bluuebunny/crossref_metadata_2025 huggingface dataset:
Symbiotic Potential, Competitiveness, and Serological Properties of
Bradyrhizobium japonicum
Indigenous to Korean Soils
Authors: Kang Ui Gum , Somasegaran Padma , Hoben Heinz J. , Bohlool B. Ben
Published: January 2020

The symbiotic potential of
Bradyrhizobium japonicum
isolates indigenous to seven Korean soils was evaluated by inoculating soybeans with 10- and 1,000-fold-diluted soil suspensions (whole-soil inocula). At both levels, significant differences in the symbiotic potential of the indigenous
B. japonicum
isolates were demonstrated. The relationship between rhizobial numbers in the whole-soil inocula (
x
) and nitrogen fixation parameters (
y
) was best predicted by a straight line (
y
=
a
+
bx
) when the numbers in the inocula were 100 to 10,000 ml
-1
, while the power curve (
y
=

ax
b

) predicted the variation when the numbers were 1 to 100 ml
-1
. Thirty isolates from three soils showed wide differences in effectiveness (measured as milligrams of shoot N per plant), and several were of equal or greater effectiveness than reference strain
B. japonicum
USDA 110 on soybean cultivars Clark and Jangbaekkong. On both of the soybean cultivars grown in a Hawaiian mollisol, the Korean
B. japonicum
isolate YCK 213 and USDA 110 were of equal effectiveness; USDA 110 was the superior strain in colonization (nodule occupancy). Korean isolates YCK 117 and YCK 141 were superior colonizers compared with USDA 110. However,
B. japonicum
USDA 123 was the superior colonizer compared with isolates YCK 213, YCK 141, and YCK 117. In an immunoblot analysis of 97 indigenous Korean isolates of
B. japonicum
, 41% fell into the USDA 110 and USDA 123 serogroups. Serogroups USDA 110 and USDA 123 were represented in six of the seven soils examined. In one Korean soil, 100% of the
B. japonicum
isolates reacted only with antisera of YCK 117, an isolate from the same soil.
Immunocytochemical Identification and Localization of Active and Inactive α-Amylase and Pullulanase in Cells of
Clostridium thermosulfurogenes
EM1
Authors: Specka U. , Spreinat A. , Antranikian G. , Mayer F.
Published: January 2020

Clostridium thermosulfurogenes
EM1 formed blebs, i.e., protrusions still in contact with the cytoplasmic membrane, that originated from the cytoplasmic membrane during growth in batch culture and continuous culture. They could be observed squeezed between the cell wall and cytoplasmic membrane in cells with seemingly intact wall layers (surface layer and peptidoglycan layer) as well as in cells with wall layers in different states of degradation caused by phosphate limitation or high dilution rates. Blebs were found to turn into membrane vesicles by constriction in cases when the cell wall was heavily degraded. Bleb and vesicle formation was also observed in the absence of substrates that induce α-amylase and pullulanase synthesis. No correlations existed between bleb formation and the presence of active enzyme. Similar blebs could also be observed in a number of other gram-positive bacteria not producing these enzymes, but they were not observed in gram-negative bacteria. For immunoelectron-microscopic localization of α-amylase and pullulanase in
C. thermosulfurogenes
EM1, two different antisera were applied. One was raised against the enzymes isolated from the culture fluid; the other was produced against a peptide synthesized, as a defined epitope, in analogy to the N-terminal amino acid sequence (21 amino acids) of the native extracellular α-amylase. By using these antisera, α-amylase and pullulanase were localized at the cell periphery in samples taken from continuous culture or batch culture. In samples prepared for electron microscopy by freeze substitution followed by ultrathin sectioning, blebs could be seen, and the immunolabel pinpointing α-amylase enzyme particles was seen not only randomly distributed in the cell periphery, but also lining the surface of the cytoplasmic membrane and the blebs. Cells exhibiting high or virtually no enzyme activity were labeled similarly with both antisera. This finding strongly suggests that α-amylase and pullulanase may occur in both active and inactive forms, depending on growth conditions.
Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins
Authors: Yu Y M , Ohba M , Gill S S
Published: January 2020

The mosquitocidal crystals of Bacillus thuringiensis subsp. fukuokaensis were isolated and bioassayed against fourth-instar larvae of two mosquito species. The 50% lethal concentration values of the crystals to Aedes aegypti and Culex quinquefasciatus were 4.1 and 2.9 micrograms/ml, respectively. In addition, the solubilized crystals had hemolytic activity; 50 micrograms/ml was the lowest detectable level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the crystals consisted of polypeptides of 90, 86, 82, 72, 50, 48, 37, and 27 kDa. When the solubilized inclusion was treated with C. quinquefasciatus midgut brush border membrane vesicles or Manduca sexta gut juice, only one major protein was detected. This protein retained mosquitocidal activity but had no detectable hemolytic activity. Immunological analysis of this subspecies and the subspecies israelensis, kyushuensis and darmstadiensis by using polyclonal antisera raised against the whole-crystal protein of B. thuringiensis subsp. fukuokaensis revealed that the proteins in subsp. fukuokaensis are distinct from proteins in the other subspecies because little cross-reaction was observed. Analysis of the plasmid pattern showed that the crystal protein genes are located on a plasmid of 130 MDa. Analysis of plasmid and chromosomal DNA from subsp. fukuokaensis showed little homology to the 72-kDa toxin gene (PG-14) of B. thuringiensis subsp. morrisoni. However, some of the proteins of B. thuringiensis subsp. fukuokaensis are homologous to other B. thuringiensis toxins because N-terminal amino acid analysis revealed that the 90-kDa protein is encoded by a cryIV gene type.
Microbial Formation of Manganese Oxides
Authors: Greene Anthony C. , Madgwick John C.
Published: January 2020

Microbial manganese oxidation was demonstrated at high Mn
2+
concentrations (5 g/liter) in bacterial cultures in the presence of a microalga. The structure of the oxide produced varied depending on the bacterial strain and mode of culture. A nonaxenic, acid-tolerant microalga, a
Chlamydomonas
sp., was found to mediate formation of manganite (γ-MnOOH). Bacteria isolated from associations with crude cultures of this alga grown in aerated bioreactors formed disordered γ-MnO
2
from Mn
2+
at concentrations of 5 g/liter over 1 month, yielding 3.3 g of a semipure oxide per liter. All algal-bacterial cultures removed Mn
2+
from solution, but only those with the highest removal rates formed an insoluble oxide. While the alga was an essential component of the reaction, a
Pseudomonas
sp. was found to be primarily responsible for the formation of a manganese precipitate. Medium components—algal biomass and urea—showed optima at 5.7 and 10 g/liters, respectively. The scaled-up culture (50 times) gave a yield of 22.3 g (53 mg/liter/day from a 15-liter culture) of semipure disordered γ-MnO
2
, identified by X-ray diffraction and Fourier transform infrared (FTIR) spectroscopy, and had a manganese oxide O/Mn ratio of 1.92. The Mn(IV) content in the oxide was low (30.5%) compared with that of mined or chemically formed γ-MnO
2
(ca. 50%). The shortfall in the bacterial oxide manganese content was due to biological and inorganic contaminants. FTIR spectroscopy, transmission electron microscopy, and electron diffraction studies have identified manganite as a likely intermediate product in the formation of disordered γ-MnO
2
.
Cloning of the structural gene for Clostridium botulinum type C1 toxin and whole nucleotide sequence of its light chain component
Authors: Kimura K , Fujii N , Tsuzuki K , Murakami T , Indoh T , Yokosawa N , Oguma K
Published: January 2020

The toxigenicity of Clostridium botulinum type C1 is mediated by specific bacteriophages. DNA was extracted from one of these phages. Two DNA fragments, 3 and 7.8 kb, which produced the protein reacting with antitoxin serum were cloned by using bacteriophage lambda gt11 and Escherichia coli. Both DNA fragments were then subcloned into pUC118 plasmids and transferred into E. coli cells. The nucleotide sequences of the cloned DNA fragments were analyzed by the dideoxy chain termination method, and their gene products were analyzed by Western immunoblot. The 7.8-kb fragment coded for the entire light chain component and the N terminus of the heavy chain component of the toxin, whereas the 3-kb fragment coded for the remaining heavy chain component. The entire nucleotide sequence for the light chain component was determined, and the derived amino acid sequence was compared with that of tetanus toxin. It was found that the light chain component of C1 toxin possessed several amino acid regions, in addition to the N terminus, that were homologous to tetanus toxin.
Characterization of Tn
5
-Induced Mutants of
Xenorhabdus nematophilus
ATCC 19061
Authors: Xu Jimin , Olson Mark E. , Kahn Michael L. , Hurlbert Ronald E.
Published: January 2020

A negative-selection vector, pHX1, was constructed for use in transposon mutagenesis of
Xenorhabdus nematophilus
ATCC 19061. pHX1 contains the
Bacillus subtilis
levansucrase gene which confers sucrose sensitivity. In addition, various Tn
5
-containing plasmids with different replication origins were transferred by conjugation from
Escherichia coli
into
X. nematophilus
ATCC 19061, and one of these plasmids, pGS9, yields Tn
5
insertion mutants of
X. nematophilus
ATCC 19061. By using these two delivery vehicles, more than 250 putative Tn
5
insertion mutants of
X. nematophilus
ATCC 19061 were isolated and were then characterized. Mutants that were altered in bromothymol blue adsorption, ability to lyse sheep erythrocytes, production of antibiotics on a variety of media, and virulence for
Galleria mellonella
were found.
Characterization of the nisin gene as part of a polycistronic operon in the chromosome of Lactococcus lactis ATCC 11454
Authors: Steen M T , Chung Y J , Hansen J N
Published: January 2020

The location and organization of the nisin locus in Lactococcus lactis ATCC 11454 were studied. Primer extension of in vivo mRNA transcripts of the gene that encodes the nisin prepropeptide sequence indicated the presence of a promoter at least 4 kb upstream from the nisin gene and that the mRNA has several processing sites. Restriction fragment patterns using rare-cutting enzymes, orthogonal pulsed-field clamped homogeneous electric field (CHEF) agarose gel electrophoresis, and hybridization with nisin gene probes showed that the nisin prepropeptide gene was located on a megabase-size restriction fragment, which was taken as proof of a chromosomal location. This is contrary to earlier reports, which had indicated that genes for nisin production were located on plasmids. There was no evidence of more than one chromosomal location or more than one copy of the nisin gene. The restriction patterns indicated that the size of the L. lactis genome is about 2,500 kb. The previously observed (G. W. Buchman, S. Banerjee, and J. N. Hansen, J. Biol. Chem. 263: 16260-16266, 1988) downstream open reading frame (ORF) was fully sequenced to reveal an 851-amino-acid coding region, an upstream putative mRNA processing site, and a putative rho-independent terminator. The ORF was analyzed for secondary structural features, and the sequence data bases were searched for homologies. The ORF contained many amphipathic helices, a C-terminal transmembrane helix, and homologies to some membrane-associated proteins. It lacked an N-terminal membrane insertion sequence and accordingly appears to be associated with, and anchored to, the cytoplasmic side of the membrane. An additional ORF that possessed a ribosome-binding sequence and tandem promoters, indicating the beginning of a new operon, was identified still farther downstream. The results were consistent with the nisin gene being part of a polycistronic operon with a size greater than 8.5 kb.
Growth and survival of Bordetella bronchiseptica in natural waters and in buffered saline without added nutrients
Authors: Porter J F , Parton R , Wardlaw A C
Published: January 2020

Bordetella bronchiseptica showed increases in viable count when incubated in phosphate-buffered saline (PBS), in reagent-grade water, and in local lake and pond waters, all without added nutrients. Within 48 to 72 h at 37 degrees C in PBS and in lake and pond waters, stationary-phase populations of around 2.7 x 10(6) CFU/ml developed from washed B. bronchiseptica inocula of around 2 x 10(3) CFU/ml. Increases in CFU on the order of five- and eightfold, respectively, were observed in reagent-grade water and in seawater from the same sizes of inocula. The organisms remained viable for at least 3 weeks in PBS and in lake waters at 37 degrees C. The possibility that carry-over of nutrients was responsible for growth was discounted by showing serial transfer of B. bronchiseptica in PBS under conditions in which Escherichia coli tested in parallel rapidly died out.
Arachidonic Acid Production by Fungi
Authors: Bajpai Pramod K. , Bajpai Pratima , Ward Owen P.
Published: January 2020

After preliminary screening,
Mortierella alpina
and
Mortierella elongata
were compared with respect to arachidonic acid content.
M. alpina
ATCC 16266 produced 2.1 g of arachidonic acid per liter in media containing 10% glucose while the highest percentage of arachidonic acid in lipid (43.3%) was observed at a glucose concentration of 2%. Arachidonic acid content in lipids increased to 66% during storage.
Purification and Characterization of
d
-Aminoacylase from
Alcaligenes faecalis
DA1
Authors: Yang Yunn-Bor , Lin Chyuan-Sheng , Tseng Ching-Ping , Wang Yng-Jiin , Tsai Ying-Chieh
Published: January 2020

A
d
-aminoacylase from
Alcaligenes faecalis
DA1 has been purified to homogeneity by a simple purification procedure with two columns, Fractogel DEAE-650 and HW-50. The specific activity of the purified enzyme was found to be 580 U/mg of protein with
N
-acetyl-
dl
-methionine as the reaction substrate. The apparent molecular weight and isoelectric point of this enzyme were determined to be 55,000 and 5.4, respectively.
Effect of Copper on
Methylomonas albus
BG8
Authors: Collins Mary Lynne Perille , Buchholz Lorie A. , Remsen Charles C.
Published: January 2020

Addition of copper to the medium for
Methylomonas albus
BG8 increased cell yield and methane monooxygenase activity. Intracytoplasmic membrane was formed only in cells grown with copper supplementation. Additionally, the abundances of two major membrane proteins were affected by copper in the growth medium. These findings indicate that effects of copper on the physiology of methanotrophic bacteria are not limited to those on types II and X.
Effects of Hydrostatic Pressure on Growth of Hyperthermophilic Archaebacteria from the Juan de Fuca Ridge
Authors: Reysenbach Anna-Louise , Deming Jody W.
Published: January 2020

Two new strains (AL1 and AL2) of hyperthermophilic, sulfur-reducing, heterotrophic archaebacteria from high-temperature (350°C) vents on the Juan de Fuca Ridge were highly barotolerant at their optimal growth temperatures (90 and 100°C, respectively). A trend towards barophily at pressures greater than those encountered in situ at the sea floor was demonstrated for the more extremely thermophilic strain (AL2), implying an ability to thrive in (unexplored) habitats well below accessible vent formations.
Transformation of a Methylotrophic Bacterium,
Methylobacterium extorquens,
with a Broad-Host-Range Plasmid by Electroporation
Authors: Ueda Shunsaku , Matsumoto Seiji , Shimizu Shoichi , Yamane Tsuneo
Published: January 2020

An efficient method for the transformation of the methylotrophic bacterium
Methylobacterium extorquens
NR-2 with a broad-host-range plasmid, pLA2917, by electroporation was examined. Transformants of
M. extorquens
NR-2 expressing resistance to kanamycin were obtained after electric pulse. These transformants were found to harbor a single plasmid which was electrophoretically identical and homologous to pLA2917 obtained from
Escherichia coli.
Several factors which determined the transformation efficiency were optimized, resulting in a transformation efficiency of up to 8 × 10
3
transformants per μg of plasmid DNA by 10 pulses at a field strength of 10 kV/cm and a pulse duration of 300 μs.
Aerobic Microbial Cometabolism of Benzothiophene and 3-Methylbenzothiophene
Authors: Fedorak Phillip M. , Grbić-Galić Dunja
Published: January 2020

A culture enriched by growth on 1-methylnaphthalene was used to study the aerobic biotransformations of benzothiophene and 3-methylbenzothiophene. Neither of the sulfur heterocyclic compounds would support growth, but they were transformed by the culture growing on 1-methylnaphthalene or glucose or peptone. Cometabolism of benzothiophene yielded benzothiophene-2,3-dione, whereas that of 3-methylbenzothiophene yielded 3-methylbenzothiophene sulfoxide and the corresponding sulfone. The identities of the dione and sulfone were verified by comparison with authentic standards. The identity of the sulfoxide was surmised from gas chromatography-mass spectrometry and gas chromatography- Fourier transform infrared spectroscopy results. Oxidation preferentially occurred at carbons 2 and 3 in benzothiophene, but when carbon 3 was substituted with a methyl group, as in 3-methylbenzothiophene, the sulfur atom was oxygenated. The predominant microorganism in the enrichment culture was a
Pseudomonas
strain, designated BT1, which mineralized aromatic but not aliphatic hydrocarbons. This isolate cometabolized benzothiophene and 3-methylbenzothiophene. There was no evidence that it could metabolize 3-methylbenzothiophene sulfone. When 3-methylbenzothiophene was added to Prudhoe Bay crude oil, the sulfur heterocycle was oxidized to its sulfoxide and sulfone by strain BT1 as it grew on the aromatic hydrocarbons in the crude oil. Benzothiophene-2,3-dione was found to be chemically unstable when incubated with Prudhoe Bay crude oil. Thus its formation from benzothiophene in the presence of crude oil could not be determined.
Lactose Uptake Driven by Galactose Efflux in
Streptococcus thermophilus
: Evidence for a Galactose-Lactose Antiporter
Authors: Hutkins Robert W. , Ponne Carina
Published: January 2020

Galactose-nonfermenting (Gal
-
)
Streptococcus thermophilus
TS2 releases galactose into the extracellular medium when grown in medium containing excess lactose. Starved and de-energized Gal
-
cells, however, could be loaded with galactose to levels approximately equal to the extracellular concentration (0 to 50 mM). When loaded cells were separated from the medium and resuspended in fresh broth containing 5 mM lactose, galactose efflux occurred. De-energized, galactose-loaded cells, resuspended in buffer or medium, accumulated [
14
C]lactose at a greater rate and to significantly higher intracellular concentrations than unloaded cells. Uptake of lactose by loaded cells was inhibited more than that by unloaded cells in the presence of extracellular galactose, indicating that a galactose gradient was involved in the exchange system. When de-energized, galactose-loaded cells were resuspended in carbohydrate-free medium at pH 6.7, a proton motive force (Δp) of 86 to 90 mV was formed, whereas de-energized, nonloaded cells maintained a Δp of about 56 mV. However, uptake of lactose by loaded cells occurred when the proton motive force was abolished by the addition of an uncoupler or in the presence of a proton-translocating ATPase inhibitor. These results support the hypothesis that galactose efflux in Gal
-
S. thermophilus
is electrogenic and that the exchange reaction (lactose uptake and galactose efflux) probably occurs via an antiporter system.
Gastrointestinal health effects associated with the consumption of drinking water produced by point-of-use domestic reverse-osmosis filtration units
Authors: Payment P , Franco E , Richardson L , Siemiatycki J
Published: January 2020

During a prospective epidemiological study of gastrointestinal health effects associated with the consumption of drinking water produced by reverse-osmosis domestic units, a correlation was demonstrated between the bacterial counts on R2A medium incubated at 35 degrees C and the reported gastrointestinal symptoms in families who used these units. A univariate correlation was found with bacterial counts on R2A medium at 20 degrees C but was confounded by the bacterial counts at 35 degrees C. Other variables, such as family size and amount of water consumed, were not independently explanatory of the rate of illness. These observations raise concerns for the possibility of increased disease associated with certain point-of-use treatment devices for domestic use when high levels of bacterial growth occur.
Use of inosine-containing oligonucleotide primers for enzymatic amplification of different alleles of the gene coding for heat-stable toxin type I of enterotoxigenic Escherichia coli
Authors: Candrian U , Furrer B , Höfelein C , Lüthy J
Published: January 2020

A method which employs the polymerase chain reaction (PCR) to identify Escherichia coli strains containing the estA gene was developed. This gene codes for heat-stable enterotoxin type I. The use of an inosine-containing pair of amplification primers allowed the amplification of a specific 175-bp DNA fragment from several different estA alleles. The amplified fragments were identified and distinguished by allele-specific oligonucleotide hybridization and characterized by restriction endonuclease analysis. An extension of the classical two-primer PCR proved to be a very simple and rapid method to identify and characterize the estA alleles. Besides the inosine-containing pair of primers, which recognized all described alleles, additional oligonucleotides were used as primers. The sequence of each of these primers was allele specific, and each was amplification compatible with one of the inosine-containing primers. Thus, in one PCR the 175-bp fragment typical for all estA alleles and an allele-specific fragment of different size were produced. These fragments could be separated by agarose gel electrophoresis and were recognized by ethidium bromide staining. Twenty-seven E. coli strains were tested with this amplification system. The presence or lack of the genetic information for production of heat-stable enterotoxin type I was perfectly consistent with the ability of these strains to produce this enterotoxin, as determined by enzyme-linked immunosorbent assay.
Reduction of nitroaromatic compounds by anaerobic bacteria isolated from the human gastrointestinal tract
Authors: Rafil F , Franklin W , Heflich R H , Cerniglia C E
Published: January 2020

Human intestinal microbial flora were screened for their abilities to reduce nitroaromatic compounds by growing them on brain heart infusion agar plates containing 1-nitropyrene. Bacteria metabolizing 1-nitropyrene, detected by the appearance of clear zones around the colonies, were identified as Clostridium leptum, Clostridium paraputrificum, Clostridium clostridiiforme, another Clostridium sp., and a Eubacterium sp. These bacteria produced aromatic amines from nitroaromatic compounds, as shown by thin-layer chromatography, high-pressure liquid chromatography, and biochemical tests. Incubation of three of these bacteria with 1-nitropyrene, 1,3-dinitropyrene, and 1,6-dinitropyrene inactivated the direct-acting mutagenicity associated with these compounds. Menadione and o-iodosobenzoic acid inhibited nitroreductase activity in all of the isolates, indicating the involvement of sulfhydryl groups in the active site of the enzyme. The optimum pH for nitroreductase activity was 8.0. Only the Clostridium sp. required added flavin adenine dinucleotide for nitroreductase activity. The nitroreductases were constitutive and extracellular. An activity stain for the detection of nitroreductase on anaerobic native polyacrylamide gels was developed. This activity stain revealed only one isozyme in each bacterium but showed that the nitroreductases from different bacteria had distinct electrophoretic mobilities.
Production, Purification, and Characterization of β-(1-4)-Endoxylanase of
Streptomyces roseiscleroticus
Authors: Grabski Anthony C. , Jeffries Thomas W.
Published: January 2020

Twelve species of
Streptomyces
that formerly belonged to the genus
Chainia
were screened for the production of xylanase and cellulase. One species,
Streptomyces roseiscleroticus
(
Chainia rosea
) NRRL B-11019, produced up to 16.2 IU of xylanase per ml in 48 h. A xylanase from
S. roseiscleroticus
was purified and characterized. The enzyme was a debranching β-(1-4)-endoxylanase showing high activity on xylan but essentially no activity against acid-swollen (Walseth) cellulose. It had a very low apparent molecular weight of 5,500 by native gel filtration, but its denatured molecular weight was 22,600 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had an isoelectric point of 9.5. The pH and temperature optima for hydrolysis of arabinoxylan were 6.5 to 7.0 and 60°C, respectively, and more than 75% of the optimum enzyme activity was retained at pH 8.0. The xylanase had a

K
m

of 7.9 mg/ml and an apparent
V
max
of 305 μmol · min
-1
· mg of protein
-1
. The hydrolysis rate was linear for xylan concentrations of less than 4 mg/ml, but significant inhibition was observed at xylan concentrations of more than 10 mg/ml. The predominant products of arabinoxylan hydrolysis included arabinose, xylobiose, and xylotriose.
Conservation of Plasmid DNA Sequences in Coronatine-Producing Pathovars of
Pseudomonas syringae
Authors: Bender Carol L. , Young Scott A. , Mitchell Robin E.
Published: January 2020

In
Pseudomonas syringae
pv. tomato PT23.2, plasmid pPT23A (101 kb) is involved in synthesis of the phytotoxin coronatine (C. L. Bender, D. K. Malvick, and R. E. Mitchell, J. Bacteriol. 171:807-812, 1989). The physical characterization of mutations that abolished coronatine production indicated that at least 30 kb of pPT23A DNA are required for toxin synthesis. In the present study,
32
P-labeled DNA fragments from the 30-kb region of pPT23A hybridized to plasmid DNAs from several coronatine-producing pathovars of
P. syringae
under conditions of high stringency. These experiments indicated that this region of pPT23A was strongly conserved in large plasmids (90 to 105 kb) that reside in
P. syringae
pv. atropurpurea, glycinea, and morsprunorum. The functional significance of the observed homology was demonstrated in marker-exchange experiments in which Tn
5
-inactivated sequences from the 30-kb region of pPT23A were used to mutate coronatine synthesis genes in the three heterologous pathovars. Physical characterization of the Tn
5
insertions generated by marker exchange indicated that genes controlling coronatine synthesis in
P. syringae
pv. atropurpurea 1304, glycinea 4180, and morsprunorum 567 and 3714 were located on the large indigenous plasmids where homology was originally detected. Therefore, coronatine biosynthesis genes are strongly conserved in the plasmid DNAs of four producing pathovars, despite their disparate origins (California, Japan, New Zealand, Great Britain, and Italy).
Biodegradation of creosote and pentachlorophenol in contaminated groundwater: chemical and biological assessment
Authors: Mueller J G , Middaugh D P , Lantz S E , Chapman P J
Published: January 2020

Shake flask studies examined the rate and extent of biodegradation of pentachlorophenol (PCP) and 42 components of coal-tar creosote present in contaminated groundwater recovered from the American Creosote Works Superfund site, Pensacola, Fla. The ability of indigenous soil microorganisms to remove these contaminants from aqueous solutions was determined by gas chromatographic analysis of organic extracts of biotreated groundwater. Changes in potential environmental and human health hazards associated with the biodegradation of this material were determined at intervals by Microtox assays and fish toxicity and teratogenicity tests. After 14 days of incubation at 30 degrees C, indigenous microorganisms effectively removed 100, 99, 94, 88, and 87% of measured phenolic and lower-molecular-weight polycyclic aromatic hydrocarbons (PAHs) and S-heterocyclic, N-heterocyclic, and O-heterocyclic constituents of creosote, respectively. However, only 53% of the higher-molecular-weight PAHs were degraded; PCP was not removed. Despite the removal of a majority of the organic contaminants through biotreatment, only a slight decrease in the toxicity and teratogenicity of biotreated groundwater was observed. Data suggest that toxicity and teratogenicity are associated with compounds difficult to treat biologically and that one may not necessarily rely on indigenous microorganisms to effectively remove these compounds in a reasonable time span; to this end, alternative or supplemental approaches may be necessary. Similar measures of the toxicity and teratogenicity of treated material may offer a simple, yet important, guide to bioremediation effectiveness.
Identification and Characterization of a
Pseudomonas
Strain Capable of Metabolizing Phenoxybenzoates
Authors: Topp Edward , Akhtar M. Humayoun
Published: January 2020

3-Phenoxybenzoate is a transient metabolite from the breakdown of a number of pyrethroid insecticides in soil. In this study, we identified and characterized a bacterium which could grow on 3-phenoxybenzoate, converting it to phenol. On the basis of morphological and biochemical features, the 3-phenoxybenzoatedegrading isolate was determined to be a
Pseudomonas
species, probably a strain of
Pseudomonas delafieldii
, now designated
Pseudomonas
strain ET1.
Pseudomonas
strain ET1 grew on 3-phenoxybenzoate with a generation time of 3 h and a specific rate of metabolism of (2.6 ± 0.9) × 10
-13
g of 3-phenoxybenzoate consumed cell
-1
h
-1
. The

K
m

for 3-phenoxybenzoate metabolism was 1.4 ± 0.8 μM. The metabolism of 3-phenoxybenzoate was constitutive and not subject to catabolite repression. The metabolism of a variety of substituted diaryl ether compounds was examined. 3- and 4-Phenoxybenzoates were metabolized, but 2-phenoxybenzoate was not. Phenoxy-substituted benzyl aldehyde was metabolized, but phenoxy-substituted benzyl alcohol, benzene, phenol, and aniline were not. Derivatives of 3-phenoxybenzoate substituted in the 4′ position with hydroxyl, methyl, or chlorine were metabolized, yielding the corresponding 4-substituted phenol. 3-(2-Hydroxyphenoxy)benzoate was not metabolized, but 3-phenoxy-4-fluorobenzoate was. These results indicate that the metabolism of the tested diaryl ether compounds was restricted to 4-phenoxybenzoate, 3-phenoxybenzyl aldehyde, and 3-phenoxybenzoate derivatives without a substitution in the 2′ position.
Behavior of Listeria monocytogenes inoculated into raw tomatoes and processed tomato products
Authors: Beuchat L R , Brackett R E
Published: January 2020

Rates of death and growth of Listeria monocytogenes inoculated onto raw whole and into chopped tomatoes stored at 10 and 21 degrees C were not influenced by prior treatment of tomatoes with chlorine or packaging under an atmosphere of 3% O2 and 97% N2. Growth of the pathogen occurred in whole tomatoes held at 21 degrees C but not at 10 degrees C, while death occurred in chopped tomatoes stored at these temperatures. Likewise, growth patterns of mesophilic aerobic microorganisms, psychrotrophic microorganisms, and yeasts and molds on whole and chopped tomatoes were essentially unaffected by chlorine and modified atmosphere packaging treatments. Populations of L. monocytogenes inoculated into commercially processed tomato juice and sauce and held at 5 degrees C remained constant for 14 days. A gradual decrease in the number of viable L. monocytogenes cells was observed in juice and sauce held at 21 degrees C. In contrast, the organism died rapidly when suspended in commercial tomato ketchup at 5 and 21 degrees C. Unlike low-acid raw salad vegetables such as lettuce, broccoli, asparagus, and cauliflower on which we have observed L. monocytogenes grow at refrigeration temperatures, tomatoes are not a good growth substrate for the organism. Nevertheless, L. monocytogens can remain viable on raw whole and chopped tomatoes and in commercial tomato juice and sauce for periods extending beyond their normal shelf-life expectancy.
Cloning, nucleotide sequence, and transcriptional analysis of the NAD(P)-dependent cholesterol dehydrogenase gene from a Nocardia sp. and its hyperexpression in Streptomyces spp
Authors: Horinouchi S , Ishizuka H , Beppu T
Published: January 2020

NAD(P)-dependent cholesterol dehydrogenases [NAD(P)-CDH], which allow easier quantification of cholesterol by means of directly measuring the A340 of NAD(P)H, are useful for clinical purposes. The amino acid sequences of the NH2 terminus and the fragments obtained by CNBr decomposition of the NAD(P)-CDH from a Nocardia sp. were determined for preparation of synthetic oligonucleotides as hybridization probes. A 4.4-kbp BamHI fragment hybridizing to these probes was cloned on pUC19 in Escherichia coli. The nucleotide sequence together with the determined amino acid sequences revealed that this enzyme consists of 364 amino acids (Mr, 39,792) and contains an NAD(P)-binding consensus sequence at its NH2-terminal portion. High-resolution S1 nuclease mapping suggested that in NAD(P)-CDH of both Nocardia and Streptomyces spp. transcription initiates at the adenine residue, which is the first position of the translational initiation triplet (AUG) of this protein. The S1 mapping experiments also showed that cholesterol-dependent regulation in the Nocardia sp. occurred at the level of transcription. In Streptomyces lividans containing the cloned fragment, however, this promoter was expressed constitutively. DNA manipulation of the cloned gene in E. coli, including the generation of a ribosome-binding sequence at an appropriate position by oligonucleotide-directed mutagenesis, led to production of this protein in a very large amount but in the enzymatically inactive form of inclusion bodies. On the other hand, a Streptomyces host-vector system was successfully used for producing 40 times as much enzymatically active NAD(P)-CDH as that produced by the original Nocardia sp.
Influence of CO2 and low concentrations of O2 on fermentative metabolism of the ruminal ciliate Polyplastron multivesiculatum
Authors: Ellis J E , McIntyre P S , Saleh M , Williams A G , Lloyd D
Published: January 2020

The effects of ruminal concentrations of CO2 and oxygen on the end products of endogenous metabolism and fermentation of D-glucose by the ruminal entodiniomorphid ciliate Polyplastron multivesiculatum were investigated. The principal metabolic products were butyric, acetic, and lactic acids, H2, and CO2. 13C nuclear magnetic resonance spectroscopy identified glycerol as a previously unknown major product of D-[1-13C]glucose fermentation by this protozoan. Metabolite formation rates were clearly influenced by the headspace gas composition. In the presence of 1 to 3 microM O2, acetate, H2, and CO2 formation was partially depressed. A gas headspace with a high CO2 content (66 kPa) was found to suppress hydrogenosomal pathways and to favor butyrate accumulation. Cytochromes were not detected (less than 2 pmol/mg of protein) in P. multivesiculatum; protozoal suspensions, however, consumed O2 for up to 3 h at 1 kPa of O2. Under gas phases of greater than 2.6 kPa of O2, the organisms rapidly became vacuolate and the cilia became inactive. The results suggest that fermentative pathways in P. multivesiculatum are influenced by the O2 and CO2 concentrations that prevail in situ in the rumen.
Mechanisms of H
2
S Production from Cysteine and Cystine by Microorganisms Isolated from Soil by Selective Enrichment
Authors: Morra Matthew J. , Dick Warren A.
Published: January 2020

Hydrogen sulfide (H
2
S) is a major component of biogenic gaseous sulfur emissions from terrestrial environments. However, little is known concerning the pathways for H
2
S production from the likely substrates, cysteine and cystine. A mixed microbial culture obtained from cystine-enriched soils was used in assays (50 min, 37°C) with 0.05 M Tris-HCl (pH 8.5), 25 μmol of
l
-cysteine, 25 μmol of
l
-cystine, and 0.04 μmol of pyridoxal 5′-phosphate. Sulfide was trapped in a center well containing zinc acetate, while pyruvate was measured by derivatization with 2,4-dinitrophenylhydrazine. Sulfide and total pyruvate production were 17.6 and 17.2 nmol mg of protein
-1
min
-1
, respectively. Dithiothreitol did not alter reaction stoichiometry or the amount of H
2
S and total pyruvate, whereas
N
-ethylmaleimide reduced both H
2
S and total pyruvate production equally. The amount of H
2
S produced was reduced by 96% when only
l
-cystine was included as the substrate in the assay and by 15% with the addition of propargylglycine, a specific suicide inhibitor of cystathionine γ-lyase. These data indicate that the substrate for the reaction was cysteine and the enzyme responsible for H
2
S and pyruvate production was cysteine desulfhydrase (EC 4.4.1.1). The enzyme had a

K
m

of 1.32 mM and was inactivated by temperatures greater than 60°C. Because cysteine is present in soil and cysteine desulfhydrase is an inducible enzyme, the potential for H
2
S production by this mechanism exists in terrestrial environments. The relative importance of this mechanism compared with other processes involved in H
2
S production from soil is unknown.
Fate of DNA encoding hygromycin resistance after meiosis in transformed strains of Gibberella fujikuroi (Fusarium moniliforme)
Authors: Leslie J F , Dickman M B
Published: January 2020

Stability of foreign DNA transformed into a novel host is an important parameter in decisions to permit the release of genetically engineered microorganisms into the environment. Meiotic instability of transformed DNA has been reported in fungi such as Ascobolus, Aspergillus, and Neurospora. We used strains of Gibberella fujikuroi (Fusarium moniliforme) transformed with the hygr gene from Escherichia coli to study meiotic stability of foreign DNA in this plant pathogenic fungus. Crosses with single-copy transformants segregated hygr:hygs in a 1:1 manner consistent with that expected for a Mendelian locus in a haploid organism. Multicopy transformants, however, segregated hygr:hygs in a 1:2 manner that was not consistent with Mendelian expectations for a chromosomal marker, even though two unrelated auxotrophic nuclear genes were segregating normally. Segregation ratios in crosses in which hygr was introduced via the male parent did not differ significantly from crosses in which the transformed strain served as the female parent. Some of the sensitive progeny from the crosses with the multicopy transformants carried hygr sequences. When these phenotypically sensitive progeny were crossed with a wild-type strain that carried no hygr sequences, some of the progeny were phenotypically hygr. Some progeny from some crosses were more resistant to hygromycin than were their sibs or the transformant strains that served as their parents. Transformants passaged through a maize plant only rarely segregated progeny with the high levels of resistance. The mechanism underlying these genetic instabilities is not clear but may involve unequal crossing over or methylation or both. Further work with cloned genes with homology to sequences already present in the Fusarium genome is warranted.
Flavonoids Released Naturally from Alfalfa Promote Development of Symbiotic
Glomus
Spores In Vitro
Authors: Tsai Siu M. , Phillips Donald A.
Published: January 2020

Because flavonoids from legumes induce transcription of nodulation genes in symbiotic rhizobial bacteria, it is reasonable to test whether these compounds alter the development of vesicular-arbuscular mycorrhizal (VAM) fungi that infect those plants. Quercetin-3-
O
-galactoside, the dominant flavonoid released naturally from alfalfa (
Medicago sativa
L.) seeds, promoted spore germination of
Glomus etunicatum
and
Glomus macrocarpum
in vitro. Quercetin produced the maximum increases in spore germination, hyphal elongation, and hyphal branching in
G. etunicatum
at 1 to 2.5 μM concentrations. Two flavonoids exuded from alfalfa roots, 4′,7-dihydroxyflavone and 4′,7-dihydroxyflavanone, also enhanced spore germination of this fungal species. Formononetin, an isoflavone that is released from stressed alfalfa roots, inhibited germination of both
Glomus
species. These in vitro results suggest that plant flavonoids may facilitate or regulate the development of VAM symbioses and offer new hope for developing pure, plant-free cultures of VAM fungi.
Conditional-Suicide Containment System for Bacteria Which Mineralize Aromatics
Authors: Contreras Asunción , Molin Soren , Ramos Juan-Luis
Published: January 2020

A model conditional-suicide system to control genetically engineered microorganisms able to degrade substituted benzoates is reported. The system is based on two elements. One element consists of a fusion between the promoter of the
Pseudomonas putida
TOL plasmid-encoded
meta
-cleavage pathway operon (P
m
) and the
lacI
gene encoding Lac repressor plus
xylS
, coding for the positive regulator of P
m
. The other element carries a fusion between the P
tac
promoter and the
gef
gene, which encodes a killing function. In the presence of XylS effectors, LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the P
tac
::
gef
cassette is no longer prevented and a high rate of cell killing is observed. The substitution of XylS for XylSthr45, a mutant regulator with altered effector specificity and increased affinity for benzoates, allows the control of populations able to degrade a wider range of benzoates at micromolar substrate concentrations. Given the wide effector specificity of the key regulators, the wild-type and mutant XylS proteins, the system should allow the control of populations able to metabolize benzoate; methyl-, dimethyl-, chloro-, dichloro-, ethyl-, and methoxybenzoates; salicylate; and methyl- and chlorosalicylates. A small population of genetically engineered microorganisms became Gef resistant; however, the mechanism of such survival remains unknown.
Total coliform detection in drinking water: comparison of membrane filtration with Colilert and Coliquik
Authors: Olson B H , Clark D L , Milner B B , Stewart M H , Wolfe R L
Published: January 2020

The Colilert (CL) and Coliquik (CQ) systems were compared in a presence-absence format against the Standard Methods membrane filtration (MF) technique to determine whether differences existed in total coliform detection. Approximately 750 water samples were collected from distribution systems, covered and uncovered storage reservoirs, well sites, and the influent to drinking water treatment plants. Samples were analyzed for total coliforms and heterotrophic bacteria with MF, CL, and CQ. The agreements between CL and MF and between CQ and MF were both greater than 94.8%, which indicates that both may be acceptable methods for total coliform detection. Disagreement between the CL and CQ methods was primarily due to false-negative results. Furthermore, laboratory and field inoculation methods were compared for CL, more than 98% agreement was obtained. This finding indicates that sampling and immediate field inoculation may be an alternative to the traditional laboratory inoculation.
Mineralization of Linear Alkylbenzene Sulfonate by a Four-Member Aerobic Bacterial Consortium
Authors: Jiménez Luis , Breen Alec , Thomas Nikki , Federle Thomas W. , Sayler Gary S.
Published: January 2020

A bacterial consortium capable of linear alkylbenzene sulfonate (LAS) mineralization under aerobic conditions was isolated from a chemostat inoculated with activated sludge. The consortium, designated KJB, consisted of four members, all of which were gram-negative, rod-shaped bacteria that grew in pairs and short chains. Three isolates had biochemical properties characteristic of
Pseudomonas
spp.; the fourth showed characteristics of the
Aeromonas
spp. Cell suspensions were grown together in minimal medium with [
14
C]LAS as the only carbon source. After 13 days of incubation, more than 25% of the [
14
C]LAS was mineralized to
14
CO
2
by the consortium. Pure bacterial cultures and combinations lacking any one member of the KJB bacterial consortium did not mineralize LAS. Three isolates carried out primary biodegradation of the surfactant, and one did not. This study shows that the four bacteria complemented each other and synergistically mineralized LAS, indicating catabolic cooperation among the four consortium members.
Phenotypic Diversity among Strains of
Bradyrhizobium japonicum
Belonging to Serogroup 110
Authors: Basit H. Abdel , Angle J. S. , Salem S. , Gewaily E. M. , Kotob S. I. , van Berkum P.
Published: January 2020

Thirty-four strains of
Bradyrhizobium japonicum
within serogroup 110 were examined for phenotypic diversity. The strains differed in their abilities to nodulate and fix dinitrogen with
Glycine max
(L.) Merr. cv. Williams. Thirteen strains expressed uptake hydrogenase activity when induced as free-living cultures in the presence of 2% hydrogen and oxygen. Six bacteriophage susceptibility reactions were observed. Each of the strains produced either a large, mucoid or a small, dry colony morphology, but colony type was not related to effectiveness for nitrogen fixation.
Characterization of exopolymers of aquatic bacteria by pyrolysis-mass spectrometry
Authors: Ford T , Sacco E , Black J , Kelley T , Goodacre R , Berkeley R C , Mitchell R
Published: January 2020

Exopolymers from a diverse collection of marine and freshwater bacteria were characterized by pyrolysis-mass spectrometry (Py-MS). Py-MS provides spectra of pyrolysis fragments that are characteristic of the original material. Analysis of the spectra by multivariate statistical techniques (principal component and canonical variate analysis) separated these exopolymers into distinct groups. Py-MS clearly distinguished characteristic fragments, which may be derived from components responsible for functional differences between polymers. The importance of these distinctions and the relevance of pyrolysis information to exopolysaccharide function in aquatic bacteria is discussed.
Rapid confirmation of Listeria monocytogenes isolated from foods by a colony blot assay using a digoxigenin-labeled synthetic oligonucleotide probe
Authors: Kim C , Swaminathan B , Cassaday P K , Mayer L W , Holloway B P
Published: January 2020

An oligodeoxyribonucleotide probe based on the sequence of a 321-bp internal fragment of the msp gene encoding a major secreted polypeptide of Listeria monocytogenes was labeled with digoxigenin by using terminal deoxynucleotidyl transferase. The specificity of the digoxigenin-labeled probe was determined by dot blot assays. The probe reacted with all strains of L. monocytogenes tested (12 of 12 strains representing five serotypes). The probe did not react with any other Listeria species or with other gram-positive bacteria (Brochothrix, Erysipelothrix, Corynebacterium, Rhodococcus, Lactobacillus, Leuconostoc, Bacillus, Staphylococcus, and Streptococcus). The probe was used to develop a colony blot assay for the rapid confirmation of L. monocytogenes on Listeria-selective agars which had been streaked with food enrichment cultures. Forty-eight food samples were tested by conventional culture and DNA colony blot assay. The sensitivity and specificity of the DNA colony blot were 100 and 97%, respectively.
Bioluminescence assay for estimating the hydrophobic properties of bacteria as revealed by hydrophobic interaction chromatography
Authors: Mafu A A , Roy D , Savoie L , Goulet J
Published: January 2020

The luciferin-luciferase bioluminescence method was used to estimate the number of bacteria retained in neutral and amphiphilic gels and those in the eluate to determine the hydrophobic surface properties of bacteria by using hydrophobic interaction chromatography. Good correlations were found between viable counts and ATP content for Escherichia coli, Pseudomonas fragi, and Listeria monocytogenes. ATP determination was more rapid than viable counts for characterizing the relative hydrophobicity of L. monocytogenes. Quantitative estimations of adsorption of L. monocytogenes on octyl-Sepharose indicate that this microorganism is hydrophilic.