bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interactions between Carbon and Nitrogen Metabolism in
Fibrobacter succinogenes
S85: a
1
H and
13
C Nuclear Magnetic Resonance and Enzymatic Study
Authors: Matheron Christelle , Delort Anne-Marie , Gaudet Genevieve , Liptaj Tibor , Forano Evelyne 
Published: December 2019

ABSTRACT

The effect of the presence of ammonia on [1-
13
C]glucose metabolism in the rumen fibrolytic bacterium
Fibrobacter succinogenes
S85 was studied by
13
C and
1
H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The
13
C enrichment of succinate and acetate was precisely quantified by
13
C-filtered spin-echo difference
1
H-NMR spectroscopy. The presence of ammonia did not modify the
13
C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the
13
C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by
13
C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in
F. succinogenes
were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and
1
H NMR. When cells were incubated in vivo with [1-
13
C]glucose, only
13
C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.
Adhesion of
Acinetobacter venetianus
to Diesel Fuel Droplets Studied with In Situ Electrochemical and Molecular Probes
Authors: Baldi Franco , Ivos̆ević Nadica , Minacci Andrea , Pepi Milva , Fani Renato , Svetlic̆ić Vesna , Z̆utić Vera 
Published: December 2019

ABSTRACT

The adhesion of a recently described species,
Acinetobacter venetianus
VE-C3 (F. Di Cello, M. Pepi, F. Baldi, and R. Fani, Res. Microbiol. 148:237–249, 1997), to diesel fuel (a mixture of C
12
to C
28
n
-alkanes) and
n
-hexadecane was studied and compared to that of
Acinetobacter
sp. strain RAG-1, which is known to excrete the emulsifying lipopolysaccharide, emulsan. Oxygen consumption rates, biomass, cell hydrophobicity, electrophoretic mobility, and zeta potential were measured for the two strains. The dropping-mercury electrode (DME) was used as an in situ adhesion sensor. In seawater, RAG-1 was hydrophobic, with an electrophoretic mobility (μ) of −0.38 × 10
−8
m
2
V
−1
s
−1
and zeta potential (ζ) of −4.9 mV, while VE-C3 was hydrophilic, with μ of −0.81 × 10
−8
m
2
V
−1
s
−1
and ζ of −10.5 mV. The microbial adhesion to hydrocarbon (MATH) test showed that RAG-1 was always hydrophobic whereas the hydrophilic VE-C3 strain became hydrophobic only after exposure to
n
-alkanes. Adhesion of VE-C3 cells to diesel fuel was partly due to the production of capsular polysaccharides (CPS), which were stained with the lectin concanavalin A (ConA) conjugated to fluorescein isothiocyanate and observed in situ by confocal microscopy. The emulsan from RAG-1, which was negative to ConA, was stained with Nile Red fluorochrome instead. Confocal microscope observations at different times showed that VE-C3 underwent two types of adhesion: (i) cell-to-cell interactions, preceding the cell adhesion to the
n
-alkane, and (ii) incorporation of nanodroplets of
n
-alkane into the hydrophilic CPS to form a more hydrophobic polysaccharide–
n
-alkane matrix surrounding the cell wall. The incorporation of
n
-alkanes as nanodroplets into the CPS of VE-C3 cells might ensure the partitioning of the bulk apolar phase between the aqueous medium and the outer cell membrane and thus sustain a continuous growth rate over a prolonged period.
Detection of
Salmonella
spp. and
Listeria monocytogenes
in Suspended Organic Waste by Nucleic Acid Extraction and PCR
Authors: Burtscher Carola , Fall Papa A. , Wilderer Peter A. , Wuertz Stefan 
Published: December 2019

ABSTRACT

A nucleic acid-based method for the detection of the bacterial pathogens
Salmonella
spp. and
Listeria monocytogenes
in biological waste was developed. The detection limits were less than 10 cells per ml of biological waste. The method does not include a phenol extraction step and can be easily performed in 1 to 2 days.
Whey Fermentation by
Anaerobiospirillum succiniciproducens
for Production of a Succinate-Based Animal Feed Additive
Authors: Samuelov Nissim S. , Datta Rathin , Jain Mahendra K. , Zeikus J. Gregory 
Published: December 2019

ABSTRACT

Anaerobic fermentation processes for the production of a succinate-rich animal feed supplement from raw whey were investigated with batch, continuous, and variable-volume fed-batch cultures with
Anaerobiospirillum succiniciproducens
. The highest succinate yield, 90%, was obtained in a variable-volume fed-batch process in comparison to 80% yield in a batch cultivation mode. In continuous culture, succinate productivity was 3 g/liter/h, and the yield was 60%. Under conditions of excess CO
2
, more than 90% of the whey-lactose was consumed, with an end product ratio of 4 succinate to 1 acetate. Under conditions of limited CO
2
, lactose was only partially consumed and lactate was the major end product, with lower levels of ethanol, succinate, and acetate. When the succinic acid in this fermentation product was added to rumen fluid, it was completely consumed by a mixed rumen population and was 90% decarboxylated to propionate on a molar basis. The whey fermentation product formed under excess CO
2
, which contained mainly organic acids and cells, could potentially be used as an animal feed supplement.
Expression of the
Escherichia coli pntA
and
pntB
Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in
Saccharomyces cerevisiae
and Its Effect on Product Formation during Anaerobic Glucose Fermentation
Authors: Anderlund Mikael , Nissen Torben L. , Nielsen Jens , Villadsen John , Rydström Jan , Hahn-Hägerdal Bärbel , Kielland-Brandt Morten C. 
Published: December 2019

ABSTRACT

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant
Saccharomyces cerevisiae
expressing the membrane-bound transhydrogenase from
Escherichia coli
. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD
+
in
S. cerevisiae
and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD
+
by NADPH and by NADH in the presence of NADP
+
, which demonstrated that an active enzyme was present. Unlike the situation in
E. coli
, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD
+
, NADPH, and NADP
+
were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD
+
.
Overexpression of the
Saccharomyces cerevisiae
Mannosylphosphodolichol Synthase-Encoding Gene in
Trichoderma reesei
Results in an Increased Level of Protein Secretion and Abnormal Cell Ultrastructure
Authors: Kruszewska Joanna S. , Butterweck Arno H. , Kurza˛tkowski Wiesław , Migdalski Andrzej , Kubicek Christian P. , Palamarczyk Graz˙yna 
Published: December 2019

ABSTRACT

Production of extracellular proteins plays an important role in the physiology of
Trichoderma reesei
and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in
T. reesei
the
DPM1
gene of
Saccharomyces cerevisiae
, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated
DPM1
, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of
cbh1
, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of the
DPM1
-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in
Trichoderma
can result in improved protein secretion.
Hyperproduction of Tryptophan by
Corynebacterium glutamicum
with the Modified Pentose Phosphate Pathway
Authors: Ikeda Masato , Katsumata Ryoichi 
Published: December 2019

ABSTRACT

A classically derived tryptophan-producing
Corynebacterium glutamicum
strain was recently significantly improved both by plasmid-mediated amplification of the genes for the rate-limiting enzymes in the terminal pathways and by construction of a plasmid stabilization system so that it produced more tryptophan. This engineered strain, KY9218 carrying pKW9901, produced 50 g of tryptophan per liter from sucrose after 80 h in fed-batch cultivation without antibiotic pressure. Analysis of carbon balances showed that at the late stage of the fermentation, tryptophan yield decreased with a concomitant increase in CO
2
yield, suggesting a transition in the distribution of carbon flow from aromatic biosynthesis toward the tricarboxylic acid cycle via glycolysis. To circumvent this transition by increasing the supply of erythrose 4-phosphate, a direct precursor of aromatic biosynthesis, the transketolase gene of
C. glutamicum
was coamplified in the engineered strain by using low- and high-copy-number plasmids which were compatible with the resident plasmid pKW9901. The presence of the gene in low copy numbers contributed to improvement of tryptophan yield, especially at the late stage, and led to accumulation of more tryptophan (57 g/liter) than did its absence, while high-copy-number amplification of the gene resulted in a tryptophan production level even lower than that resulting from the absence of the gene due to reduced growth and sugar consumption. In order to assemble all the cloned genes onto a low-copy-number plasmid, the high-copy-number origin of pKW9901 was replaced with the low-copy-number one, generating low-copy-number plasmid pSW9911, and the transketolase gene was inserted to yield pIK9960. The pSW9911-carrying producer showed almost the same fermentation profiles as the pKW9901 carrier in fed-batch cultivation without antibiotic pressure. Under the same culture conditions, however, the pIK9960 carrier achieved a final tryptophan titer of 58 g/liter, which represented a 15% enhancement over the titers achieved by the pKW9901 and pSW9911 carriers.
Characterization of a
Pseudomonas putida
Allylic Alcohol Dehydrogenase Induced by Growth on 2-Methyl-3-Buten-2-ol
Authors: Malone Vincent F. , Chastain Amy J. , Ohlsson John T. , Poneleit Loelle S. , Nemecek-Marshall Michele , Fall Ray 
Published: December 2019

ABSTRACT

We have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-MB), which is produced and emitted by certain pines. To this end we have isolated the soil bacterium
Pseudomonas putida
MB-1, which uses 232-MB as a sole carbon source. Strain MB-1 contains inducible 3-methyl-2-buten-1-ol (321-MB) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-MB is metabolized by isomerization to 321-MB followed by oxidation. 321-MB dehydrogenase was purified to near-homogeneity and found to be a tetramer (151 kDa) with a subunit mass of 37,700 Da. It catalyzes NAD
+
-dependent, reversible oxidation of 321-MB to 3-methyl-2-buten-1-al. The optimum pH for the oxidation reaction was 10.0, while that for the reduction reaction was 5.4. 321-MB dehydrogenase oxidized a wide variety of aliphatic and aromatic alcohols but exhibited the highest catalytic specificity with allylic or benzylic substrates, including 321-MB, 3-chloro-2-buten-1-ol, and 3-aminobenzyl alcohol. The N-terminal sequence of the enzyme contained a region of 64% identity with the TOL plasmid-encoded benzyl alcohol dehydrogenase of
P. putida
. The latter enzyme and the chromosomally encoded benzyl alcohol dehydrogenase of
Acinetobacter calcoaceticus
were also found to catalyze 321-MB oxidation. These findings suggest that 321-MB dehydrogenase and other bacterial benzyl alcohol dehydrogenases are broad-specificity allylic and benzylic alcohol dehydrogenases that, in conjunction with a 232-MB isomerase, might be useful in an enzyme-linked assay for 232-MB.
Optimization of Simultaneous Chemical and Biological Mineralization of Perchloroethylene
Authors: Büyüksönmez Fati˙h , Hess Thomas F. , Crawford Ronald L. , Paszczynski Andrzej , Watts Richard J. 
Published: December 2019

ABSTRACT

Optimization of the simultaneous chemical and biological mineralization of perchloroethylene (PCE) by modified Fenton’s reagent and
Xanthobacter flavus
was investigated by using a central composite rotatable experimental design. Concentrations of PCE, hydrogen peroxide, and ferrous iron and the microbial cell number were set as variables. Percent mineralization of PCE to CO
2
was investigated as a response. A second-order, quadratic response surface model was generated and fit the data adequately, with a correlation coefficient of 0.72. Analysis of the results showed that the PCE concentration had no significant effect within the tested boundaries of the model, while the other variables, hydrogen peroxide and iron concentrations and cell number, were significant at α = 0.05 for the mineralization of PCE. The
14
C radiotracer studies showed that the simultaneous chemical and biological reactions increased the extent of mineralization of PCE by more than 10% over stand-alone Fenton reactions.
Delineation of Key Amino Acid Side Chains and Peptide Domains for Antimicrobial Properties of Divercin V41, a Pediocin-Like Bacteriocin Secreted by
Carnobacterium divergens
V41
Authors: Bhugaloo-Vial Parwin , Douliez Jean-Paul , Mollé Daniel , Dousset Xavier , Boyaval Patrick , Marion Didier 
Published: December 2019

ABSTRACT

Divercin V41 (DV41) is a class IIa bacteriocin produced by
Carnobacterium divergens
V41. This antilisterial peptide is homologous to pediocin PA-1 and contains two disulfide bonds. To establish the structure-activity relationships of this specific family of bacteriocin, chemical modifications and enzymatic hydrolysis were performed on DV41. Alteration of the net charge of this cationic bacteriocin by succinylation and acetylation revealed that, in a certain range, the electrostatic interactions were surprisingly not necessary for the activity of DV41. Cleavage of DV41 by endoproteinase Asp-N released two fragments N1[1–17] and N2[18–43] corresponding to the conserved hydrophilic N-terminal and the variable hydrophobic C-terminal sequences, respectively. Inhibitory assays showed that only the C-terminal fragment was active, and after trypsin cleavage at Lys42 or disulfide reduction it lost its inhibitory activity. These results suggested that both hydrophobicity and folding imposed by the Cys25-Cys43 disulfide bond were essential for antilisterial activity of the C-terminal hydrophobic peptide. Chemical oxidation of tryptophan residues by N-bromosuccinimide demonstrated that these residues were crucial for inhibitory activity since modification of any one of them rendered DV41 inactive. On the contrary, only the modification of all the three tyrosine residues caused a total loss of antilisterial activity. These latter results strengthened previous results suggesting that the N-terminal domain containing the YGNGV consensus sequence was not involved in the binding of DV41 to a potential specific receptor on listerial cells.
Evaluation of Bottlenecks in the Late Stages of Protein Secretion in
Bacillus subtilis
Authors: Bolhuis Albert , Tjalsma Harold , Smith Hilde E. , de Jong Anne , Meima Rob , Venema Gerard , Bron Sierd , van Dijl Jan Maarten 
Published: December 2019

ABSTRACT

Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by
Bacillus subtilis
is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins:
Bacillus lichenifomis
α-amylase (AmyL),
Escherichia coli
TEM β-lactamase (Bla), human pancreatic α-amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of
B. subtilis
cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.
Isolation and Characterization of a Sulfate-Reducing Bacterium That Anaerobically Degrades Alkanes
Authors: So Chi Ming , Young L. Y. 
Published: December 2019

ABSTRACT

An alkane-degrading, sulfate-reducing bacterial strain, AK-01, was isolated from an estuarine sediment with a history of chronic petroleum contamination. The bacterium is a short, nonmotile, non-spore-forming, gram-negative rod. It is mesophilic and grows optimally at pH 6.9 to 7.0 and at an NaCl concentration of 1%. Formate, fatty acids (C
4
to C
16
) and hydrogen were readily utilized as electron donors. Sulfate, sulfite, and thiosulfate were used as electron acceptors, but sulfur, nitrite, and nitrate were not. Phenotypic characterization and phylogenetic analysis based on 16S rRNA gene sequence indicate that AK-01 is most closely related to the genera
Desulfosarcina
,
Desulfonema
, and
Desulfococcus
in the delta subdivision of the class
Proteobacteria
. It is phenotypically and phylogenetically different from strains Hxd3 and TD3, two previously reported isolates of alkane-degrading, sulfate-reducing bacteria. The alkanes tested to support growth of AK-01 had chain lengths of C
13
to C
18
. 1-Alkenes (C
15
and C
16
) and 1-alkanols (C
15
and C
16
) also supported growth. The doubling time for growth on hexadecane was 3 days, about four times longer than that for growth on hexadecanoate. Mineralization of hexadecane was indicated by the recovery of
14
CO
2
from cultures grown on [1-
14
C]hexadecane. Degradation of hexadecane was dependent on sulfate reduction. The stoichiometric ratio (as moles of sulfate reduced per mole of hexadecane degraded) was 10.6, which is very close to the theoretical ratio of 12.25, assuming a complete oxidation to CO
2
. Anaerobic alkane degradation by sulfate reducers may be a more widespread phenomenon than was previously thought.
Composition of Toluene-Degrading Microbial Communities from Soil at Different Concentrations of Toluene
Authors: Hubert Casey , Shen Yin , Voordouw Gerrit 
Published: December 2019

ABSTRACT

Toluene-degrading bacteria were isolated from hydrocarbon-contaminated soil by incubating liquid enrichment cultures and agar plate cultures in desiccators in which the vapor pressure of toluene was controlled by dilution with vacuum pump oil. Incubation in desiccators equilibrated with either 100, 10, or 1% (wt/wt) toluene in vacuum pump oil and testing for genomic cross-hybridization resulted in four genomically distinct strains (standards) capable of growth on toluene (strains Cstd1, Cstd2, Cstd5, and Cstd7). The optimal toluene concentrations for growth of these standards on plating media differed considerably. Cstd1 grew best in an atmosphere equilibrated with 0.1% (wt/wt) toluene, but Cstd5 failed to grow in this atmosphere. Conversely, Cstd5 grew well in the presence of 10% (wt/wt) toluene, which inhibited growth of Cstd1. 16S ribosomal DNA sequencing and cross-hybridization analysis indicated that both Cstd1 and Cstd5 are members of the genus
Pseudomonas
. An analysis of the microbial communities in soil samples that were incubated with 10% (wt/wt) toluene with reverse sample genome probing indicated that
Pseudomonas
strain Cstd5 was the dominant community member. However, incubation of soil samples with 0.1% (wt/wt) toluene resulted in a community that was dominated by
Pseudomonas
strain Q7, a toluene degrader that has been described previously (Y. Shen, L. G. Stehmeier, and G. Voordouw, Appl. Environ. Microbiol. 64:637–645, 1998). Q7 was not able to grow by itself in an atmosphere equilibrated with 0.1% (wt/wt) toluene but grew efficiently in coculture with Cstd1, suggesting that toluene or metabolic derivatives of toluene were transferred from Cstd1 to Q7.
Nisin Production by a Mixed-Culture System Consisting of
Lactococcus lactis
and
Kluyveromyces marxianus
Authors: Shimizu Hiroshi , Mizuguchi Taiji , Tanaka Eiji , Shioya Suteaki 
Published: December 2019

ABSTRACT

To control the pH during antimicrobial peptide (nisin) production by a lactic acid bacterium,
Lactococcus lactis
subsp.
lactis
(ATCC11454), a novel method involving neither addition of alkali nor a separation system such as a ceramic membrane filter and electrodialyzer was developed. A mixed culture of
L. lactis
and
Kluyveromyces marxianus
, which was isolated from kefir grains, was utilized in the developed system. The interaction between lactate production by
L. lactis
and its assimilation by
K. marxianus
was used to control the pH. To utilize the interaction of these microorganisms to maintain high-level production of nisin, the kinetics of growth of, and production of lactate, acetate, and nisin by,
L. lactis
were investigated. The kinetics of growth of and lactic acid consumption by
K. marxianus
were also investigated. Because the pH of the medium could be controlled by the lactate consumption of
K. marxianus
and the specific lactate consumption rate of
K. marxianus
could be controlled by changing the dissolved oxygen (DO) concentration, a cascade pH controller coupled with DO control was developed. As a result, the pH was kept constant because the lactate level was kept low and nisin accumulated in the medium to a high level compared with that attained using other pH control strategies, such as with processes lacking pH control and those in which pH is controlled by addition of alkali.