66 (1) ◽  
pp. 206-212 ◽  
Author(s):  
Kaare M. Nielsen ◽  
Kornelia Smalla ◽  
Jan D. van Elsas
Keyword(s):  
Pseudomonas Fluorescens ◽  
Burkholderia Cepacia ◽  
Natural Transformation ◽  
Bacterial Species ◽  
Physical Stability ◽  
Biological Significance ◽  
Bacterial Cells ◽  
Chromosomal Dna ◽  
Cell Lysates

ABSTRACT To elucidate the biological significance of dead bacterial cells in soil to the intra- and interspecies transfer of gene fragments by natural transformation, we have exposed the kanamycin-sensitive recipient Acinetobacter sp. strain BD413(pFG4) to lysates of the kanamycin-resistant donor bacteria Acinetobacterspp., Pseudomonas fluorescens, and Burkholderia cepacia. Detection of gene transfer was facilitated by the recombinational repair of a partially (317 bp) deleted kanamycin resistance gene in the recipient bacterium. The investigation revealed a significant potential of these DNA sources to transformAcinetobacter spp. residing both in sterile and in nonsterile silt loam soil. Heat-treated (80°C, 15 min) cell lysates were capable of transforming strain BD413 after 4 days of incubation in sterile soil and for up to 8 h in nonsterile soil. Transformation efficiencies obtained in vitro and in situ with the various lysates were similar to or exceeded those obtained with conventionally purified DNA. The presence of cell debris did not inhibit transformation in soil, and the debris may protect DNA from rapid biological inactivation. Natural transformation thus providesAcinetobacter spp. with an efficient mechanism to access genetic information from different bacterial species in soil. The relatively short-term biological activity (e.g., transforming activity) of chromosomal DNA in soil contrasts the earlier reported long-term physical stability of DNA, where fractions have been found to persist for several weeks in soil. Thus, there seems to be a clear difference between the physical and the functional significance of chromosomal DNA in soil.

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