bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cytoplasmic Membrane Lipoprotein LppC of
Streptococcus equisimilis
Functions as an Acid Phosphatase
Authors: Malke Horst 
Published: December 2019

ABSTRACT

The function of the streptococcal cytoplasmic membrane lipoprotein, LppC, was identified with isogenic
Streptococcus equisimilis
H46A and
Escherichia coli
JM109 strain pairs differing in whether they contained [H46A and JM109(pLPP2)] or lacked (H46A
lppC
::pLPP10 and JM109) the functional
lppC
gene for comparative phosphatase determinations under acidic conditions.
lppC
-directed acid phosphatase activity was demonstrated zymographically and by specific enzymatic activity assays, with whole cells or cell membrane preparations as enzyme sources. LppC acid phosphatase showed optimum activity at pH 5, and the enzyme activity was unaffected by Triton X-100,
l
-(+)-tartaric acid, or EDTA. Database searches revealed significant structural homology of LppC to the
Streptococcus pyogenes
LppA,
Flavobacterium meningosepticum
OplA,
Helicobacter pylori
HP1285, and
Haemophilus influenzae
Hel [
e
(P4)] proteins. These results suggest a possible function for these proteins and establish a novel function of streptococcal cell membrane lipoproteins.
Molecular Cloning, Nucleotide Sequence, and Expression in
Escherichia coli
of a Hemolytic Toxin (Aerolysin) Gene from
Aeromonas trota
Authors: Khan Ashraf A. , Kim Eungbin , Cerniglia Carl E. 
Published: December 2019

ABSTRACT

Aeromonas trota
AK2, which was derived from ATCC 49659 and produces the extracellular pore-forming hemolytic toxin aerolysin, was mutagenized with the transposon mini-Tn
5Km1
to generate a hemolysin-deficient mutant, designated strain AK253. Southern blotting data indicated that an 8.7-kb
Not
I fragment of the genomic DNA of strain AK253 contained the kanamycin resistance gene of mini-Tn
5Km1
. The 8.7-kb
Not
I DNA fragment was cloned into the vector pGEM5Zf(−) by selecting for kanamycin resistance, and the resultant clone, pAK71, showed aerolysin activity in
Escherichia coli
JM109. The nucleotide sequence of the
aerA
gene, located on the 1.8-kb
Apa
I-
Eco
RI fragment, was determined to consist of 1,479 bp and to have an ATG initiation codon and a TAA termination codon. An in vitro coupled transcription-translation analysis of the 1.8-kb region suggested that the
aerA
gene codes for a 54-kDa protein, in agreement with nucleotide sequence data. The deduced amino acid sequence of the
aerA
gene product of
A. trota
exhibited 99% homology with the amino acid sequence of the
aerA
product of
Aeromonas sobria
AB3 and 57% homology with the amino acid sequences of the products of the
aerA
genes of
Aeromonas salmonicida
17-2 and
A. sobria
33.
Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds
Bacillus cereus
Spores
Authors: Koo Kai , Foegeding Peggy M. , Swaisgood Harold E. 
Published: December 2019

ABSTRACT

Control of microorganisms such as
Bacillus cereus
spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of
B. cereus
T spores. The genes that encode
B. cereus
T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in
Escherichia coli
BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of
B. cereus
spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 10
4
B. cereus
spores per ml. Greater than 90% of the
B. cereus
spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.
Sequencing and Characterization of the
xyl
Operon of a Gram-Positive Bacterium,
Tetragenococcus halophila
Authors: Takeda Yasuo , Takase Kazuma , Yamato Ichiro , Abe Keietsu 
Published: December 2019

ABSTRACT

The
xyl
operon of a gram-positive bacterium,
Tetragenococcus halophila
(previously called
Pediococcus halophilus
), was cloned and sequenced. The DNA was about 7.7 kb long and contained genes for a ribose binding protein and part of a ribose transporter,
xylR
(a putative regulatory gene), and the
xyl
operon, along with its regulatory region and transcription termination signal, in this order. The DNA was AT rich, the GC content being 35.8%, consistent with the GC content of this gram-positive bacterium. The
xyl
operon consisted of three genes,
xylA
, encoding a xylose isomerase,
xylB
, encoding a xylulose kinase, and
xylE
, encoding a xylose transporter, with predicted molecular weights of 49,400, 56,400, and 51,600, respectively. The deduced amino acid sequences of the XylR, XylA, XylB, and XylE proteins were similar to those of the corresponding proteins in other gram-positive and -negative bacteria, the similarities being 37 to 64%. Each polypeptide of XylB and XylE was expressed functionally in
Escherichia coli
. XylE transported
d
-xylose in a sodium ion-dependent manner, suggesting that it is the first described xylose/Na
+
symporter. The XylR protein contained a consensus sequence for binding catabolites of glucose, such as glucose-6-phosphate, which has been discovered in glucose and fructose kinases in bacteria. Correspondingly, the regulatory region of this operon contained a putative binding site of XylR with a palindromic structure. Furthermore, it contained a consensus sequence, CRE (catabolite-responsive element), for binding CcpA (catabolite control protein A). We speculate that the transcriptional regulation of this operon resembles the regulation of catabolite-repressible operons such as the
amy
,
lev
,
xyl
, and
gnt
operons in various gram-positive bacteria. We discuss the significance of the regulation of gene expression of this operon in
T. halophila
.
A Highly Selective PCR Protocol for Detecting 16S rRNA Genes of the Genus
Pseudomonas
(Sensu Stricto) in Environmental Samples
Authors: Widmer Franco , Seidler Ramon J. , Gillevet Patrick M. , Watrud Lidia S. , Di Giovanni George D. 
Published: December 2019

ABSTRACT

Pseudomonas
species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation. Specific detection of
Pseudomonas
species in the environment may help us gain a more complete understanding of the ecological significance of these microorganisms. The objective of this study was to develop a PCR protocol for selective detection of
Pseudomonas
(sensu stricto) in environmental samples. Extensive database searches identified a highly selective PCR primer pair for amplification of
Pseudomonas
16S rRNA genes. A protocol that included PCR amplification and restriction analysis, a general cloning and sequencing strategy, and phylogenetic analyses was developed. The PCR protocol was validated by testing 50 target and 14 nontarget pure cultures, which confirmed the selectivity to 100%. Further validation used amplification of target sequences from purified bulk soil DNA followed by cloning of PCR products. Restriction analysis with
Hae
III revealed eight different fragmentation patterns among 36 clones. Sequencing and phylogenetic analysis of 8 representative clones indicated that 91.7% of the products were derived from target organisms of the PCR protocol. Three patterns, representing only 8.3% of the 36 clones, were derived from non-
Pseudomonas
or chimeric PCR artifacts. Three patterns, representing 61.1% of the clones, clustered with sequences of confirmed
Pseudomonas
species, whereas two patterns, representing 30.6% of the clones, formed a novel phylogenetic cluster closely associated with
Pseudomonas
species. The results indicated that the
Pseudomonas
-selective PCR primers were highly specific and may represent a powerful tool for
Pseudomonas
population structure analyses and taxonomic confirmations.
Cloning and Expression of the Inositol Monophosphatase Gene from
Methanococcus jannaschii
and Characterization of the Enzyme
Authors: Chen Liangjing , Roberts Mary F. 
Published: December 2019

ABSTRACT

Inositol monophosphatase (EC
3.1.3.25
) plays a pivotal role in the biosynthesis of di-
myo
-inositol-1,1′-phosphate, an osmolyte found in hyperthermophilic archaea. Given the sequence homology between the MJ109 gene product of
Methanococcus jannaschii
and human inositol monophosphatase, the MJ109 gene was cloned and expressed in
Escherichia coli
and examined for inositol monophosphatase activity. The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (

K
m

= 0.091 ± 0.016 mM;
V
max
= 9.3 ± 0.45 μmol of P
i
min
−1
mg of protein
−1
) comparable to those of mammalian and
E. coli
enzymes. Its substrate specificity, Mg
2+
requirement, Li
+
inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases. The lack of inhibition by low concentrations of Li
+
and high concentrations of Mg
2+
and the high rates of hydrolysis of glucose-1-phosphate and
p
-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources. The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between
M. jannaschii
and human inositol monophosphatase and the crystal structure of the mammalian enzyme.
Development of a Lipase Fermentation Process That Uses a Recombinant
Pseudomonas alcaligenes
Strain
Authors: Gerritse Gijs , Hommes Ronald W. J. , Quax Wim J. 
Published: December 2019

ABSTRACT

Pseudomonas alcaligenes
M-1 secretes an alkaline lipase, which has excellent characteristics for the removal of fatty stains under modern washing conditions. A fed-batch fermentation process based on the secretion of the alkaline lipase from
P. alcaligenes
was developed. Due to the inability of
P. alcaligenes
to grow on glucose, citric acid and soybean oil were applied as substrates in the batch phase and feed phase, respectively. The gene encoding the high-alkaline lipase from
P. alcaligenes
was isolated and characterized. Amplification of lipase gene copies in
P. alcaligenes
with the aid of low- and high-copy-number plasmids resulted in an increase of lipase expression that was apparently colinear with the gene copy number. It was found that overexpression of the lipase helper gene,
lipB
, produced a stimulating effect in strains with high copy numbers (>20) of the lipase structural gene,
lipA
. In strains with
lipA
on a low-copy-number vector, the
lipB
gene did not show any effect, suggesting that LipB is required in a low ratio to LipA only. During scaling up of the fermentation process to 100 m
3
, severe losses in lipase productivity were observed. Simulations have identified an increased level of dissolved carbon dioxide as the most probable cause for the scale-up losses. A large-scale fermentation protocol with a reduced dissolved carbon dioxide concentration resulted in a substantial elimination of the scale-up loss.
Lyme Disease
Borrelia
Species in Northeastern China Resemble Those Isolated from Far Eastern Russia and Japan
Authors: Li Muqing , Masuzawa Toshiyuki , Takada Nobuhiro , Ishiguro Fubito , Fujita Hiromi , Iwaki Atsue , Wang Haipeng , Wang Jichun , Kawabata Masato , Yanagihara Yasutake 
Published: December 2019

ABSTRACT

Fifty-nine
Borrelia burgdorferi
sensu lato culture isolates collected from northeastern China were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism (RFLP) analysis and reactivity with monoclonal antibodies (MAbs). Among 59 culture isolates, 30 (50.8%) were
Borrelia garinii
and 17 (28.8%) were
Borrelia afzelii
, 2 were mixtures composed of
B. garinii
with RFLP pattern B and
B. garinii
with pattern C, and 9 were mixtures composed of
B. garinii
and
B. afzelii
. One isolate, ChY13p, produced a unique pattern and was identified as
B. garinii
based on analyses of 16S rRNA gene sequence, flagellin PCR-RFLP typing, and MAb reactivities. No
Borrelia burgdorferi
sensu stricto or
Borrelia japonica
isolates were detected. The results indicate that Lyme disease
Borrelia
species in northeastern China resemble those of
Borrelia
isolates from far eastern Russia and Japan.
Phanerochaete flavido-alba
Laccase Induction and Modification of Manganese Peroxidase Isoenzyme Pattern in Decolorized Olive Oil Mill Wastewaters
Authors: Pérez J. , de la Rubia T. , Hamman O. Ben , Martínez J. 
Published: December 2019

ABSTRACT

Lignin-degrading enzymes were partially purified from supernatant solutions obtained from
Phanerochaete flavido-alba
-decolorized olive oil mill wastewaters (OMW). The dominant enzymes, manganese peroxidases, exhibited different isoform patterns in decolorized OMW-containing cultures than in residue-free samples. Laccase induction was also detected in OMW-containing cultures but not in control cultures.
A Chromosomally Based
tod-luxCDABE
Whole-Cell Reporter for Benzene, Toluene, Ethybenzene, and Xylene (BTEX) Sensing
Authors: Applegate B. M. , Kehrmeyer S. R. , Sayler G. S. 
Published: December 2019

ABSTRACT

A
tod-luxCDABE
fusion was constructed and introduced into the chromosome of
Pseudomonas putida
F1, yielding the strain TVA8. This strain was used to examine the induction of the
tod
operon when exposed to benzene, toluene, ethylbenzene, and xylene (BTEX) compounds and aqueous solutions of JP-4 jet fuel constituents. Since this system contained the complete
lux
cassette (
luxCDABE
), bacterial bioluminescence in response to putative chemical inducers of the
tod
operon was measured on-line in whole cells without added aldehyde substrate. There was an increasing response to toluene concentrations from 30 μg/liter to 50 mg/liter, which began to saturate at higher concentrations. The detection limit was 30 μg/liter. There was a significant light response to benzene,
m
- and
p
-xylenes, phenol, and water-soluble JP-4 jet fuel components, but there was no bioluminescence response upon exposure to
o
-xylene. The transposon insertion was stable and had no negative effect on cell growth.
Gene Cloning and Characterization of a Novel Cellulose-Binding β-Glucosidase from
Phanerochaete chrysosporium
Authors: Li Bin , Renganathan V. 
Published: December 2019

ABSTRACT

Analysis of a 2.4-kb cDNA of the cellulose-binding extracellular β-glucosidase (CBGL) from
Phanerochaete chrysosporium
suggested that CBGL is organized into two domains, an N-terminal cellulose-binding domain and a C-terminal catalytic domain. Genomic sequence analysis suggested that
cbgl
is encoded by 30 exons. Southern analysis of DNA from homokaryotic cultures indicated that CBGL is encoded by two alleles,
cbgl-1
and
cbgl-2
, of a single gene.
Expression and Secretion of Defined Cutinase Variants by
Aspergillus awamori
Authors: van Gemeren I. A. , Beijersbergen A. , van den Hondel C. A. M. J. J. , Verrips C. T. 
Published: December 2019

ABSTRACT

Several cutinase variants derived by molecular modelling and site-directed mutagenesis of a cutinase gene from
Fusarium solani pisi
are poorly secreted by
Saccharomyces cerevisiae
. The majority of these variants are successfully produced by the filamentous fungus
Aspergillus awamori
. However, the L51S and T179Y mutations caused reductions in the levels of extracellular production of two cutinase variants by
A. awamori
. Metabolic labelling studies were performed to analyze the bottleneck in enzyme production by the fungus in detail. These studies showed that because of the single L51S substitution, rapid extracellular degradation of cutinase occurred. The T179Y substitution did not result in enhanced sensitivity towards extracellular proteases. Presumably, the delay in the extracellular accumulation of this cutinase variant is caused by the enhanced hydrophobicity of the molecule. Overexpression of the
A. awamori
gene encoding the chaperone BiP in the cutinase-producing
A. awamori
strains had no significant effect on the secretion efficiency of the cutinases. A cutinase variant with the amino acid changes G28A, A85F, V184I, A185L, and L189F that was known to aggregate in the endoplasmic reticulum of
S. cerevisiae
, resulting in low extracellular protein levels, was successfully produced by
A. awamori
. An initial bottleneck in secretion occurred before or during translocation into the endoplasmic reticulum but was rapidly overcome by the fungus.
Biochemical Characterization of Chloromethane Emission from the Wood-Rotting Fungus
Phellinus pomaceus
Authors: Saxena Deepti , Aouad Saleh , Attieh Jihad , Saini Hargurdeep S. 
Published: December 2019

ABSTRACT

Many wood-rotting fungi, including
Phellinus pomaceus
, produce chloromethane (CH
3
Cl).
P. pomaceus
can be cultured in undisturbed glucose mycological peptone liquid medium to produce high amounts of CH
3
Cl. The biosynthesis of CH
3
Cl is catalyzed by a methyl chloride transferase (MCT), which appears to be membrane bound. The enzyme is labile upon removal from its natural location and upon storage at low temperature in its bound state. Various detergents failed to solubilize the enzyme in active form, and hence it was characterized by using a membrane fraction. The enzyme had a sharp pH optimum between 7 and 7.2. Its apparent

K
m

for Cl
−
(ca. 300 mM) was much higher than that for I
−
(250 μM) or Br
−
(11 mM). A comparison of these

K
m

values to the relative in vivo methylation rates for different halides suggests that the real

K
m

for Cl
−
may be much lower, but the calculated value is high because the CH
3
Cl produced is used immediately in a coupled reaction. Among various methyl donors tested,
S
-adenosyl-
l
-methionine (SAM) was the only one that supported significant methylation by MCT. The reaction was inhibited by
S
-adenosyl-
l
-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings advance our comprehension of a poorly understood metabolic sector at the origin of biogenic emissions of halomethanes, which play an important role in atmospheric chemistry.
Degradation of 1,3-Dichloropropene by
Pseudomonas cichorii
170
Authors: Poelarends Gerrit J. , Wilkens Marga , Larkin Michael J. , van Elsas Jan Dirk , Janssen Dick B. 
Published: December 2019

ABSTRACT

The gram-negative bacterium
Pseudomonas cichorii
170, isolated from soil that was repeatedly treated with the nematocide 1,3-dichloropropene, could utilize low concentrations of 1,3-dichloropropene as a sole carbon and energy source. Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-
n
-alkanes. This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for
cis
-3-chloroacrylic acid and the other specific for
trans
-3-chloroacrylic acid. The haloalkane dehalogenase and the
trans
-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the
cis
-3-chloroacrylic acid dehalogenase was inducible. The presence of these enzymes indicates that 1,3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid. The latter compound is then dehalogenated, probably forming malonic acid semialdehyde. The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studied
dhaA
gene of the gram-positive bacterium
Rhodococcus rhodochrous
NCIMB13064. Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth. PCR analysis showed that these mutants had lost at least part of the
dhaA
gene.
Influence of Carbon Source on Nitrate Removal by Nitrate-Tolerant
Klebsiella oxytoca
CECT 4460 in Batch and Chemostat Cultures
Authors: Piñar Guadalupe , Kovárová Karin , Egli Thomas , Ramos Juan L. 
Published: December 2019

ABSTRACT

The nitrate-tolerant organism
Klebsiella oxytoca
CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h
−1
, whereas with glycerol it was 0.45 h
−1
. In batch cultures
K. oxytoca
cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures
K. oxytoca
CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (Y
C
) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (Y
N
) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h
−1
the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h
−1
the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.