bluuebunny/crossref_metadata_2025 huggingface dataset:
A Competitive Microflora Increases the Resistance of
Salmonella typhimurium
to Inimical Processes: Evidence for a Suicide Response
Authors: Aldsworth Timothy G. , Sharman Rachel L. , Dodd Christine E. R. , Stewart Gordon S. A. B.
Published: December 2019

The presence of a viable competitive microflora at cell densities of 10
8
CFU ml
−1
protects an underlying population of 10
5
CFU of
Salmonella typhimurium
ml
−1
against freeze injury. The mechanism of enhanced resistance was initially postulated to be via an RpoS-mediated adaptive response. By using an
spvRA::luxCDABE
reporter we have shown that although the onset of RpoS-mediated gene expression was brought forward by the addition of a competitive microflora, the time taken for induction was measured in hours. Since the protective effect of a competitive microflora is essentially instantaneous, the stationary-phase adaptive response is excluded as the physiological mechanism. The only instantaneous effect of the competitive microflora was a reduction in the percent saturation of oxygen from 100% to less than 10%. For both mild heat treatment (55°C) and freeze injury this change in oxygen tension affords
Salmonella
a substantive (2 orders of magnitude) enhancement in survival. By reducing the levels of dissolved oxygen through active respiration, a competitive microflora reduces oxidative damage to exponential-phase cells irrespective of the inimical treatment. These results have led us to propose a suicide hypothesis for the destruction of rapidly growing cells by inimical processes. In essence, the suicide hypothesis proposes that a mild inimical process leads to the growth arrest of exponential-phase cells and to the decoupling of anabolic and catabolic metabolism. The result of this is a free radical burst which is lethal to unadapted cells.
Evidence that a New Antibiotic Flavone Glycoside Chemically Defends the Sea Grass
Thalassia testudinum
against Zoosporic Fungi
Authors: Jensen P. R. , Jenkins K. M. , Porter D. , Fenical W.
Published: December 2019

Significantly fewer thraustochytrid protists (zoosporic fungi) were observed in association with healthy leaf tissue of the marine angiosperm
Thalassia testudinum
than in association with sterilized samples that were returned to the collection site for 48 h. In support of the hypothesis that sea grass secondary metabolites were responsible for these differences, extracts of healthy
T. testudinum
leaf tissues inhibited the growth of the co-occurring thraustochytrid
Schizochytrium aggregatum
and deterred the attachment of
S. aggregatum
motile zoospores to an extract-impregnated substrate. By using
S. aggregatum
for bioassay-guided chemical fractionation, a new flavone glycoside was isolated and structurally characterized as luteolin 7-
O
-β-
d
-glucopyranosyl-2"-sulfate. Whole-leaf tissue concentrations of this metabolite (4 mg/ml of wet leaf tissue) inhibited
S. aggregatum
attachment, and a significantly lower concentration (270 μg/ml) reduced thraustochytrid growth by 50%, suggesting that natural concentrations are at least 15 times greater than that needed for significant microbiological effects. These results offer the first complete chemical characterization of a sea grass sulfated flavone glycoside and provide evidence that a secondary metabolite chemically defends
T. testudinum
against fouling microorganisms.
Anaerobic Oxidation of [1,2-
14
C]Dichloroethene under Mn(IV)-Reducing Conditions
Authors: Bradley Paul M. , Landmeyer James E. , Dinicola Richard S.
Published: December 2019

Anaerobic oxidation of [1,2-
14
C]dichloroethene to
14
CO
2
under Mn(IV)-reducing conditions was demonstrated. The results indicate that oxidative degradation of partially chlorinated solvents like dichloroethene can be significant even under anoxic conditions and demonstrate the potential importance of Mn(IV) reduction for remediation of chlorinated groundwater contaminants.
Mechanism of the Citrate Transporters in Carbohydrate and Citrate Cometabolism in
Lactococcus
and
Leuconostoc
Species
Authors: Bandell M. , Lhotte M. E. , Marty-Teysset C. , Veyrat A. , Prévost H. , Dartois V. , Diviès C. , Konings W. N. , Lolkema J. S.
Published: December 2019

Citrate metabolism in the lactic acid bacterium
Leuconostoc mesenteroides
generates an electrochemical proton gradient across the membrane by a secondary mechanism (C. Marty-Teysset, C. Posthuma, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Bacteriol. 178:2178–2185, 1996). Reports on the energetics of citrate metabolism in the related organism
Lactococcus lactis
are contradictory, and this study was performed to clarify this issue. Cloning of the membrane potential-generating citrate transporter (CitP) of
Leuconostoc mesenteroides
revealed an amino acid sequence that is almost identical to the known sequence of the CitP of
Lactococcus lactis
. The cloned gene was expressed in a
Lactococcus lactis
Cit
−
strain, and the gene product was functionally characterized in membrane vesicles. Uptake of citrate was counteracted by the membrane potential, and the transporter efficiently catalyzed heterologous citrate-lactate exchange. These properties are essential for generation of a membrane potential under physiological conditions and show that the
Leuconostoc
CitP retains its properties when it is embedded in the cytoplasmic membrane of
Lactococcus lactis
. Furthermore, using the same criteria and experimental approach, we demonstrated that the endogenous CitP of
Lactococcus lactis
has the same properties, showing that the few differences in the amino acid sequences of the CitPs of members of the two genera do not result in different catalytic mechanisms. The results strongly suggest that the energetics of citrate degradation in
Lactococcus lactis
and
Leuconostoc mesenteroides
are the same; i.e., citrate metabolism in
Lactococcus lactis
is a proton motive force-generating process.
Identification and Analysis of Genes Involved in Anaerobic Toluene Metabolism by Strain T1: Putative Role of a Glycine Free Radical
Authors: Coschigano Peter W. , Wehrman Thomas S. , Young L. Y.
Published: December 2019

The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain
tutD
and
tutE
, two genes in the T1 toluene metabolic pathway, have been generated. The
tutD
gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The
tutE
gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.
Antibiotic Production by
Erwinia herbicola
Eh1087: Its Role in Inhibition of
Erwinia amylovora
and Partial Characterization of Antibiotic Biosynthesis Genes
Authors: Kearns L. P. , Mahanty H. K.
Published: December 2019

Mutants of
Erwinia herbicola
Eh1087 (Ant
−
), which did not produce antibiotic activity against
Erwinia amylovora
, the fire blight pathogen, were selected after Tn
phoA
mutagenesis. In immature pear fruit Ant
−
mutants grew at the same rate as wild-type strain Eh1087 but did not suppress development of the disease caused by
E. amylovora
. These results indicated that antibiosis plays an important role in the suppression of disease by strain Eh1087. All of the Ant
−
mutations obtained were located in a 2.2-kb region on a 200-kb indigenous plasmid. Sequence analysis of the mutated DNA region resulted in identification of six open reading frames, designated ORF1 through ORF6, four of which were essential to antibiotic expression. One gene was identified as a gene which encodes a translocase protein which is probably involved in antibiotic secretion. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasmid proteins produced in
Escherichia coli
minicells confirmed the presence of proteins whose sizes corresponded to the sizes of the predicted open reading frame products.
New
Bradyrhizobium japonicum
Strains That Possess High Copy Numbers of the Repeated Sequence RSα
Authors: Minamisawa Kiwamu , Isawa Tsuyoshi , Nakatsuka Yoko , Ichikawa Norikazu
Published: December 2019

In a survey of DNA fingerprints of indigenous
Bradyrhizobium japonicum
with the species-specific repeated sequences RSα and RSβ, 21 isolates from three field sites showed numerous RS-specific hybridization bands. The isolates were designated highly reiterated sequence-possessing (HRS) isolates, and their DNA hybridization profiles were easily distinguished from the normal patterns. Some HRS isolates from two field sites possessed extremely high numbers of RSα copies, ranging from 86 to 175 (average, 128), and showed shifts and duplications of
nif
- and
hup
-specific hybridization bands. The HRS isolates exhibited slower growth than normal isolates, although no difference in symbiotic properties was detected between the HRS and normal isolates. Nucleotide sequence analysis of 16S rRNA genes showed that HRS isolates were strains of
B. japonicum
. There was no difference in the spectra of serological and hydrogenase groupings of normal and HRS isolates. Some HRS isolates possessed a tandem repeat RSα dimer that is similar to the structure of (IS
30
)
2
, which was shown to cause a burst of transpositional rearrangements in
Escherichia coli
. The results suggest that HRS isolates are derived from normal isolates in individual fields by genome rearrangements that may be mediated by insertion sequences such as RSα.
Improved Dechlorinating Performance of Upflow Anaerobic Sludge Blanket Reactors by Incorporation of
Dehalospirillum multivorans
into Granular Sludge
Authors: Hörber Christine , Christiansen Nina , Arvin Erik , Ahring Birgitte K.
Published: December 2019

Dechlorination of tetrachloroethene, also known as perchloroethylene (PCE), was investigated in an upflow anaerobic sludge blanket (UASB) reactor after incorporation of the strictly anaerobic, reductively dechlorinating bacterium
Dehalospirillum multivorans
into granular sludge. This reactor was compared to the reference 1 (R1) reactor, where the granules were autoclaved to remove all dechlorinating abilities before inoculation, and to the reference 2 (R2) reactor, containing only living granular sludge. All three reactors were fed mineral medium containing 3 to 57 μM PCE, 2 mM formate, and 0.5 mM acetate and were operated under sterile conditions. In the test reactor, an average of 93% (mole/mole) of the effluent chloroethenes was dichloroethene (DCE), compared to 99% (mole/mole) in the R1 reactor. The R2 reactor, with no inoculation, produced only trichloroethene (TCE), averaging 43% (mole/mole) of the effluent chloroethenes. No dechlorination of PCE was observed in an abiotic control consisting of sterile granules without inoculum. During continuous operation with stepwise-reduced hydraulic retention times (HRTs), both the test reactor and the R1 reactor showed conversion of PCE to DCE, even at HRTs much lower than the reciprocal maximum specific growth rate of
D. multivorans
, indicating that this bacterium was immobilized in the living and autoclaved granular sludge. In contrast, the R2 reactor, with no inoculation of
D. multivorans
, only converted PCE to TCE under the same conditions. Immobilization could be confirmed by using fluorescein-labeled antibody probes raised against
D. multivorans
. In granules obtained from the R1 reactor,
D. multivorans
grew mainly in microcolonies located in the centers of the granules, while in the test reactor, the bacterium mainly covered the surfaces of granules.
arfI
and
arfII
, Two Genes Encoding α-
l
-Arabinofuranosidases in
Cytophaga xylanolytica
Authors: Kim Kwi S. , Lilburn Timothy G. , Renner Michael J. , Breznak John A.
Published: December 2019

arfI
encoded the 57.7-kDa subunit of
Cytophaga xylanolytica
arabinofuranosidase I (ArfI).
arfII
encoded a 59.2-kDa subunit of ArfII. Products of both cloned genes liberated arabinose from arabinan and arabinoxylan. The deduced amino acid sequences of ArfI and ArfII revealed numerous regions that were identical to each other and to regions of homologous proteins from
Bacteroides ovatus
,
Bacillus subtilis
, and
Clostridium stercorarium.
Cloning of the Ribosomal Protein L41 Gene of
Phaffia rhodozyma
and Its Use as a Drug Resistance Marker for Transformation
Authors: Kim I.-G. , Nam S.-K. , Sohn J.-H. , Rhee S.-K. , An G.-H. , Lee S.-H. , Choi E.-S.
Published: December 2019

The ribosomal protein L41 gene of
Phaffia rhodozyma
was cloned and used as a dominant selectable marker for cycloheximide resistance in the transformation of
P. rhodozyma
. Electrotransformation with a plasmid containing a ribosomal DNA fragment as a targeting signal typically yielded 800 to 1,200 transformants/μg of DNA with an integrated copy number of about seven per haploid genome.
Differentiation of
Cryptosporidium parvum
Isolates by a Simplified Randomly Amplified Polymorphic DNA Technique
Authors: Deng Ming Qi , Cliver Dean O.
Published: December 2019

Genomic DNA was isolated from
Cryptosporidium parvum
oocysts by a specific immunomagnetic separation-in vitro excystation procedure and subjected to randomly amplified polymorphic DNA analysis using sequence-independent primers. An estuary
C. parvum
isolate was easily differentiated from several bovine isolates, while five bovine isolates of the same origin were indistinguishable from each other.
Small, Acid-Soluble Spore Proteins of the α/β Type Do Not Protect the DNA in
Bacillus subtilis
Spores against Base Alkylation
Authors: Setlow Barbara , Tautvydas Kes J. , Setlow Peter
Published: December 2019

Ethyl methanesulfonate (EMS) killed wild-type
Bacillus subtilis
spores as rapidly as spores lacking small, acid-soluble proteins (SASP) of the α/β type (α
−
β
−
spores), and 20% of the survivors had obvious mutations. A
recA
mutation increased the EMS sensitivity of wild-type and α
−
β
−
spores similarly but reduced their mutagenesis; EMS treatment of dormant spores also resulted in the induction of RecA synthesis during spore germination. EMS generated similar levels of alkylated bases in wild-type and α
−
β
−
spore DNAs, in purified DNA, or in DNA saturated with α/β-type SASP. Ethylene oxide (EtO) also generated similar levels of base alkylation in wild-type and α
−
β
−
spore DNAs. These data indicate that EMS and EtO kill spores at least in part by DNA damage but that α/β-type SASP, which protect DNA against many types of damage, do not protect spore DNA from base alkylation.
Regulation of Lipid Synthesis in
Bradyrhizobium japonicum
: Low Oxygen Concentrations Trigger Phosphatidylinositol Biosynthesis
Authors: Tang Yin , Hollingsworth Rawle I.
Published: December 2019

Lowering oxygen tension in free-living
Bradyrhizobium japonicum
resulted in a dramatic switch of membrane chemistry in which phosphatidylcholine, the predominant lipid in aerated cultures, was no longer synthesized and phosphatidylethanolamine became the major lipid. Besides this change, phosphatidylinositol, a typical plant lipid rarely found in bacteria, was also synthesized.
Endopolygalacturonase PG1 in Different Formae Speciales of
Fusarium oxysporum
Authors: Di Pietro Antonio , García-Maceira Fe I. , Huertas-González M. Dolores , Ruíz-Roldan M. Carmen , Caracuel Zaira , Barbieri Andrea S. , Roncero M. Isabel G.
Published: December 2019

PG1, the major endopolygalacturonase of the vascular wilt pathogen
Fusarium oxysporum
, was secreted during growth on pectin by 10 of 12 isolates belonging to seven formae speciales, as determined with isoelectric focusing zymograms and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. A Southern analysis of genomic DNA and PCR performed with gene-specific primers revealed that the
pg1
locus was highly conserved structurally in most isolates. Two PG1-deficient isolates were identified; one lacked the encoding gene, and the other carried a
pg1
allele disrupted by a 3.2-kb insertion with sequence homology to
hAT
transposases. The virulence for muskmelon of different
F. oxysporum
f. sp.
melonis
isolates was not correlated with PG1 production in vitro. We concluded that PG1 is widely distributed in
F. oxysporum
and that it is not essential for pathogenicity.
Bsn-t
Alleles from French Field Strains of
Agaricus bisporus
Authors: Callac Philippe , Hocquart Sophie , Imbernon Micheline , Desmerger Christophe , Olivier Jean-Marc
Published: December 2019

In the
Agaricus bisporus
desert population in California, the dominant
Bsn-t
allele determines the production of tetrasporic basidia and homokaryotic spores (
n
) that characterize a heterothallic life cycle. Strains belonging to a French population have the
Bsn-b/b
genotype that results in bisporic basidia that produce heterokaryotic spores (
n + n
) which characterize a pseudohomothallic life cycle. More recombination occurs in the tetrasporic population than in the bisporic population. In France, tetrasporic strains are rare. For two such isolates, Bs 261 and Bs 423, we determined the life cycle, the heritability of the tetrasporic trait, the amount of variation in the recombination rate, and the haploid fruiting ability. We found that (i) Bs 261 was heterothallic, (ii) Bs 423 was homokaryotic and homothallic, (iii) Bs 261 was
Bsn-t/b
, (iv) recombination on a segment of chromosome
I
depended on the genotype at
BSN
, (v) some of the homokaryotic offspring of Bs 261 and all of the progeny of Bs 423 were able to fruit, (vi) Bs 261 and Bs 423 were closely related, and (vii) Bs 423 was partially intersterile with other strains of the species.
Enzymological Characteristics of the Hyperthermostable NAD-Dependent Glutamate Dehydrogenase from the Archaeon
Pyrobaculum islandicum
and Effects of Denaturants and Organic Solvents
Authors: Kujo Chizu , Ohshima Toshihisa
Published: December 2019

NAD-dependent glutamate dehydrogenase (
l
-glutamate:NAD oxidoreductase, deaminating; EC
1.4.1.2
) was purified to homogeneity from a crude extract of the continental hyperthermophilic archaeon
Pyrobaculum islandicum
by two successive Red Sepharose CL-4B affinity chromatographies. The enzyme is the most thermostable NAD-dependent dehydrogenase found to date; the activity was not lost after incubation at 100°C for 2 h. The enzyme activity increased linearly with temperature, and the maximum was observed at ca. 90°C. The enzyme has a molecular mass of about 220 kDa and consists of six subunits with identical molecular masses of 36 kDa. The enzyme required NAD as a coenzyme for
l
-glutamate deamination and was different from the NADP-dependent glutamate dehydrogenase from other hyperthermophiles. The

K
m

values for NAD,
l
-glutamate, NADH, 2-oxoglutarate, and ammonia were 0.025, 0.17, 0.0050, 0.066, and 9.7 mM, respectively. The enzyme activity was significantly increased by the addition of denaturants such as guanidine hydrochloride and some water-miscible organic solvents such as acetonitrile and tetrahydrofuran. When fluorescence of the enzyme was measured in the presence of guanidine hydrochloride, a significant emission spectrum change and a shift in the maximum were observed but not in the presence of urea. These results indicate that this hyperthermophilic enzyme may have great potential in applications to biosensor and bioreactor processes.
Structure of a Microbial Community in Soil after Prolonged Addition of Low Levels of Simulated Acid Rain
Authors: Pennanen Taina , Fritze Hannu , Vanhala Pekka , Kiikkilä Oili , Neuvonen Seppo , Bååth Erland
Published: December 2019

Humus samples were collected 12 growing seasons after the start of a simulated acid rain experiment situated in the subarctic environment. The acid rain was simulated with H
2
SO
4
, a combination of H
2
SO
4
and HNO
3
, and HNO
3
at two levels of moderate acidic loads close to the natural anthropogenic pollution levels of southern Scandinavia. The higher levels of acid applications resulted in acidification, as defined by humus chemistry. The concentrations of base cations decreased, while the concentrations of exchangeable H
+
, Al, and Fe increased. Humus pH decreased from 3.83 to 3.65. Basal respiration decreased with decreasing humus pH, and total microbial biomass, measured by substrate-induced respiration and total amount of phospholipid fatty acids (PLFA), decreased slightly. An altered PLFA pattern indicated a change in the microbial community structure at the higher levels of acid applications. In general, branched fatty acids, typical of gram-positive bacteria, increased in the acid plots. PLFA analysis performed on the bacterial community growing on agar plates also showed that the relative amount of PLFA specific for gram-positive bacteria increased due to the acidification. The changed bacterial community was adapted to the more acidic environment in the acid-treated plots, even though bacterial growth rates, estimated by thymidine and leucine incorporation, decreased with pH. Fungal activity (measured as acetate incorporation into ergosterol) was not affected. This result indicates that bacteria were more affected than fungi by the acidification. The capacity of the bacterial community to utilize 95 different carbon sources was variable and only showed weak correlations to pH. Differences in the toxicities of H
2
SO
4
and HNO
3
for the microbial community were not found.
Rapid and Reliable Identification of Food-Borne Yeasts by Fourier-Transform Infrared Spectroscopy
Authors: Kümmerle Michael , Scherer Siegfried , Seiler Herbert
Published: December 2019

Computer-based Fourier-transform infrared spectroscopy (FT-IR) was used to identify food-borne, predominantly fermentative yeasts. Dried yeast suspensions provided the films suitable for FT-IR measurement. Informative windows in the spectrum were selected and combined to achieve optimal results. A reference spectrum library was assembled, based on 332 defined yeast strains from international yeast collections and our own isolates. All strains were identified with conventional methods using physiological and morphological characteristics. In order to assess identification quality, another 722 unknown yeast isolates not included in the reference spectrum library were identified both by classical methods and by comparison of their FT-IR spectra with those of the reference spectrum library. Ninety-seven and one-half percent of these isolates were identified correctly by FT-IR. Easy handling, rapid identification within 24 h when starting from a single colony, and a high differentiation capacity thus render FT-IR technology clearly superior to other routine methods for the identification of yeasts.
Isolation and Characterization of a Dibenzofuran-Degrading Yeast: Identification of Oxidation and Ring Cleavage Products
Authors: Hammer Elke , Krowas Dirk , Schäfer Annett , Specht Michael , Francke Wittko , Schauer Frieder
Published: December 2019

We characterized the ability of a yeast to cleave the aromatic structure of the dioxin-like compound dibenzofuran. The yeast strain was isolated from a dioxin-contaminated soil sample and identified as
Trichosporon mucoides
. During incubation of glucose-pregrown cells with dibenzofuran, six major metabolites were detected by high-performance liquid chromatography. The formation of four different monohydroxylated dibenzofurans was proven by comparison of analytical data (gas chromatography-mass spectrometry) with that for authentic standards. Further oxidation produced 2,3-dihydroxydibenzofuran and its ring cleavage product 2-(1-carboxy methylidene)-2,3-dihydrobenzo[
b
]furanylidene glycolic acid, which were characterized by mass spectrometry and
1
H nuclear magnetic resonance spectroscopy. These two metabolites are derived from 2-hydroxydibenzofuran and 3-hydroxydibenzofuran, as shown by incubation experiments using these monohydroxylated dibenzofurans as substrates.
Myoglobin as an Inhibitor of Exopeptidases from
Lactobacillus sake
Authors: Sanz Yolanda , Toldrá Fidel
Published: December 2019

The effects of myoglobin on exopeptidases of
Lactobacillus sake
were determined. Inhibition of the aminopeptidases increased as the myoglobin concentration increased; aminopeptidase 3 was the most affected (90% inhibition). Aminopeptidases 1, 2, and 4 showed similar inhibition levels (around 60%). Myoglobin did not affect tripeptidase activity. Thus, myoglobin could limit amino acid generation in meat systems.
Isolation of a Bacterial Strain with the Ability To Utilize the Sulfonated Azo Compound 4-Carboxy-4′-Sulfoazobenzene as the Sole Source of Carbon and Energy
Authors: Blümel Silke , Contzen Matthias , Lutz Martina , Stolz Andreas , Knackmuss Hans-Joachim
Published: December 2019

A bacterial strain (strain S5) which grows aerobically with the sulfonated azo compound 4-carboxy-4′-sulfoazobenzene as the sole source of carbon and energy was isolated. This strain was obtained by continuous adaptation of “
Hydrogenophaga palleronii
” S1, which has the ability to grow aerobically with 4-aminobenzenesulfonate. Strain S5 probably cleaves 4-carboxy-4′-sulfoazobenzene reductively under aerobic conditions to 4-aminobenzoate and 4-aminobenzene-sulfonate, which are mineralized by previously established degradation pathways.
Cytoplasmic Membrane Lipoprotein LppC of
Streptococcus equisimilis
Functions as an Acid Phosphatase
Authors: Malke Horst
Published: December 2019

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.