bluuebunny/crossref_metadata_2025 huggingface dataset:
Biodegradation of Aromatic Hydrocarbons in an Extremely Acidic Environment
Authors: Stapleton Raymond D. , Savage Dwayne C. , Sayler Gary S. , Stacey Gary
Published: December 2019

The potential for biodegradation of aromatic hydrocarbons was evaluated in soil samples recovered along gradients of both contaminant levels and pH values existing downstream of a long-term coal pile storage basin. pH values for areas greatly impacted by runoff from the storage basin were 2.0. Even at such a reduced pH, the indigenous microbial community was metabolically active, showing the ability to oxidize more than 40% of the parent hydrocarbons, naphthalene and toluene, to carbon dioxide and water. Treatment of the soil samples with cycloheximide inhibited mineralization of the aromatic substrates. DNA hybridization analysis indicated that whole-community nucleic acids recovered from these samples did not hybridize with genes, such asnahA,nahG,nahH,todC1C2, andtomA, that encode common enzymes from neutrophilic bacteria. Since these data suggested that the degradation of aromatic compounds may involve a microbial consortium instead of individual acidophilic bacteria, experiments using microorganisms isolated from these samples were initiated. While no defined mixed cultures were able to evolve14CO2from labeled substrates in these mineralization experiments, an undefined mixed culture including a fungus, a yeast, and several bacteria successfully metabolized approximately 27% of supplied naphthalene after 1 week. This study shows that biodegradation of aromatic hydrocarbons can occur in environments with extremely low pH values.
Purification and Characterization of a Trehalose Synthase from the Basidiomycete
Grifola frondosa
Authors: Saito Koki , Kase Toshiya , Takahashi Eiichi , Takahashi Eisaku , Horinouchi Sueharu
Published: December 2019

A trehalose synthase (TSase) that catalyzes the synthesis of trehalose from
d
-glucose and α-
d
-glucose 1-phosphate (α-
d
-glucose 1-P) was detected in a basidiomycete,
Grifola frondosa
. TSase was purified 106-fold to homogeneity with 36% recovery by ammonium sulfate precipitation and several steps of column chromatography. The native enzyme appears to be a dimer since it has apparent molecular masses of 120 kDa, as determined by gel filtration column chromatography, and 60 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although TSase catalyzed the phosphorolysis of trehalose to
d
-glucose and α-
d
-glucose 1-P, in addition to the synthesis of trehalose from the two substrates, the TSase equilibrium strongly favors trehalose synthesis. The optimum temperatures for phosphorolysis and synthesis of trehalose were 32.5 to 35°C and 35 to 37.5°C, respectively. The optimum pHs for these reactions were 6.5 and 6.5 to 6.8, respectively. The substrate specificity of TSase was very strict: among eight disaccharides examined, only trehalose was phosphorolyzed, and only α-
d
-glucose 1-P served as a donor substrate with
d
-glucose as the acceptor in trehalose synthesis. Two efficient enzymatic systems for the synthesis of trehalose from sucrose were identified. In system I, the α-
d
-glucose 1-P liberated by 1.05 U of sucrose phosphorylase was linked with
d
-glucose by 1.05 U of TSase, generating trehalose at the initial synthesis rate of 18 mmol/h in a final yield of 90 mol% under optimum conditions (300 mM each sucrose and glucose, 20 mM inorganic phosphate, 37.5°C, and pH 6.5). In system II, we added 1.05 U of glucose isomerase and 20 mM MgSO
4
to the reaction mixture of system I to convert fructose, a by-product of the sucrose phosphorylase reaction, into glucose. This system generated trehalose at the synthesis rate of 4.5 mmol/h in the same final yield.
Analysis of Airborne Actinomycete Spores with Fluorogenic Substrates
Authors: Gazenko S. V. , Reponen T. A. , Grinshpun S. A. , Willeke K.
Published: December 2019

The reactions between seven fluorogenic substrates and different groups of enzymes, esterases, lipases, phosphatases, and dehydrogenases, were studied in a search for a new method for the detection of actinomycete spores. Fluorescence measurement was chosen as a fast and sensitive method for microbial analysis. The focus of the research was on the spores of important air contaminants:
Streptomyces albus
and
Thermoactinomyces vulgaris
. For the measurement of the enzymatic activity, the chosen fluorogenic substrate was added to a mixture of spores and nutrient media, and the resulting fluorescence was measured with a spectrofluorometer. Fluorogenic substrates were found to show enzymatic activities even for dormant spores. Comparison of the enzymatic activities of dormant spores with those of vegetative cells showed similarity of the enzymatic profiles but higher activity for vegetative cells. The increase of enzymatic activity from dormant spores to vegetative cells was not linear but fluctuating. The largest fluctuations were found after 4 to 5 h of incubation. The enzymatic activities of
S. albus
were 10 to 50 times lower than those of
T. vulgaris
, except for the dehydrogenase activity, which was seven times higher. These results indicate that analysis with fluorogenic substrates has the potential for becoming a fast and sensitive method for the enumeration and identification of airborne actinomycete spores.
Molecular Characterization and Expression of a Phytase Gene from the Thermophilic Fungus
Thermomyces lanuginosus
Authors: Berka Randy M. , Rey Michael W. , Brown Kimberly M. , Byun Tony , Klotz Alan V.
Published: December 2019

The
phyA
gene encoding an extracellular phytase from the thermophilic fungus
Thermomyces lanuginosus
was cloned and heterologously expressed, and the recombinant gene product was biochemically characterized. The
phyA
gene encodes a primary translation product (PhyA) of 475 amino acids (aa) which includes a putative signal peptide (23 aa) and propeptide (10 aa). The deduced amino acid sequence of PhyA has limited sequence identity (ca. 47%) with
Aspergillus niger
phytase. The
phyA
gene was inserted into an expression vector under transcriptional control of the
Fusarium oxysporum
trypsin gene promoter and used to transform a
Fusarium venenatum
recipient strain. The secreted recombinant phytase protein was enzymatically active between pHs 3 and 7.5, with a specific activity of 110 μmol of inorganic phosphate released per min per mg of protein at pH 6 and 37°C. The
Thermomyces
phytase retained activity at assay temperatures up to 75°C and demonstrated superior catalytic efficiency to any known fungal phytase at 65°C (the temperature optimum). Comparison of this new
Thermomyces
catalyst with the well-known
Aspergillus niger
phytase reveals other favorable properties for the enzyme derived from the thermophilic gene donor, including catalytic activity over an expanded pH range.
Characterization of a Strain of Infectious Hepatitis E Virus Isolated from Sewage in an Area where Hepatitis E Is Not Endemic
Authors: Pina Sonia , Jofre Joan , Emerson Suzanne U. , Purcell Robert H. , Girones Rosina
Published: December 2019

Raw sewage samples from an area where hepatitis E is not endemic (Barcelona, Spain) were analyzed by reverse transcriptase-PCR followed by nested PCR. One of the 37 tested samples showed a positive result for hepatitis E virus (HEV). The detected strain was amplified by inoculation into rhesus monkeys, and the course of the infection was studied by analyzing serological and biochemical parameters and by monitoring the presence of HEV in serum and feces. Fecal suspensions from the rhesus monkeys were used as the source of viral particles for sequence analysis. Eighty percent of the genome of the isolated strain, named BCN, was sequenced and found to be phylogenetically related to Asian (Indian) strains, with a 98% nucleotide identity with an isolate from Madras, India. Since this was a single isolation we cannot conclude that HEV is regularly present in the sewage. However, the finding of viable HEV in sewage has implications for contamination of the environment and shellfish by HEV and must be considered in the diagnosis of viral hepatitis in regions of nonendemic hepatitis.
Effect of Prior Exposure to Noninfected Ticks on Susceptibility of Mice to Lyme Disease Spirochetes
Authors: Richter Dania , Spielman Andrew , Matuschka Franz-Rainer
Published: December 2019

To determine whether prior exposure to Nearctic
Ixodes
vector ticks protects native reservoir mice from tick-borne infection by Lyme disease spirochetes, we compared their infectivities for white-footed mice and laboratory mice that had been repeatedly infested by noninfected deer ticks. Nymphal ticks readily engorged on tick-exposed laboratory mice, but their feeding success on white-footed mice progressively declined. Tick-borne spirochetes readily infected previously tick-infested mice. Thus, prior infestation by Nearctic ticks does not protect sympatric reservoir mice or Palearctic laboratory mice from infection by sympatric tick-borne spirochetes.
Degradation of Polychlorinated Biphenyl Metabolites by Naphthalene-Catabolizing Enzymes
Authors: Barriault Diane , Durand Jacinthe , Maaroufi Halim , Eltis Lindsay D. , Sylvestre Michel
Published: December 2019

The ability of the dehydrogenase and ring cleavage dioxygenase of the naphthalene degradation pathway to transform 3,4-dihydroxylated biphenyl metabolites was investigated. 1,2-Dihydro-1,2-dihydroxynaphthalene dehydrogenase was expressed as a histidine-tagged protein. The purified enzyme transformed 2,3-dihydro-2,3-dihydroxybiphenyl, 3,4-dihydro-3,4-dihydroxybiphenyl, and 3,4-dihydro-3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl to 2,3-dihydroxybiphenyl, 3,4-dihydroxybiphenyl (3,4-DHB), and 3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl (3,4-DH-2,2′,5,5′-TCB), respectively. Our data also suggested that purified 1,2-dihydroxynaphthalene dioxygenase catalyzed the
meta
cleavage of 3,4-DHB in both the 2,3 and 4,5 positions. This enzyme cleaved 3,4-DH-2,2′,5,5′-TCB and 3,4-DHB at similar rates. These results demonstrate the utility of the naphthalene catabolic enzymes in expanding the ability of the
bph
pathway to degrade polychlorinated biphenyls.
Geographic Risk for Lyme Disease and Human Granulocytic Ehrlichiosis in Southern New York State
Authors: Daniels Thomas J. , Boccia Theresa M. , Varde Shobha , Marcus Jonathan , Le Jianhua , Bucher Doris J. , Falco Richard C. , Schwartz Ira
Published: December 2019

Ixodes scapularis, the tick vector of Lyme disease and human granulocytic ehrlichiosis (HGE), is prevalent in much of southern New York state. The distribution of this species has increased, as have reported cases of both Lyme disease and HGE. The unreliability of case reports, however, demonstrates the need for tick and pathogen surveillance in order to accurately define areas of high risk. In this study, a total of 89,550 m2at 34 study sites was drag sampled in 1995 and a total of 51,540 m2at 40 sites was sampled in 1996 to determine tick and pathogen distribution in southern New York state.I. scapulariswas collected from 90% of the sites sampled, and regionally, a 2.5-fold increase in nymphal abundance occurred from 1995 to 1996.I. scapularisindividuals from all sites were infected withBorrelia burgdorferiin 1995, while an examination of ticks for bothB. burgdorferiand the agent of HGE in 1996 confirmed that these organisms were present in all counties; the average coinfection rate was 1.9%. No correlation was found between estimated risk and reported cases of Lyme disease. The geographic disparity of risk observed among sites in this study underscores the need for vector and pathogen surveillance on a regional level. An entomologic risk index can help identify sites for targeted tick control efforts.
Isolation of Marine Polycyclic Aromatic Hydrocarbon (PAH)-Degrading
Cycloclasticus
Strains from the Gulf of Mexico and Comparison of Their PAH Degradation Ability with That of Puget Sound
Cycloclasticus
Strains
Authors: Geiselbrecht Allison D. , Hedlund Brian P. , Tichi Mary A. , Staley J. T.
Published: December 2019

Phenanthrene- and naphthalene-degrading bacteria were isolated from four offshore and nearshore locations in the Gulf of Mexico by using a modified most-probable-number technique. The concentrations of these bacteria ranged from 10
2
to 10
6
cells per ml of wet surficial sediment in mildly contaminated and noncontaminated sediments. A total of 23 strains of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were obtained. Based on partial 16S ribosomal DNA sequences and phenotypic characteristics, these 23 strains are members of the genus
Cycloclasticus
. Three representatives were chosen for a complete phylogenetic analysis, which confirmed the close relationship of these isolates to type strain
Cycloclasticus pugetii
PS-1, which was isolated from Puget Sound. PAH substrate utilization tests which included high-molecular-weight PAHs revealed that these isolates had similar, broad substrate ranges which included naphthalene, substituted naphthalenes, phenanthrene, biphenyl, anthracene, acenaphthene, and fluorene. Degradation of pyrene and fluoranthene occurred only when the strains were incubated with phenanthrene. Two distinct partial PAH dioxygenase iron sulfur protein (ISP) gene sequences were PCR amplified from Puget Sound and Gulf of Mexico
Cycloclasticus
strains. Phylogenetic analyses of these sequences revealed that one ISP type is related to the
bph
type of ISP sequences, while the other ISP type is related to the
nah
type of ISP sequences. The predicted ISP amino acid sequences for the Gulf of Mexico and Puget Sound strains are identical, which supports the hypothesis that these geographically separated isolates are closely related phylogentically.
Cycloclasticus
species appear to be numerically important and widespread PAH-degrading bacteria in both Puget Sound and the Gulf of Mexico.
Purification and Characterization of Gallic Acid Decarboxylase from
Pantoea agglomerans
T71
Authors: Zeida Mitsuhiro , Wieser Marco , Yoshida Toyokazu , Sugio Tsuyoshi , Nagasawa Toru
Published: December 2019

Oxygen-sensitive gallic acid decarboxylase from
Pantoea
(formerly
Enterobacter
)
agglomerans
T71 was purified from a cell extract after stabilization by reducing agents. This enzyme has a molecular mass of approximately 320 kDa and consists of six identical subunits. It is highly specific for gallic acid. Gallic acid decarboxylase is unique among similar decarboxylases in that it requires iron as a cofactor, as shown by plasma emission spectroscopy (which revealed an iron content of 0.8 mol per mol of enzyme subunit), spectrophotometric analysis (absorption shoulders at 398 and 472 nm), and inhibition of the enzyme activity by 2,2′-bipyridyl,
o
-phenanthroline, and EDTA. Another interesting feature of this strain is the fact that it contains a tannase, which is used together with the gallic acid decarboxylase in a two-enzyme resting cell bioconversion to synthesize valuable pyrogallol from readily available tannic acid.
Assessment of Changes in Microbial Community Structure during Operation of an Ammonia Biofilter with Molecular Tools
Authors: Sakano Y. , Kerkhof L.
Published: December 2019

Biofiltration has been used for two decades to remove odors and various volatile organic and inorganic compounds in contaminated off-gas streams. Although biofiltration is widely practiced, there have been few studies of the bacteria responsible for the removal of air contaminants in biofilters. In this study, molecular techniques were used to identify bacteria in a laboratory-scale ammonia biofilter. Both 16S rRNA and ammonia monooxygenase (
amoA
) genes were used to characterize the heterotrophic and ammonia-oxidizing bacteria collected from the biofilter during a 102-day experiment. The overall diversity of the heterotrophic microbial population appeared to decrease by 38% at the end of the experiment. The community structure of the heterotrophic population also shifted from predominantly members of two subdivisions of the
Proteobacteria
(the beta and gamma subdivisions) to members of one subdivision (the gamma subdivision). An overall decrease in the diversity of ammonia monooxygenase genes was not observed. However, a shift from groups dominated by organisms containing
Nitrosomonas
-like and
Nitrosospira
-like
amoA
genes to groups dominated by organisms containing only
Nitrosospira
-like
amoA
genes was observed. In addition, a new
amoA
gene was discovered. This new gene is the first freshwater
amoA
gene that is closely affiliated with
Nitrosococcus oceanus
and the particulate methane monooxygenase gene from the methane oxidizers belonging to the gamma subdivision of the
Proteobacteria
.
Purification and Genetic Characterization of Enterocin I from
Enterococcus faecium
6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins
Authors: Floriano Belén , Ruiz-Barba José L. , Jiménez-Díaz Rufino
Published: December 2019

Enterocin I (ENTI) is a novel bacteriocin produced by
Enterococcus faecium
6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and
Listeria monocytogenes
. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C
2
/C
18
reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the
entI
structural gene, which is located on the 23-kb plasmid pEF1 of
E. faecium
6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of
entI
, encodes a putative protein that is 72.7% identical to ENTI.
entI
and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1.
Species-Specific Oligonucleotides for Enumeration of
Pseudomonas putida
F1,
Burkholderia
sp. Strain JS150, and
Bacillus subtilis
ATCC 7003 in Biodegradation Experiments
Authors: DuTeau Nancy M. , Rogers Julia D. , Bartholomay Christian T. , Reardon Kenneth F.
Published: December 2019

Species-specific sequences were identified within the V4 variable region of 16S rRNA of two bacterial species capable of aromatic hydrocarbon metabolism,
Pseudomonas putida
F1 and
Burkholderia
sp. strain JS150, and a third,
Bacillus subtilis
ATCC 7003, that can function as a secondary degrader. Fluorescent in situ hybridization (FISH) with species-specific oligonucleotides was used for direct counting of these species throughout a phenol biodegradation experiment in batch culture. Traditional differential plate counting methods could not be used due to the similar metabolism and interactions of the primary degraders and difficulties in selecting secondary degraders in mixed culture. In contrast, the FISH method provided reliable quantitative results without interference from those factors.
Effect of Metabolites Produced by
Trichoderma harzianum
Biotypes and
Agaricus bisporus
on Their Respective Growth Radii in Culture
Authors: Mumpuni A. , Sharma H. S. S. , Brown Averil E.
Published: December 2019

Trichoderma harzianum
biotypes Th1, Th2, and Th3 produced volatile metabolites in vitro which had similar fungistatic effects on the growth of
Agaricus bisporus
. Metabolites present in agar colonized by these strains also inhibited mycelial growth of
A. bisporus
, although the reduction in growth was less in the presence of metabolites produced by biotype Th2 than that in the presence of metabolites produced by Th1 or Th3.
A. bisporus
produced metabolites in liquid culture that inhibited the growth of Th1 and Th3 but stimulated the growth of Th2. A compound(s) responsible for the inhibition and stimulation was extracted from
A. bisporus
culture filtrate and from compost-grown fruit bodies with
n
-butanol, but the identity of the compound(s) was not determined. We suggest that the stimulation of Th2 by metabolites produced by
A. bisporus
and the relatively low level of inhibition of
A. bisporus
by Th2 facilitate colonization of compost by both fungi. However, as compost colonization reaches a maximum, a change in the competitive balance in favor of Th2 results in the inhibition of fruit body production by
A. bisporus
and the devastating green mold epidemics affecting mushroom production.
Optimization of Bacteriocin Release Protein (BRP)-Mediated Protein Release by
Escherichia coli
: Random Mutagenesis of the pCloDF13-Derived BRP Gene To Uncouple Lethality and Quasi-Lysis from Protein Release
Authors: van der Wal Fimme J. , Koningstein G. , ten Hagen C. M. , Oudega Bauke , Luirink Joen
Published: December 2019

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the
Escherichia coli
periplasm into the culture medium. However, high-level expression of BRP causes apparent lysis of the host cells in liquid cultures (quasi-lysis) and inhibition of growth on broth agar plates (lethality). To optimize BRP-mediated protein release, the pCloDF13 BRP gene was subjected to random mutagenesis by using PCR techniques. Mutated BRPs with a strongly reduced capacity to cause growth inhibition on broth agar plates were selected, analyzed by nucleotide sequencing, and further characterized by performing growth and release experiments in liquid cultures. A subset of these BRP derivatives did not cause quasi-lysis and had only a small effect on growth but still functioned in the release of the periplasmic protein β-lactamase and the periplasmic K88 molecular chaperone FaeE and in the release of the bacteriocin cloacin DF13 into the culture medium. These BRP derivatives can be more efficiently used for extracellular production of proteins by
E. coli
than can the original BRP.
Lacticin 3147, a Broad-Spectrum Bacteriocin Which Selectively Dissipates the Membrane Potential
Authors: McAuliffe Olivia , Ryan Maire P. , Ross R. Paul , Hill Colin , Breeuwer Pieter , Abee Tjakko
Published: December 2019

Lacticin 3147 is a broad-spectrum bacteriocin produced by
Lactococcus lactis
subsp.
lactis
DPC3147 (M. P. Ryan, M. C. Rea, C. Hill, and R. P. Ross, Appl. Environ. Microbiol. 62:612–619, 1996). Partial purification of the bacteriocin by hydrophobic interaction chromatography and reverse-phase fast protein liquid chromatography revealed that two components are required for full activity. Lacticin 3147 is bactericidal against
L. lactis
,
Listeria monocytogenes
, and
Bacillus subtilis
; at low concentrations of the bacteriocin, bactericidal activity is enhanced when target cells are energized. This finding suggests that the presence of a proton motive force promotes the interaction of the bacteriocin with the cytoplasmic membrane, leading to the formation of pores at these low lacticin 3147 concentrations. These pores were shown to be selective for K
+
ions and inorganic phosphate. The loss of these ions resulted in immediate dissipation of the membrane potential and hydrolysis of internal ATP, leading to an eventual collapse of the pH gradient at the membrane and ultimately to cell death. Our results suggest that lacticin 3147 is a pore-forming bacteriocin which acts on a broad range of gram-positive bacteria.
Alcaligenes eutrophus
as a Bacterial Chromate Sensor
Authors: Peitzsch Nicola , Eberz Günther , Nies Dietrich H.
Published: December 2019

In
Alcaligenes eutrophus
CH34, determinants encoding inducible resistance to chromate (
chr
) and to cobalt and nickel (
cnr
) are located adjacent to each other on plasmid pMOL28. To develop metal-sensing bacterial strains, a cloned part of plasmid pMOL28, which contains both determinants, was mutated with Tn
5-lacZ
. The
chr
::
lacZ
fusions were specifically induced by chromium;
cnr
was induced best by Ni
2+
but was also induced by Co
2+
, Mn
2+
, chromate, Cu
2+
, Cd
2+
, and Zn
2+
. The broad-host-range IncP1 plasmid pEBZ141, which contains a
chr
::
lux
fusion, was constructed.
A. eutrophus
AE104(pEBZ141), carrying a
chr
::
lux
transcriptional fusion, could be used as a biosensor for chromate when cultivated in glycerol as an optimal carbon source. Chromate and bichromate were the best inducers; induction by Cr
3+
was 10 times lower, and other ions induced only a little or not at all. Interactions among induction of the
chr
resistance determinant, chromate reduction, chromate accumulation, and the sulfate concentration of the growth medium were demonstrated.
Characterization of Marine Temperate Phage-Host Systems Isolated from Mamala Bay, Oahu, Hawaii
Authors: Jiang Sunny C. , Kellogg Christina A. , Paul John H.
Published: December 2019

To understand the ecological and genetic role of viruses in the marine environment, it is critical to know the infectivity of viruses and the types of interactions that occur between marine viruses and their hosts. We isolated four marine phages from turbid plaques by using four indigenous bacterial hosts obtained from concentrated water samples from Mamala Bay, Oahu, Hawaii. Two of the rod-shaped bacterial hosts were identified as
Sphingomonas paucimobilis
and
Flavobacterium
sp. All of the phage isolates were tailed phages and contained double-stranded DNA. Two of the phage isolates had morphologies typical of the family
Siphoviridae
, while the other two belonged to the families
Myoviridae
and
Podoviridae
. The head diameters of these viruses ranged from 47 to 70.7 nm, and the tail lengths ranged from 12 to 146 nm. The burst sizes ranged from 7.8 to 240 phage/bacterial cell, and the genome sizes, as determined by restriction digestion, ranged from 36 to 112 kb. The members of the
Siphoviridae
, T-φHSIC, and T-φD0, and the member of the
Myoviridae
, T-φD1B, were found to form lysogenic associations with their bacterial hosts, which were isolated from the same water samples. Hybridization of phage T-φHSIC probe with lysogenic host genomic DNA was observed in dot blot hybridization experiments, indicating that prophage T-φHSIC was integrated within the host genome. These phage-host systems are available for use in studies of marine lysogeny and transduction.
Effect of Low Temperatures on Growth, Structure, and Metabolism of
Campylobacter coli
SP10
Authors: Höller Christiane , Witthuhn Doris , Janzen-Blunck Birgit
Published: December 2019

The effect of low temperatures on the survival, structure, and metabolism of
Campylobacter coli
SP10, a virulent strain, was investigated.
C. coli
became nonculturable rapidly at 20 and 10°C and slightly later at 4°C. Incubation in a microaerobic atmosphere improved survival, but after day 8, campylobacters were detectable by direct-count procedures only. The increase in the number of coccoid cells was most pronounced at 37°C but also was noticeable at 20 and 10°C. Two forms of coccoid cells were seen electron microscopically, but only one (20 and 10°C) seemed to be a degenerative form. The flagella were shorter at 20 and 10°C, a result which correlates well with the observed slight changes in the 62-kDa protein band. The fatty acid composition of bacterial cells was influenced significantly by low temperatures. An increase in the short-chain and unsaturated acids was noted; above all, a drastic increase in C
19:0
cyc at 20°C with a concomitant decrease in C
18:1
trans9,cis11 was seen. The concentrations of excreted metabolites were analyzed to obtain information on metabolic activity. Depending on the magnitude of the temperature downshift, the production of organic acids decreased, but it was always observable after a temperature-specific lag phase and regardless of ability to be cultured. Under optimal conditions, succinate, lactate, and acetate were the main metabolites, other acids being of less importance. The pattern changed significantly at lower temperatures. Succinate was never detected at 20°C and was only occasionally detected at 10 and 4°C. At the same time, fumarate concentrations, which are normally not detectable at 37°C, were highest at 20°C and reduced at 10 and 4°C. Inactivation of fumarate reductase was considered to be a possible explanation.
Endophytic Fungi in Indigenous Australasian Grasses Associated with Toxicity to Livestock
Authors: Miles Christopher O. , di Menna Margaret E. , Jacobs Surrey W. L. , Garthwaite Ian , Lane Geoffrey A. , Prestidge Ron A. , Marshall Sergio L. , Wilkinson Heather H. , Schardl Christopher L. , Ball Olivier J.-P. , Latch Garrick C. M.
Published: December 2019

Grazing ofEchinopogonspp. by livestock in Australia has caused symptoms similar to those of perennial ryegrass staggers. We observed an endophytic fungus in the intercellular spaces of the leaves and seeds of New Zealand and Australian specimens ofEchinopogon ovatus. Culture of surface-sterilized seeds from New Zealand specimens yielded a slow-growing fungus. An examination in which immunoblotting and an enzyme-linked immunosorbent assay (ELISA) were used indicated thatE. ovatusplants from Australia and New Zealand were infected with fungi serologically related toNeotyphodium lolii(the endophyte of perennial ryegrass) and otherEpichloeandNeotyphodiumspp. endophytic in pooid grasses. No lolitrems (the indole–diterpenoids implicated as the causative agents of perennial ryegrass staggers), peramine analogs, or ergot alkaloids were detected in the infected specimens by high-performance liquid chromatography or ELISA. However, in endophyte-infectedE. ovatusplants from New Zealand, analogs of the indole–diterpenoid paxilline (thought to be a biosynthetic precursor of the lolitrems and related tremorgens) were detected by ELISA, andN-formylloline was detected by gas chromatography. Endophyte-free specimens of New ZealandE. ovatusdid not contain detectable paxilline analogs or lolines and were more palatable than infected specimens to adults of the pasture pestListronotus bonariensis(Argentine stem weevil). Hyphae similar to those of theE. ovatusendophyte were also found in herbarium specimens ofEchinopogon nutansvar.major,Echinopogon intermedius,Echinopogon caespitosus, andEchinopogon cheeli. This appears to be the first time that an endophyticNeotyphodiumspecies has been identified in grasses endemic to New Zealand or Australia.
Identification of Hydrocarbon-Degrading Bacteria in Soil by Reverse Sample Genome Probing
Authors: Shen Yin , Stehmeier Lester G. , Voordouw Gerrit
Published: December 2019

Bacteria with limited genomic cross-hybridization were isolated from soil contaminated with C5+, a mixture of hydrocarbons, and identified by partial 16S rRNA sequencing. Filters containing denatured genomic DNAs were used in a reverse sample genome probe (RSGP) procedure for analysis of the effect of an easily degradable compound (toluene) and a highly recalcitrant compound (dicyclopentadiene [DCPD]) on community composition. Hybridization with labeled total-community DNA isolated from soil exposed to toluene indicated enrichment of several
Pseudomonas
spp., which were subsequently found to be capable of toluene mineralization. Hybridization with labeled total-community DNA isolated from soil exposed to DCPD indicated enrichment of a
Pseudomonas
sp. or a
Sphingomonas
sp. These two bacteria appeared capable of producing oxygenated DCPD derivatives in the soil environment, but mineralization could not be shown. These results demonstrate that bacteria, which metabolize degradable or recalcitrant hydrocarbons, can be identified by the RSGP procedure.
Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis
Authors: Loferer-Krößbacher M. , Klima J. , Psenner R.
Published: December 2019

We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (
V
) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW of
Escherichia coli
DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and
V
(in cubic micrometers) of bacteria (DW = 435 ·
V
0.86
); i.e., smaller bacteria had a higher ratio of DW to
V
than larger cells. The measured DW of
E. coli
cells ranged from 83 to 1,172 fg, and
V
ranged from 0.1 to 3.5 μm
3
(
n
= 678). Bacterial cells from Piburger See and Gossenköllesee (
n
= 465) had DWs from 3 fg (
V
= 0.003 μm
3
) to 1,177 fg (
V
= 3.5 μm
3
). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to
V
of individual cells varied from 466 fg of C μm
−3
for
V
s of 0.001 to 0.01 μm
3
to 397 fg of C μm
−3
(0.01 to 0.1 μm
3
) and 288 fg of C μm
−3
(0.1 to 1 μm
3
). Exponentially growing and stationary cells of
E. coli
DSM 613 showed conversion factors of 254 fg of C μm
−3
(0.1 to 1 μm
3
) and 211 fg of C μm
−3
(1 to 4 μm
3
), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.
Oxygen Consumption byDesulfovibrioStrains with and without Polyglucose
Authors: van Niel Ed W. J. , Gottschal Jan C.
Published: December 2019

The kinetics of oxygen reduction byDesulfovibrio salexigensMast1 and the role of polyglucose in this activity were examined and compared with those of strains ofD. desulfuricansandD. gigas. Oxidation rates were highest at air saturation (up to 40 nmol of O2min−1mg of protein−1) and declined with decreasing oxygen concentrations. Studies with cell extracts (CE) indicated that NADH oxidase was entirely responsible for the oxygen reduction in strain Mast1. InD. desulfuricansCSN, at least three independent systems appeared to reduce oxygen. Two were active at all oxygen concentrations (NADH oxidase and NADPH oxidase), and one was maximally active at less than 10 μM oxygen. In contrast toD. gigasandD. salexigensstrains, theD. desulfuricansstrains also contained NADH peroxidase and NADPH peroxidase activities and did not accumulate polyglucose under nonlimiting growth conditions. At air saturation, initial activities of the oxidases and peroxidases of cells harvested at the end of the log phase were on the order of 20 to 140 nmol of O2min−1mg of protein−1. In all strains, these enzymes were relatively stable but were susceptible to inactivation as soon as substrates were added to the assay mixture. Under those conditions, all oxidation activity disappeared after ca. 1 h of incubation. The same finding was observed with whole cells ofD. desulfuricansCSN andD. desulfuricansATCC 27774, but inactivation was less pronounced with cells ofD. salexigensMast1. It appeared that the presence of polyglucose in the whole cells retarded the process of inactivation of NADH oxidase, but this property was lost in crude CE. In spite of the effect of polyglucose on the oxidative potential, oxygen-dependent growth ofD. salexigensMast1 could be demonstrated neither in batch nor in continuous culture.
Dibiphytanyl Ether Lipids in Nonthermophilic Crenarchaeotes
Authors: DeLong Edward F. , King Linda L. , Massana Ramon , Cittone Henry , Murray Alison , Schleper Christa , Wakeham Stuart G.
Published: December 2019

The kingdom
Crenarchaeota
is now known to include archaea which inhabit a wide variety of low-temperature environments. We report here lipid analyses of nonthermophilic crenarchaeotes, which revealed the presence of cyclic and acyclic dibiphytanylglycerol tetraether lipids. Nonthermophilic crenarchaeotes appear to be a major biological source of tetraether lipids in marine planktonic environments.
Hydrostatic Pressure Enhances Vital Staining with Carboxyfluorescein or Carboxydichlorofluorescein in
Saccharomyces cerevisiae
: Efficient Detection of Labeled Yeasts by Flow Cytometry
Authors: Abe Fumiyoshi
Published: December 2019

The extent of intracellular accumulation of the fluorescent dye carboxyfluorescein or carboxydichlorofluorescein (CDCF) in
Saccharomyces cerevisiae
was found to be increased 5- to 10-fold under a nonlethal hydrostatic pressure of 30 to 50 MPa. This observation was confirmed by analysis of individual labeled cells by flow cytometry. The pressure-induced enhancement of staining with CDCF required
d
-glucose and was markedly inhibited by 2-deoxy-
d
-glucose, suggesting that glucose metabolism has a role in the process.
Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in
Bacillus subtilis
: Streptokinase with Longer Functional Half-Life
Authors: Wu Xu-Chu , Ye Ruiqiong , Duan Yanjun , Wong Sui-Lam
Published: December 2019

The short in vivo half-life of streptokinase limits its efficacy as an efficient blood clot-dissolving agent. During the clot-dissolving process, streptokinase is processed to smaller intermediates by plasmin. Two of the major processing sites are Lys59 and Lys386. We engineered two versions of streptokinase with either one of the lysine residues changed to glutamine and a third version with both mutations. These mutant streptokinase proteins (muteins) were produced by secretion with the protease-deficient
Bacillus subtilis
WB600 as the host. The purified muteins retained comparable kinetics parameters in plasminogen activation and showed different degrees of resistance to plasmin depending on the nature of the mutation. Muteins with double mutations had half-lives that were extended 21-fold when assayed in a 1:1 molar ratio with plasminogen in vitro and showed better plasminogen activation activity with time in the radial caseinolysis assay. This study indicates that plasmin-mediated processing leads to the inactivation of streptokinase and is not required to convert streptokinase to its active form. Plasmin-resistant forms of streptokinase can be engineered without affecting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.
Regulation of the
Aspergillus nidulans
Penicillin Biosynthesis Gene
acvA
(
pcbAB
) by Amino Acids: Implication for Involvement of Transcription Factor PACC
Authors: Bergh Katharina Then , Brakhage Axel A.
Published: December 2019

The β-lactam antibiotic penicillin is produced as an end product by some filamentous fungi only. It is synthesized from the amino acid precursors
l
-α-aminoadipic acid,
l
-cysteine, and
l
-valine. Previous data suggested that certain amino acids play a role in the regulation of its biosynthesis. Therefore, in this study the effects of externally added amino acids on both
Aspergillus
(
Emericella
)
nidulans
penicillin production and expression of the bidirectionally oriented biosynthesis genes
acvA
(
pcbAB
) and
ipnA
(
pcbC
) were comprehensively investigated. Different effects caused by amino acids on the expression of penicillin biosynthesis genes and penicillin production were observed. Amino acids with a major negative effect on the expression of
acvA-uidA
and
ipnA-lacZ
gene fusions, i.e., histidine, valine, lysine, and methionine, led to a decreased ambient pH during cultivation of the fungus. An analysis of deletion clones lacking binding sites for the pH-dependent transcriptional factor PACC in the intergenic regions between
acvA-uidA
and
ipnA-lacZ
gene fusions and in a
pacC5
mutant (PacC5-5) suggested that the negative effects of histidine and valine on
acvA-uidA
expression were due to reduced activation by PACC under acidic conditions. These data also implied that PACC regulates the expression of
acvA
, predominantly through PACC binding site ipnA3. The repressing effect caused by lysine and methionine on
acvA
expression, however, was even enhanced in one of the deletion clones and the
pacC5
mutant strain, suggesting that regulators other than PACC are also involved.
Use of an Oxygen-Insensitive Microscale Biosensor for Methane To Measure Methane Concentration Profiles in a Rice Paddy
Authors: Damgaard Lars R. , Revsbech Niels Peter , Reichardt Wolfgang
Published: December 2019

An oxygen-insensitive microscale biosensor for methane was constructed by furnishing a previously described biosensor with an oxygen guard. The guard consisted of a glass capillary containing heterotrophic bacteria, which consumed oxygen diffusing through the tip membrane, thus preventing it from diffusing into the methane-sensing unit. Oxygen microprofiles were measured through the oxygen guard capillary, demonstrating the principle and limitations of the method. When the tip of the guard capillary was exposed to 100% oxygen at 21°C, heterotrophic oxygen consumption prevented oxygen from diffusing further than 170 μm into the capillary, whereas atmospheric levels of oxygen were consumed within 50 μm. The capacity of the oxygen guard for scavenging oxygen decreased with decreasing temperature, and atmospheric levels of oxygen caused oxygen penetration to 200 μm at 5°C. The sensors could be manufactured with tip diameters as small as 25 μm, and response times were about 1 min at room temperature. Pore water profiles of methane concentrations in a rice paddy soil were measured, and a strong correlation between the depths of oxygen penetration and methane appearance was observed as a function of the light regimen; this finding confirmed the role of microbenthic photosynthesis in limiting methane emissions from surfaces of waterlogged sediments and soils.
PCR Amplification of Ribosomal DNA for Species Identification in the Plant Pathogen Genus
Phytophthora
Authors: Ristaino Jean B. , Madritch Michael , Trout Carol L. , Parra Gregory
Published: December 2019

We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genus
Phytophthora
. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymes
Rsa
I,
Msp
I, and
Hae
III. Restriction fragment patterns were similar after digestions with
Rsa
I for the following species:
P. capsici
and
P. citricola
;
P. infestans
,
P. cactorum
, and
P. mirabilis
;
P. fragariae
,
P. cinnamomi
, and
P. megasperma
from peach;
P. palmivora
,
P. citrophthora
,
P. erythroseptica
, and
P. cryptogea
; and
P. megasperma
from raspberry and
P. sojae
. Restriction digests with
Msp
I separated
P. capsici
from
P. citricola
and separated
P. cactorum
from
P. infestans
and
P. mirabilis
. Restriction digests with
Hae
III separated
P. citrophthora
from
P. cryptogea
,
P. cinnamomi
from
P. fragariae
and
P. megasperma
on peach,
P. palmivora
from
P. citrophthora
, and
P. megasperma
on raspberry from
P. sojae. P. infestans
and
P. mirabilis
digests were identical and
P. cryptogea
and
P. erythroseptica
digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in
P. capsici
was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of
P. capsici
,
P. citricola
, and
P. citrophthora
and not 13 other species in the genus. Restriction digests with
Msp
I separated
P. capsici
from the other two species. PCR was superior to traditional isolation methods for detection of
P. capsici
in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genus
Phytophthora.
Pyruvate Decarboxylase Catalyzes Decarboxylation of Branched-Chain 2-Oxo Acids but Is Not Essential for Fusel Alcohol Production by
Saccharomyces cerevisiae
Authors: ter Schure Eelko G. , Flikweert Marcel T. , van Dijken Johannes P. , Pronk Jack T. , Verrips C. Theo
Published: December 2019

The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of pyruvate decarboxylase (EC
4.1.1.1
) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type
Saccharomyces cerevisiae
strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three
PDC
genes had been disrupted. Experiments with cell extracts from
S. cerevisiae
mutants expressing a single
PDC
gene demonstrated that both
PDC1
- and
PDC5
-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type
S. cerevisiae
and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production.
The
Desulfuromonas acetoxidans
Triheme Cytochrome
c
7
Produced in
Desulfovibrio desulfuricans
Retains Its Metal Reductase Activity
Authors: Aubert Corinne , Lojou Elisabeth , Bianco Pierre , Rousset Marc , Durand Marie-Claire , Bruschi Mireille , Dolla Alain
Published: December 2019

Multiheme cytochrome
c
proteins that belong to class III have been recently shown to exhibit a metal reductase activity, which could be of great environmental interest, especially in metal bioremediation. To get a better understanding of these activities, the gene encoding cytochrome
c
7
from the sulfur-reducing bacterium
Desulfuromonas acetoxidans
was cloned from genomic DNA by PCR and expressed in
Desulfovibrio desulfuricans
G201. The expression system was based on the
cyc
transcription unit from
Desulfovibrio vulgaris
Hildenborough and led to the synthesis of holocytochrome
c
7
when transferred by electrotransformation into the sulfate reducer
Desulfovibrio desulfuricans
G201. The produced cytochrome was indistinguishable from the protein purified from
Desulfuromonas acetoxidans
cells with respect to several biochemical and biophysical criteria and exhibited the same metal reductase activities as determined from electrochemical experiments. This suggests that the molecule was correctly folded in the host organism.
Desulfovibrio desulfuricans
produces functional multiheme
c
-type cytochromes from bacteria belonging to a different genus and may be considered a suitable host for the heterologous biogenesis of multiheme
c
-type cytochromes for either structural or engineering studies. This report, which presents the first example of the transformation of a
Desulfovibrio desulfuricans
strain by electrotransformation, describes work that is the first necessary step of a protein engineering program that aims to specify the structural features that are responsible for the metal reductase activities of multiheme cytochrome
c
7
.