Papers by Patrick Hallenbeck
Indigenous strains were screened for utilization of glycerol and xylose. Yields of lipids and bio... more Indigenous strains were screened for utilization of glycerol and xylose. Yields of lipids and biomass are likely to be improved in future optimization studies. Lipid productivity from glycerol of some was higher than previously reported. One strain gained increased biomass in the light with both xylose and glycerol. For the first time xylose utilization by a microalga is shown. a b s t r a c t Microalgae are a promising alternative for sustainable biofuel production, but production yields and costs present a significant bottleneck. Here, the use of glycerol and xylose to boost the lipid yield was evaluated using ten strains from the Université de Montréal collection of microalgae. This report shows that some microalgal strains are capable of mixotrophic and heterotrophic growth on xylose, the major carbon source found in wastewater streams from pulp and paper industries, with an increase in growth rate of 2.8-fold in comparison to photoautotrophic growth, reaching up to l = 1.1/d. On glycerol, growth rates reached as high as l = 1.52/d. Lipid productivity increased up to 370% on glycerol and 180% on xylose for the strain LB1H10, showing the suitability of this strain for further development of biofuels production through mixotrophic cultivation.
Journal of Biological Chemistry
The soluble form of a bacteriophage-induced endo-N-acetylneuraminidase (Endo-N) specific for hydr... more The soluble form of a bacteriophage-induced endo-N-acetylneuraminidase (Endo-N) specific for hydrolyzing oligo-or poly-a-2,s-linked sialosyl units in sources as disparate as bacterial and neural membrane glycoconjugates was purified approximately 10,000fold and characterized. The enzyme appears homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a subunit M , 105,000. This corresponds to one of the higher M , phage proteins which comprises 7.6% (by weight) of the total phage protein. The holoenzyme is active at neutral pH and has a M , by gel filtration of 328,000, suggesting that the active enzyme is a trimer. Endo-N requires a minimum of 6 sialyl residues (DP6, where DP represents degree of polymerization) for activity. The limit digest products from the a-2,s-linked polysialic acid capsule of Escherichia coli K1 are DP4 with some DP3 and DP1,2. DP2-4 do not appear to inhibit depolymerization of polysialic acid. Endo-N digestion of the polysialosyl moiety on neural cell adhesion molecules yields sialyl oligomers with DP3 and DP4. The presence of a terminal sialitol changes both the distribution of limit digestion products and the apparent minimum substrate size. Higher M . a-2,s-linked sialyl polymers (-DP200) are better substrates (K,,, 60-70 PM) than sialyl oligomers of -DP10-20 ( K , 1.2 mM). Endo-N activity is inhibited by DNA and several other polyanions tested. An examination of the distribution of intermediate products shows that Endo-N binds and cleaves at random sites on the polysialosyl chains, in contrast to initiating cleavage at one end and depolymerizing processively. Endo-Ncan serve as a specific molecular probe to detect and selectively modify polya-2,s-sialosyl carbohydrate units which have been implicated in bacterial meningitis and neural cell adhesion.
Analytical Biochemistry, 1987
A rapid procedure utilizing high-performance liquid chromatography was developed for the separati... more A rapid procedure utilizing high-performance liquid chromatography was developed for the separation of homooligomers of sialic acid (N-acetylneuraminic acid). The method utilizes the anion exchanger Mono-Q HR 5/5 and can resolve sialyl oligomers with degrees of polymerization (DP) from 2 to 20 in 25 min. Previous methods required 1 to 9 days. Recoveries are quantitative and the method can be used either analytically to analyze the enzymatic digestion products of polysialic acid or semipreparatively to prepare sialyl oligomers of defined length. The method is potentially useful for analyzing other anionic oligosaccharides.
Journal of Bacteriology, 2007
Both Rhodobacter capsulatus PII homologs GlnB and GlnK were found to be necessary for the proper ... more Both Rhodobacter capsulatus PII homologs GlnB and GlnK were found to be necessary for the proper regulation of nitrogenase activity and modification in response to an ammonium shock. As previously reported for several other bacteria, ammonium addition triggered the AmtB-dependent association of GlnK with the R. capsulatus membrane. Native polyacrylamide gel electrophoresis analysis indicates that the modification/ demodification of one PII homolog is aberrant in the absence of the other. In a glnK mutant, more GlnB was found to be membrane associated under these conditions. In a glnB mutant, GlnK fails to be significantly sequestered by AmtB, even though it appears to be fully deuridylylated. Additionally, the ammonium-induced enhanced sequestration by AmtB of the unmodifiable GlnK variant GlnK-Y51F follows the wild-type GlnK pattern with a high level in the cytoplasm without the addition of ammonium and an increased level in the membrane fraction after ammonium treatment. These results suggest that factors other than PII modification are driving its association with AmtB in the membrane in R. capsulatus.
Journal of Bacteriology, 2008
Analytical Biochemistry, 1998
Archives of Microbiology, 2000
Rhodobacter capsulatus modulates its in vivo nitrogenase activity in the light in response to the... more Rhodobacter capsulatus modulates its in vivo nitrogenase activity in the light in response to the addition of NH4+ in a variety of ways: with ADP-ribosylation of the Fe-protein of nitrogenase, with a switch-off response that is independent of ADP-ribosylation, and with a "magnitude response." In the light, these responses are differentially shown by cultures that differ in the degree of their nitrogen limitation. Here we examined the response of these culture types to the addition of NH4+ under dark, microoxic conditions and found that all three responses can be observed under these conditions. However, the magnitude response was much more sensitive to the ammonium concentration, and the ADP-ribosylation response correlated only poorly with activity changes, similar to results obtained in the light. In contrast to previous reports, Fe-protein was not ADP-ribosylated in response to the presence of oxygen.
Biochimica Et Biophysica Acta-bioenergetics, 1998
Pyruvate:ferredoxin (flavodoxin) oxidoreductase (POR) was purified 3050-fold to apparent homogene... more Pyruvate:ferredoxin (flavodoxin) oxidoreductase (POR) was purified 3050-fold to apparent homogeneity from the photosynthetic bacterium Rhodobacter capsulatus using ion-exchange, Reactive Red, and gel filtration chromatography. The isolated enzyme was sensitive to dilution and oxygen (especially when in dilute solution). The molecular mass of the native enzyme was determined by high performance liquid chromatography gel filtration to be 270+/-20 kDa. Since a subunit molecular mass of 130+/-5 kDa was found by denaturing gel electrophoresis, POR from R. capsulatus thus appears to be a homodimer. Electron paramagnetic resonance analysis showed that a free radical was formed upon the addition of pyruvate. This POR is shown to be an indiscriminate electron donor causing the full reduction of R. capsulatus flavodoxin (Fld), R. capsulatus ferredoxin I (FdI), R. capsulatus ferredoxin II (FdII), as well as the major plant-type ferredoxin (FdI) from Anabaena variabilis. The purified enzyme can couple the oxidation of pyruvate to the reduction of nitrogenase in a coupled system with either R. capsulatus ferredoxins or nif-specific flavodoxin, NifF; (Fld>FdI>FdII). Immunoblot analysis shows that R. capsulatus POR is constitutively synthesized, with synthesis augmented under nitrogen-fixing conditions (34+/-13%) and decreased in acetate and aerobically grown cells.
Microbiology-sgm, 2003
In most bacteria, nitrogen metabolism is tightly regulated and P II proteins play a pivotal role ... more In most bacteria, nitrogen metabolism is tightly regulated and P II proteins play a pivotal role in the regulatory processes. Rhodobacter capsulatus possesses two genes (glnB and glnK ) encoding P II -like proteins. The glnB gene forms part of a glnB-glnA operon and the glnK gene is located immediately upstream of amtB, encoding a (methyl-) ammonium transporter. Expression of glnK is activated by NtrC under nitrogen-limiting conditions. The synthesis and activity of the molybdenum and iron nitrogenases of R. capsulatus are regulated by ammonium on at least three levels, including the transcriptional activation of nifA1, nifA2 and anfA by NtrC, the regulation of NifA and AnfA activity by two different NtrC-independent mechanisms, and the post-translational control of the activity of both nitrogenases by reversible ADP-ribosylation of NifH and AnfH as well as by ADP-ribosylation independent switch-off. Mutational analysis revealed that both P II -like proteins are involved in the ammonium regulation of the two nitrogenase systems. A mutation in glnB results in the constitutive expression of nifA and anfA. In addition, the post-translational ammonium inhibition of NifA activity is completely abolished in a glnB-glnK double mutant. However, AnfA activity was still suppressed by ammonium in the glnB-glnK double mutant. Furthermore, the P II -like proteins are involved in ammonium control of nitrogenase activity via ADP-ribosylation and the switch-off response. Remarkably, in the glnB-glnK double mutant, all three levels of the ammonium regulation of the molybdenum (but not of the alternative) nitrogenase are completely circumvented, resulting in the synthesis of active molybdenum nitrogenase even in the presence of high concentrations of ammonium.
Journal of Bacteriology, 2007
Molecular Microbiology, 2009
Members of the Amt/Rh family of transporters are found almost ubiquitously in all forms of life. ... more Members of the Amt/Rh family of transporters are found almost ubiquitously in all forms of life. However, the molecular state of the substrate (NH3 or NH4+) has been the subject of active debate. At least for bacterial Amt proteins, the model emerging from computational, X-ray crystal and mutational analysis is that NH4+ is deprotonated at the exterior, conducted through the membrane as NH3, and reprotonated at the cytoplasmic interface. A proton concomitantly is transferred from the exterior to the interior, although the mechanism is unclear. Here we discuss recent evidence indicating that an important function of at least some eukaryotic and bacterial Amts is to act as ammonium sensors and regulate cellular metabolism in response to changes in external ammonium concentrations. This is now well documented in the regulation of yeast pseudohyphal development and filamentous growth. As well, membrane sequestration of GlnK, a PII signal transduction protein, by AmtB has been shown to regulate nitrogenase in some diazotrophs, and nitrogen metabolism in some Gram-positive bacteria. Formation of GlnK–AmtB membrane complexes might have other, as yet undiscovered, regulatory roles. This possibility is emphasized by the discovery in some genomes of genes for chimeric Amts with fusions to various regulatory elements.
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Papers by Patrick Hallenbeck