bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and Characterization of Pediocin AcH Chimeric Protein Mutants with Altered Bactericidal Activity
Authors: Miller Kurt W. , Schamber Robin , Osmanagaoglu Ozlem , Ray Bibek 
Published: December 2019

ABSTRACT

A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an
Escherichia coli malE
plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against
Lactobacillus plantarum
NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14–20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains
L. plantarum
NCDO955,
Listeria innocua
Lin11,
Enterococcus faecalis
M1,
Pediococcus acidilactici
LB42, and
Leuconostoc mesenteroides
Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to ∼60% of wild-type activity when assayed against
L. innocua
Lin11. One mutant, K11E, displayed ∼2.8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.
Influence of Reduced Water Activity on Lactose Metabolism byLactococcus lactissubsp.cremorisat Different pH Values
Authors: Liu S.-Q. , Asmundson R. V. , Gopal P. K. , Holland R. , Crow V. L. 
Published: December 2019

ABSTRACTThe influence of reduced water activity (aw) on lactose metabolism byLactococcus lactissubsp.cremoris2254 and 2272 was studied at different pH values. In control incubations (aw, 0.99) with nongrowing cells in pH-controlled phosphate buffer, the levels of carbon recovered asl-(+)-lactate were 92% at pH 6.1 and 5.3 and 78% at pH 4.5. However, the levels of recovery decreased to ∼50% at all pH values tested when the awwas 0.88 (with glycerol as the humectant). When growing cells in broth controlled at pH 6.3 were used, a reduction in the awfrom 0.99 to 0.96 resulted in a decrease in the level of lactose carbon recovered asl-(+)-lactate from 100 to 71%. Low levels ofl-(+)-lactate carbon recovery (<50%) were also observed with cells resuspended in pH-uncontrolled reconstituted skim milk at awvalues of 0.99 and 0.87 and in young cheese curds. The missing lactose carbon could not be accounted for by acetate, ethanol, formate, acetaldehyde, or pyruvate. Attempts were made to determine where the missing lactose carbon was diverted to under the stress conditions used. Some of the missing lactose carbon was recovered as galactose (0.1 to 2.5 mM) in culture supernatants. Decreasing either the awor the pH resulted in increased galactose accumulation by nongrowing cells; adjusting both environmental factors together potentiated the effect. The sensitivities of the two lactococcal strains tested were different; strain 2272 was more prone to accumulate galactose under stress conditions. A methyl pentose(s) and additional galactose were found in acid-hydrolyzed supernatants from cultures containing both growing and nongrowing cells, indicating that a saccharide(s) rich in these components was formed by lactococci under low-awand low-pH stress conditions.
Fate of Residual Lignin during Delignification of Kraft Pulp by
Trametes versicolor
Authors: Reid Ian D. 
Published: December 2019

ABSTRACT

The fungus
Trametes versicolor
can delignify and brighten kraft pulps. To better understand the mechanism of this biological bleaching and the by-products formed, I traced the transformation of pulp lignin during treatment with the fungus. Hardwood and softwood kraft pulps containing
14
C-labelled residual lignin were prepared by laboratory pulping of lignin-labelled aspen and spruce wood and then incubated with
T. versicolor
. After initially polymerizing the lignin, the fungus depolymerized it to alkali-extractable forms and then to soluble forms. Most of the labelled carbon accumulated in the water-soluble pool. The extractable and soluble products were oligomeric; single-ring aromatic products were not detected. The mineralization of the lignin carbon to CO
2
varied between experiments, up to 22% in the most vigorous cultures. The activities of the known enzymes laccase and manganese peroxidase did not account for all of the lignin degradation that took place in the
T. versicolor
cultures. This fungus may produce additional enzymes that could be useful in enzyme bleaching systems.
Low-Frequency Horizontal Transfer of an Element Containing the Chlorocatechol Degradation Genes from
Pseudomonas
sp. Strain B13 to
Pseudomonas putida
F1 and to Indigenous Bacteria in Laboratory-Scale Activated-Sludge Microcosms
Authors: Ravatn Roald , Zehnder Alexander J. B. , van der Meer Jan Roelof 
Published: December 2019

ABSTRACT

The possibilities for low-frequency horizontal transfer of the self-transmissible chlorocatechol degradative genes (
clc
) from
Pseudomonas
sp. strain B13 were investigated in activated-sludge microcosms. When the
clc
genes were transferred into an appropriate recipient bacterium such as
Pseudomonas putida
F1, a new metabolic pathway for chlorobenzene degradation was formed by complementation which could be selected for by the addition of mono- or 1,4-dichlorobenzene (CB). Under optimized conditions with direct donor-recipient filter matings, very low transfer frequencies were observed (approximately 3.5 × 10
−8
per donor per 24 h). In contrast, in matings on agar plate surfaces, transconjugants started to appear after 8 to 10 days, and their numbers then increased during prolonged continuous incubation with CB. In activated-sludge microcosms, CB-degrading (CB
+
) transconjugants of strain F1 which had acquired the
clc
genes were detected but only when strain B13 cell densities of more than 10
5
CFU/ml could be maintained by the addition of its specific growth substrate, 3-chlorobenzoate (3CBA). The CB
+
transconjugants reached final cell densities of between 10
2
and 10
3
CFU/ml. When strain B13 was inoculated separately (without the designated recipient strain F1) into an activated-sludge microcosm, CB
+
transconjugants could not be detected. However, in this case a new 3CBA-degrading strain appeared which had acquired the
clc
genes from strain B13. The effects of selective substrates on the survival and growth of and gene transfer between bacteria degrading aromatic pollutants in a wastewater ecosystem are discussed.
Variants of Smooth
Salmonella enterica
Serovar Enteritidis That Grow to Higher Cell Density Than the Wild Type Are More Virulent
Authors: Guard-Petter Jean 
Published: December 2019

ABSTRACT

Salmonella enterica
serovar Enteritidis that grows to a higher cell density (SE-HCD) than wild type while retaining O-chain lipopolysaccharide was isolated by transforming wild type serovar Enteritidis with the cell density sensor plasmid pSB402 and selecting for bioluminescence. A luminescent strain, SE-HCD, that emitted light in proportion with cell density and opacity through stationary phase was isolated. After a peak cell density of 1.5 × 10
11
CFU/ml was observed, luminescence decreased, although opacity continued to increase. Scanning electron microscopy revealed that changes in luminescence and opacity past peak cell density were associated with lysis of a swarming hyperflagellated coccobacillary cell type and emergence of a 10-to-30-fold-elongated rod cell type that lacked cell surface structures. Vigorous aeration was required to induce this dramatic cellular differentiation. The virulence of two isogenic variants with different patterns of light emission at an opacity of 0.2 after the culture was diluted 10-fold (1/10 OD) was assessed in animal models. Whereas SE-HCD1 killed 70% of 6-day-old chicks challenged subcutaneously, the same dose of SE-HCD2 did not kill any chicks. Conversely, subcutaneous challenge of hens with SE-HCD2 contaminated eggs five and seven times more often, respectively, than did SE-HCD1 or wild type serovar Enteritidis. Intravenous challenge with SE-HCD2 contaminated 22% of eggs versus 0.5% with wild type, depressed egg production for 4 weeks, and caused clinical signs of Gallinarum Disease (Fowl Typhoid) in hens. SE-HCD2 produced no contaminated eggs following oral infection, whereas wild type contaminated 1.3% of eggs. Thus, SE-HCD2 is better at contaminating eggs than wild type, but only by parenteral challenge. These results suggest that it may be possible to separate luminescent serovar Enteritidis into groups that infect different age groups and organs and contaminate eggs.
Structure of the β-Galactosidase Gene from
Thermus
sp. Strain T2: Expression in
Escherichia coli
and Purification in a Single Step of an Active Fusion Protein
Authors: Vian Alejandro , Carrascosa Alfonso V. , García José L. , Cortés Estrella 
Published: December 2019

ABSTRACT

The nucleotide sequence of both the
bgaA
gene, coding for a thermostable β-galactosidase of
Thermus
sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (
M
r
, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the
bgaA
gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the β-galactosidase of
Bacillus stearothermophilus
allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in
Escherichia coli
, but more interestingly, an active chimeric β-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and a

K
m

value of 1.6 mM with
o
-nitrophenyl-β-
d
-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C.
Hemoglobin Biosynthesis in
Vitreoscilla stercoraria
DW: Cloning, Expression, and Characterization of a New Homolog of a Bacterial Globin Gene
Authors: Joshi Meenal , Mande Shekhar , Dikshit Kanak L. 
Published: December 2019

ABSTRACT

In the strictly aerobic, gram-negative bacterium
Vitreoscilla
strain C1, oxygen-limited growth conditions create a more than 50-fold increase in the expression of a homodimeric heme protein which was recognized as the first bacterial hemoglobin (Hb). The recently determined crystal structure of
Vitreoscilla
Hb has indicated that the heme pocket of microbial globins differs from that of eukaryotic Hbs. In an attempt to understand the diverse functions of Hb-like proteins in prokaryotes, we have cloned and characterized the gene (
vgb
) encoding an Hb-like protein from another strain of
Vitreoscilla
,
V. stercoraria
DW. Several silent changes were observed within the coding region of the
V. stercoraria vgb
gene. Apart from that,
V. stercoraria
Hb exhibited interesting differences between the A and E helices. Compared to its Hb counterpart from
Vitreoscilla
strain C1, the purified preparation of
V. stercoraria
Hb displays a slower autooxidation rate. The differences between
Vitreoscilla
Hb and
V. stercoraria
Hb were mapped onto the three-dimensional structure of
Vitreoscilla
Hb, which indicated that the four changes, namely, Ile7Val, Ile9Thr, Ile10Ser, and Leu62Val, present within the
V. stercoraria
Hb fall in the region where the A and E helices contact each other. Therefore, alteration in the relative orientation of the A and E helices and the corresponding conformational change in the heme binding pocket of
V. stercoraria
Hb can be correlated to its slower autooxidation rate. In sharp contrast to the oxygen-regulated biosynthesis of Hb in
Vitreoscilla
strain C1, production of Hb in
V. stercoraria
has been found to be low and independent of oxygen control, which is supported by the absence of a fumarate and nitrate reductase regulator box within the
V. stercoraria vgb
promoter region. Thus, the regulation mechanisms of the Hb-encoding gene appear to be quite different in the two closely related species of
Vitreoscilla
. The relatively slower autooxidation rate of
V. stercoraria
Hb, lack of oxygen sensitivity, and constitutive production of Hb suggest that it may have some other function(s) in the cellular physiology of
V. stercoraria
DW, together with facilitated oxygen transport, predicted for earlier reported
Vitreoscilla
Hb.
Effects of Manganese Peroxidase on Residual Lignin of Softwood Kraft Pulp
Authors: Reid Ian D. , Paice Michael G. 
Published: December 2019

ABSTRACT
Manganese peroxidase treatment lowered the kappa number of kraft pulp and increased the alkali extractability of the residual lignin but did not directly solubilize it. This indicates that MnP partially oxidizes the lignin in the pulp but does not degrade it to soluble fragments.
Phosphoenolpyruvate Phosphomutase Activity in an
l
-Phosphonoalanine-Mineralizing Strain of
Burkholderia cepacia
Authors: Ternan Nigel G. , McGrath John W. , Quinn John P. 
Published: December 2019

ABSTRACT

A strain of
Burkholderia cepacia
isolated by enrichment culture utilized
l
-2-amino-3-phosphonopropionic acid (phosphonoalanine) at concentrations up to 20 mM as a carbon, nitrogen, and phosphorus source in a phosphate-insensitive manner. Cells contained phosphoenolpyruvate phosphomutase activity, presumed to be responsible for cleavage of the C—P bond of phosphonopyruvate, the transamination product of
l
-phosphonoalanine; this was inducible in the presence of phosphonoalanine.
Kinetics of Acetate Oxidation by Two Sulfate Reducers Isolated from Anaerobic Granular Sludge
Authors: Elferink Stefanie J. W. H. Oude , Luppens Suzanne B. I. , Marcelis Christian L. M. , Stams Alfons J. M. 
Published: December 2019

ABSTRACT

Kinetic parameters of acetate oxidation were determined for the sulfate reducers
Desulforhabdus amnigenus
and
Desulfobacca acetoxidans
. Based on these parameters, both sulfate reducers seem to be able to outcompete
Methanosaeta
spp. for acetate in acetate-fed anaerobic bioreactors. Mixed-substrate studies showed that
D. amnigenus
degraded acetate and hydrogen simultaneously but preferred lactate, propionate, and ethanol over acetate.
Impact on Arbuscular Mycorrhiza Formation of
Pseudomonas
Strains Used as Inoculants for Biocontrol of Soil-Borne Fungal Plant Pathogens
Authors: Barea J. M. , Andrade G. , Bianciotto V. , Dowling D. , Lohrke S. , Bonfante P. , O’Gara F. , Azcon-Aguilar C. 
Published: December 2019

ABSTRACT

The arbuscular mycorrhizal symbiosis, a key component of agroecosystems, was assayed as a rhizosphere biosensor for evaluation of the impact of certain antifungal
Pseudomonas
inoculants used to control soil-borne plant pathogens. The following three
Pseudomonas
strains were tested: wild-type strain F113, which produces the antifungal compound 2,4-diacetylphloroglucinol (DAPG); strain F113G22, a DAPG-negative mutant of F113; and strain F113(pCU203), a DAPG overproducer. Wild-type strain F113 and mutant strain F113G22 stimulated both mycelial development from
Glomus mosseae
spores germinating in soil and tomato root colonization. Strain F113(pCU203) did not adversely affect
G. mosseae
performance. Mycelial development, but not spore germination, is sensitive to 10 μM DAPG, a concentration that might be present in the rhizosphere. The results of scanning electron and confocal microscopy demonstrated that strain F113 and its derivatives adhered to
G. mosseae
spores independent of the ability to produce DAPG.
Desulfurization of Dibenzothiophene and Diesel Oils by a Newly Isolated
Gordona
Strain, CYKS1
Authors: Rhee Sung-Keun , Chang Je Hwan , Chang Yong Keun , Chang Ho Nam 
Published: December 2019

ABSTRACT

A dibenzothiophene (DBT)-desulfurizing bacterial strain was isolated and identified as
Gordona
strain CYKS1. Strain CYKS1 was found to transform DBT to 2-hydroxybiphenyl via the 4S pathway and to be able to also use organic sulfur compounds other than DBT as a sole sulfur source. Its desulfurization activity was susceptible to sulfate repression. Active resting cells for desulfurization could be prepared only in the early growth phase. When two types of diesel oils, middle distillate unit feed (MDUF) and light gas oil (LGO) containing various organic sulfur compounds including DBT, were treated with resting cells of strain CYKS1 for 12 h, the total sulfur content significantly decreased, from 0.15% (wt/wt) to 0.06% (wt/wt) for MDUF and from 0.3% (wt/wt) to 0.25% (wt/wt) for LGO. The newly isolated strain CYKS1 is considered to have good potential for application in the biodesulfurization of fossil fuels.
Structure-Activity Study of the Lantibiotic Mutacin II from
Streptococcus mutans
T8 by a Gene Replacement Strategy
Authors: Chen Ping , Novak Jan , Kirk Marion , Barnes Stephen , Qi FengXia , Caufield Page W. 
Published: December 2019

ABSTRACT

Mutacin II, elaborated by group II
Streptococcus mutans
, is a ribosomally synthesized and posttranslationally modified polypeptide antibiotic containing unusual thioether and didehydro amino acids. To ascertain the role of specific amino acid residues in mutacin II antimicrobial activity, we developed a streptococcal expression system that facilitates the replacement of the
mutA
gene with a single copy of a mutated variant gene. As a result, variants of mutacin II can be designed and expressed. The system was tested by constructing the following mutant peptides: ΔN1, V7A, P9A, T10A, T10S, C15A, C26A, and C27A. All of these mutacin II variants except ΔN1 and T10A, which were not secreted, were isolated, and their identities were verified by mass spectrometry. Variants P9A, C15A, C26A, and C27A failed to exert antimicrobial activity. Because the P9A and T10A variants comprise the “hinge” region of mutacin II, these observations suggest that in addition to the thioether and didehydro amino acids, the hinge region is essential for biological activity and biosynthesis or export of the peptide. Tandem mass spectrometry of the N-terminal part of the wild-type molecule and its C15A variant confirmed that the threonine at position 10 is dehydrated and present as a didehydrobutyrine residue. This analysis of the active T10S variant further suggested that a didehydro amino acid at this position is specific for antimicrobial activity and that the biosynthetic machinery does not discriminate between threonine and serine. In contrast, the lack of production of mutacin variants with alanine substituted for threonine at position 10, as well as the deletion of asparagine at the N terminus (ΔN1), indicates that specific residues in the propeptide may be crucial for certain steps in the biosynthetic pathway of this lantibiotic.
Thermotoga neapolitana
Homotetrameric Xylose Isomerase Is Expressed as a Catalytically Active and Thermostable Dimer in
Escherichia coli
Authors: Hess J. Michael , Tchernajenko Vladimir , Vieille Claire , Zeikus J. Gregory , Kelly Robert M. 
Published: December 2019

ABSTRACT

The
xylA
gene from
Thermotoga neapolitana
5068 was expressed in
Escherichia coli
. Gel filtration chromatography showed that the recombinant enzyme was both a homodimer and a homotetramer, with the dimer being the more abundant form. The purified native enzyme, however, has been shown to be exclusively tetrameric. The two enzyme forms had comparable stabilities when they were thermoinactivated at 95°C. Differential scanning calorimetry revealed thermal transitions at 99 and 109.5°C for both forms, with an additional shoulder at 91°C for the tetramer. These results suggest that the association of the subunits into the tetrameric form may have little impact on the stability and biocatalytic properties of the enzyme.
Atmospheric Methane Consumption by Forest Soils and Extracted Bacteria at Different pH Values
Authors: Amaral John A. , Ren Tie , Knowles Roger 
Published: December 2019

ABSTRACT

The effect of pH on atmospheric methane (CH
4
) consumption was studied with slurries of forest soils and with bacteria extracted from the same soils. Soil samples were collected from a mixed hardwood stand in New Hampshire, from jackpine and aspen stands at the BOREAS (Boreal Ecosystem Atmosphere Study) site near Thompson, northern Manitoba, from sites in southern Québec, including a beech stand and a meadow, and from a site in Ontario (cultivated humisol). Consumption of atmospheric CH
4
(concentration, approximately 1.8 ppm) occurred at depths of >5 cm in both acidic (pH 4.5 to 5.2) and alkaline (pH 7.2 to 7.8) soils. In slurries of acidic soils, maximum activity occurred at different pH values (pH 4.0 to 6.5). Bacteria extracted from these soils by high-speed blending and density gradient centrifugation showed pH responses different from the pH responses of the slurries. In all cases, these bacteria had a methanotrophy pH optimum of 5.8 and exhibited no activity at pH 6.8 to 7.0, the pH optimum range for known methanotrophs. This difference in pH responses could be useful in modifying media currently used for isolation of these organisms. Methanotrophic activity was induced in previously non-CH
4
-consuming soils by preincubation with 5% (vol/vol) CH
4
(50,000 μl of CH
4
per liter) or by liquid enrichment with 20% CH
4
. The bacteria showed pH responses typical of known methanotrophs and not typical of preexisting consumers of ambient CH
4
. Furthermore, methanotrophs induced by high CH
4
levels were more readily extracted from soil than preexisting ambient CH
4
consumers were. In the alkaline soils, preexisting activity either was destroyed or resisted extraction by the procedure used. The results support the hypothesis that consumers of ambient CH
4
in soils are physiologically distinct from the known methanotrophs.
Heterologous Protein Secretion Directed by a Repressible Acid Phosphatase System of
Kluyveromyces lactis
: Characterization of Upstream Region-Activating Sequences in the
KIPHO5
Gene
Authors: Fermiñán Encarnación , Domínguez Angel 
Published: December 2019

ABSTRACT

Transcription of the repressible acid phosphatase gene (
KIPHO5
) in
Kluyveromyces lactis
is strongly regulated in response to the level of inorganic phosphate (P
i
) present in the growth medium. We have begun a study of the promoter region of this gene in order to identify sequences involved in the phosphate control of
KIPHO5
expression and to design new expression-secretion systems in
K. lactis
. Deletion analysis and directed mutagenesis revealed two major identical upstream activating sequences (UAS) CACGTG at positions −430 (UAS1) and −192 (UAS2) relative to the ATG initiation codon. These sequences are identical to those described for
Saccharomyces cerevisiae
for the binding of Pho4p. Deletion or directed mutagenesis of either one or both UAS reduce
KIPHO5
expression by the same amount (approximately 80%). When fused to the coding region of trout growth hormone cDNA (
tGH-II
), the promoter and signal peptide-encoding region of the phosphate-repressible
KIPHO5
gene drives the expression of this gene and the secretion of the tGHII protein. Synthesis of tGHIIp in
K. lactis
transformants carrying this construct was found to be regulated by the P
i
present in the medium; derepression of heterologous protein expression can therefore be achieved by lowering the P
i
concentration.